G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
heat shock 60kDa protein 1 (chaperonin)
G00000275 (Mus musculus)

Databases (8)

ENSG00000144381 (Ensembl human gene)
3329 (Entrez Gene)
613 (G2Cdb plasticity & disease)
HSPD1 (GeneCards)
118190 (OMIM)
Marker Symbol
HGNC:5261 (HGNC)
Protein Expression
1523 (human protein atlas)
Protein Sequence
P10809 (UniProt)

Synonyms (2)

  • HSP60

Literature (183)

Pubmed - other

  • No authors listed

  • The promoter polymorphism of the IL-6 gene is associated with levels of antibodies to 60-kDa heat-shock proteins.

    Veres A, Prohászka Z, Kilpinen S, Singh M, Füst G and Hurme M

    Third Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Kútvölgyi út 4, 1125 Budapest, Hungary.

    Elevated levels of antibodies to 60 kDa heat shock proteins are associated with severe coronary heart disease and carotid atherosclerosis. The presence of self hsp60-reacting antibodies can only be partially explained by microbial infections and induction by bacterial hsp65 proteins, since important differences (including the epitope specificity and complement activating ability) between hsp60 and hsp65 reacting antibodies have been shown. The aim of this study was to investigate the possible effects of genetic polymorphisms of different genes of proinflammatory cytokines on anti-hsp60 autoantibody levels. One hundred and seventy-six male blood donors were recruited and antibody levels to human hsp60 and Mycobacterium bovis hsp65 were determined by ELISA. Also in these donors, polymorphisms of the promoter of the IL-6 gene at position -174, the biallelic base exchange of the IL-1 beta gene at the -511 position and the IL-1 alpha gene at position -889 were investigated by PCR. A strong association between IL-6 -174 polymorphism and anti-hsp60 antibody levels was seen; the effect on anti-hsp65 antibody was less marked. Carriers of allele C at this position had significantly lower levels of anti-hsp60 and anti-hsp65 antibodies. A lack of associations between IL-1 beta and IL-1 alpha gene polymorphisms and antibody levels was detected. This is the first study in which associations between genetic polymorphisms and autoantibody levels have been described in healthy subjects. Further studies are needed to gain insight into the detailed mechanism of how the IL-6 gene polymorphism at position -174 influences anti-hsp60 autoantibody levels.


  • Extracellular heat shock protein 60, cardiac myocytes, and apoptosis.

    Kim SC, Stice JP, Chen L, Jung JS, Gupta S, Wang Y, Baumgarten G, Trial J and Knowlton AA

    Molecular & Cellular Cardiology, University of California-Davis, One Shields Ave, Davis, CA 95616, USA.

    Rationale: Previously, we have found that changes in the location of intracellular heat shock protein (HSP)60 are associated with apoptosis. HSP60 has been reported to be a ligand of toll-like receptor (TLR)-4.

    Objective: We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR4.

    Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR4 and by nuclear factor kappaB binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis.

    Conclusions: This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma.

    Funded by: NHLBI NIH HHS: HL077281, HL079071, HL085440-02/05, HL089792-10/04, R01 HL077281, R01 HL077281-01, R01 HL077281-02, R01 HL077281-03, R01 HL077281-04, R01 HL077281-05, R01 HL077281-06, R01 HL077281-07, R01 HL079071, R01 HL079071-01A2, R01 HL079071-02, R01 HL079071-03, R01 HL079071-04, R01 HL085440, R01 HL089792, R01 HL089792-03

    Circulation research 2009;105;12;1186-95

  • The MitCHAP-60 disease is due to entropic destabilization of the human mitochondrial Hsp60 oligomer.

    Parnas A, Nadler M, Nisemblat S, Horovitz A, Mandel H and Azem A

    Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, 69778 Tel Aviv, Israel.

    The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the co-chaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, MitCHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Man 1f40 del, H. (2008) Am. J. Hum. Genet. 83, 30-42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.

    The Journal of biological chemistry 2009;284;41;28198-203

  • Heat shock proteins in children and young adults on chronic hemodialysis.

    Musiał K, Szprynger K, Szczepańska M and Zwolińska D

    Department of Pediatric Nephrology, Wrocław Medical University, 50-369 Wrocław, Poland.

    Chronic inflammation, lipid and autoimmune disorders are hallmarks of atherogenesis, and hemodialysis per se may be an additional factor predisposing to accelerated atherosclerosis. Elevated levels of heat shock proteins (HSP) and antibodies against these HSP have been described in adults with atherosclerotic lesions and cardiovascular events, but to date there has been a scarcity of investigations on these parameters in adult and pediatric patients on hemodialysis (HD). We have investigated the HSP profile in hemodialyzed children and the impact of a single HD session on those proteins and their correlations with known risk factors for atherosclerosis. The study group consisted of 17 children and young adults undergoing HD with polysulfone membranes. The control group comprised 15 age-matched subjects with normal kidney function. The serum concentrations of Hsp60, Hsp90alpha, anti-Hsp60, anti-Hsp70, and sE-selectin were assessed by an enzyme-linked immunosorbent assay, and serum concentration of high-sensitivity-C-reactive protein was assayed by nephelometry. The serum lipid profile [total cholesterol (CHOL), high-density lipoprotein-CHOL, low-density lipoprotein-CHOL, triglycerides] was also estimated. Compared to the control values, the median values of Hsp60 before the HD session were lower, whereas those of Hsp90alpha and anti-Hsp60 were higher. A single HD session raised the median values of Hsp60 and Hsp90alpha and decreased the concentrations of anti-Hsp60 and anti-Hsp70. In addition, the concentrations of HSPs and the antibodies against them correlated with the lipid markers both before and after HD. The altered HSP and anti-HSP concentrations in HD children, which correlated with the lipid profile and the endothelial markers, suggest a dysfunctional HSP system in this population and the possibility of HSPs being classified as new markers of atherosclerosis.

    Pediatric nephrology (Berlin, Germany) 2009;24;10;2029-34

  • Sequence variants in SPAST, SPG3A and HSPD1 in hereditary spastic paraplegia.

    Svenstrup K, Bross P, Koefoed P, Hjermind LE, Eiberg H, Born AP, Vissing J, Gyllenborg J, Nørremølle A, Hasholt L and Nielsen JE

    Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity and weakness in the lower limbs. The most common forms of autosomal dominant HSP, SPG4 and SPG3, are caused by sequence variants in the SPAST and SPG3A genes, respectively. The pathogenic variants are scattered all over these genes and many variants are unique to a specific family. The phenotype in SPG4 patients can be modified by a variant in SPAST (p.Ser44Leu) and recently, a variant in HSPD1, the gene underlying SPG13, was reported as a second genetic modifier in SPG4 patients. In this study HSP patients were screened for variants in SPG3A, SPAST and HSPD1 in order to identify disease causing variations. SPAST was sequenced in all patients whereas subsets were sequenced in HSPD1 and in selected exons of SPG3A. SPG4 patients and their HSP relatives were genotyped for the modifying variant in HSPD1. We report six new sequence variants in SPAST including a fourth non synonymous sequence v 347 ariant in exon 1 and two synonymous changes of which one has been found in a HSP patient previously, but never in controls. Of the novel variants in SPAST four were interpreted as disease causing. In addition one new disease causing sequence variant and one non pathogenic non synonymous variant were found in SPG3A. In HSPD1 we identified a sporadic patient homozygote for the potential modifying variation. The effect of the modifying HSPD1 variation was not supported by identification in one SPG4 family.

    Journal of the neurological sciences 2009;284;1-2;90-5

  • Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia.

    Molvarec A, Derzsy Z, Kocsis J, Boze T, Nagy B, Balogh K, Makó V, Cervenak L, Mézes M, Karádi I, Prohászka Z and Rigó J

    1st Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary. molvarec@freemail.hu

    It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer's kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.

    Cell stress & chaperones 2009;14;5;491-8

  • Mitochondrial heat shock protein (HSP) 70 synergizes with HSP60 in transducing endothelial cell apoptosis induced by anti-HSP60 autoantibody.

    Alard JE, Dueymes M, Mageed RA, Saraux A, Youinou P and Jamin C

    Université Européenne de Bretagne, Brest, France.

    Heat shock protein (HSP) 60, up-regulated by endothelial cells (ECs) to resist stress, is the target of a subgroup of apoptosis-inducing anti-EC autoantibodies (Abs) in human vasculitides. Given that HSP60 is not a transmembrane protein, the mechanism by which these auto-Abs induces apoptosis is unclear. EC membrane proteins were analyzed using bidimensional electrophoresis and Far Western blot, and the HSP60 receptor was identified by mass spectrometry. Heat stress-dependent synthesis of HSP60 and receptor was examined by semiquantitative RT-PCR, and expression was examined by flow cytometry and indirect immunofluorescence. Interaction was demonstrated by coimmunoprecipitations. Lipid rafts were purified to evaluate specific localization, and the apoptotic response was investigated by blocking monoclonal Ab. Mitochondrial HSP70 (mtHSP70) was identified as an HSP60 receptor. Stress was required for ECs to up-regulate mRNA and express mtHSP70 on their surface. HSP60 and mtHSP70 colocalized and interacted within lipid rafts. They were associated with chemokine CC motif receptor 5 (CCR5), also induced at the mRNA and protein levels in stressed ECs. CCR5 was involved in the anti-HSP60-triggered apoptosis of ECs. These results provide new insights into the mechanism by which anti-EC auto-Abs from vasculitides induce apoptosis of ECs.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;8;2772-9

  • Interaction of hepatitis C virus core protein with Hsp60 triggers the production of reactive oxygen species and enhances TNF-alpha-mediated apoptosis.

    Kang SM, Kim SJ, Kim JH, Lee W, Kim GW, Lee KH, Choi KY and Oh JW

    Department of Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, South Korea.

    The hepatitis C virus (HCV) core protein is the primary protein component of the nucleocapsid that encapsidates the viral RNA genome. Besides its role as a viral structural protein, the core protein is implicated in HCV chronic infection-associated liver diseases by induction of reactive oxygen species (ROS) production and modulation of apoptosis. Here, we show that interaction of the core protein, through its N-terminal domain (amino acids 1-75), with heat shock protein (Hsp60) is critical for the induction of ROS production, leading to sensitization of core protein-expressing cells to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). Moreover, overexpression of Hsp60 rescued the core protein-expressing cells from cell death by reducing ROS production. Collectively, our results suggest that impairment of Hsp60 function through binding of HCV core protein contributes to HCV viral pathogenesis by ROS generation and amplification of the apoptotic effect of TNF-alpha.

    Cancer letters 2009;279;2;230-7

  • Increased circulating heat shock protein 60 induced by menopause, stimulates apoptosis of osteoblast-lineage cells via up-regulation of toll-like receptors.

    Kim YS, Koh JM, Lee YS, Kim BJ, Lee SH, Lee KU and Kim GS

    Asan Institute for Life Sciences, Seoul, Republic of Korea.

    Postmenopausal osteoporosis is a heterogeneous disorder characterized by accelerated bone loss after natural or surgical menopause and an increased risk of fractures. The bone loss in estrogen deficiency results from the increased bone resorption and impaired ability of osteoblastic bone formation. Previous studies have reported that the HSP60 stimulates osteoclast formation and bone resorption. Here we found that plasma HSP60 levels were significantly higher in postmenopausal (median 1152.4 ng/ml; range 724.7-2123.4 ng/ml) than in premenopausal (median 316.3 ng/ml; range 164.6-638.4 ng/ml) women. In primary human bone marrow stromal cells (hBMSC) and the HS-5 hBMSC cell line, HSP60 significantly reduced cell viability and increased caspase-dependent apoptosis. Consistent with these observations, HSP60 activated caspase-3 and -9, but not caspase-8 in HS-5 cells, and increased the release of mitochondrial cytochrome c into the cytosol. In addition, HSP60 activated p38 and NFkappaB, but not ERK or JNK; importantly, inhibitors of p38 (SB203580) and NFkappaB (PDTC) abolished HSP60-induced apoptosis. Furthermore, Western blotting showed that HSP60 up-regulated TLR-2 and TLR-4 expression, and pretreatment with blocking antibodies for TLR-2 and TLR-4 almost completely eliminated the effects of HSP60 on apoptosis, caspase-3 and -9 activation, and activation of NFkappaB and p38 MAPK. Most notably, ovariectomy-induced bone loss was attenuated in TLR-2 KO mice. In conclusion, up-regulation of TLR-2 by HSP60 may play a critical role in promoting bone loss in the estrogen-deficient state.

    Bone 2009;45;1;68-76

  • Myocardial heat shock protein 60 expression is upregulated following acute cardiac rejection.

    Sarri S, Shaw SM, Gieschen-Krische MA, Archer L, Yonan N and Fildes JE

    The Transplant Centre, University Hospital of South Manchester NHS Foundation Trust, Wythenshawe Hospital, Manchester, UK.

    Background: Heat shock proteins (HSPs) are endogenous adjuvants which are upregulated following cellular injury. HSP60 may be upregulated in response to myocardial injury, inducing activation via TLR ligation on monocytes, dendritic cells and T cells. On these grounds, this study was designed to assess HSP60 expression during the rejection process following cardiac transplantation.

    Methods: Intracellular and cell surface endomyocardial concentration of HSP60 was quantified longitudinally in a cohort of heart transplant recipients (n = 17, 54 biopsy samples) using competitive sandwich ELISA.

    Results: HSP 60 concentration was low before and during an acute rejection episode. Surprisingly an increase of HSP60 was observed in the period following rejection during recovery (p = 0.026).

    Conclusions: This novel data demonstrates that human endomyocardial HSP60 is increased following an acute rejection episode. This may occur following endomyocardial damage as a result of immune cell infiltration and graft cell damage. However, in contrast to the general assumption that this molecule represents a danger signal, our findings suggest HSP60 expression may be induced as part of a protective response following tissue damage.

    Transplant immunology 2009;21;3;140-2

  • Pan-DR-binding Hsp60 self epitopes induce an interleukin-10-mediated immune response in rheumatoid arthritis.

    de Jong H, Lafeber FF, de Jager W, Haverkamp MH, Kuis W, Bijlsma JW, Prakken BJ and Albani S

    University Medical Centre Utrecht, Utrecht, The Netherlands.

    Objective: Human Hsp60 is expressed in the joints of patients with rheumatoid arthritis (RA) and can elicit a regulatory T cell response in the peripheral blood and synovial fluid. However, Hsp60 can also trigger strong proinflammatory pathways. Thus, to understand the nature of these Hsp60-directed responses in RA, it is necessary to study such responses at the molecular, epitope-specific level. This study was undertaken to characterize the disease specificity and function of pan-DR-binding Hsp60-derived epitopes as possible modulators of autoimmune inflammation in RA.

    Methods: Lymphocyte proliferation assays (using (3)H-thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester [CFSE] staining) and measurement of cytokine production (using multiplex immunoassay and intracellular staining) were performed after in vitro activation of peripheral blood mononuclear cells from patients with RA, compared with healthy controls.

    Results: A disease (RA)-specific immune eb1 recognition, characterized by T cell proliferation as well as increased production of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-10, was found for 3 of the 8 selected peptides in patients with RA as compared with healthy controls (P < 0.05). Intracellular cytokine staining and CFSE labeling showed that CD4+ T cells were the subset primarily responsible for both the T cell proliferation and the cytokine production in RA. Interestingly, the human peptides had a remarkably different phenotype, with a 5-10-fold higher IL-10:TNFalpha ratio, compared with that of the microbial peptides.

    Conclusion: These results suggest a disease-specific immune-modulatory role of epitope-specific T cells in the inflammatory processes of RA. Therefore, these pan-DR-binding epitopes could be used as a tool to study the autoreactive T cell response in RA and might be suitable candidates for use in immunotherapy.

    Arthritis and rheumatism 2009;60;7;1966-76

  • Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia.

    Martins-de-Souza D, Gattaz WF, Schmitt A, Maccarrone G, Hunyadi-Gulyás E, Eberlin MN, Souza GH, Marangoni S, Novello JC, Turck CW and Dias-Neto E

    Laboratório de Neurociências, Instituto de Psiquiatria, Faculdade de Medicina da USP, Rua Dr. Ovídio Pires de Campos, SP, Brazil. martins@mpipsykl.mpg.de

    Schizophrenia is likely to be a consequence of serial alterations in a number of genes that, together with environmental factors, will lead to the establishment of the illness. The dorsolateral prefrontal cortex (Brodmann's Area 46) is implicated in schizophrenia and executes high functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts, correct social behavior and personality expression. We performed a comparative proteome analysis using two-dimensional gel electrophoresis of pools from 9 schizophrenia and 7 healthy control patients' dorsolateral prefrontal cortex aiming to identify, by mass spectrometry, alterations in protein expression that could be related to the disease. In schizophrenia-derived samples, our analysis revealed 10 downregulated and 14 upregulated proteins. These included alterations previously implicated in schizophrenia, such as oligodendrocyte-related proteins (myelin basic protein and transferrin), as well as malate dehydrogenase, aconitase, ATP synthase subunits and cytoskeleton-related proteins. Also, six new putative disease markers were identified, including energy metabolism, cytoskeleton and cell signaling proteins. Our data not only reinforces the involvement of proteins previously implicated in schizophrenia, but also suggests new markers, providing further information to foster the comprehension of this important disease.

    Journal of psychiatric research 2009;43;11;978-86

  • Expression of heat shock protein 60 kDa is upregulated in cervical cancer.

    Hwang YJ, Lee SP, Kim SY, Choi YH, Kim MJ, Lee CH, Lee JY and Kim DY

    Division of Biological Science, Gachon University of Medicine and Science, 534-2 Yeonsu-dong, Yeonsu-gu,Incheon, Korea.

    Purpose: Cervical cancer caused by the human papilloma virus (HPV) continues to be the cause of yearly death among women. However, it is a curable disease when diagnosed at an early stage. Recently, several researches have reported that heat shock protein (HSP) 60, a chaperone protein of molecular weight of 60 kDa, is involved in carcinogenesis and apoptosis. In order to evaluate the prognostic significance of HSP60 in cervical cancer, we examined differences in the HSP60 expression between cervical cancer and normal tissues in women.

    Tissue samples were collected from 20 cervical cancer patients and 20 normal controls. HSP60 expression of cervical cancer and normal tissues were verified by the 2D gel proteomics, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analyses.

    Results: In 2D proteomic analysis, an increase of HSP60 expression was detected in cervical cancer tissues and confirmed by Western blot analysis (p < 0.05). However, messenger RNA (mRNA) levels of HSP60 did not display any significant differences between cervical cancer and normal tissues.

    Conclusion: These results suggest that HSP60 may be involved in the development of cervical cancer and have profound biological and prognostic significance.

    Yonsei medical journal 2009;50;3;399-406

  • Integrated associations of genotypes with multiple blood biomarkers linked to coronary heart disease risk.

    Drenos F, Talmud PJ, Casas JP, Smeeth L, Palmen J, Humphries SE and Hingorani AD

    Division of Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School, 5 University St, London WC1E 6JF, UK.

    Individuals at risk of coronary heart disease (CHD) show multiple correlations across blood biomarkers. Single nucleotide polymorphisms (SNPs) indexing biomarker differences could help distinguish causal from confounded associations because of their random allocation prior to disease. We examined the association of 948 SNPs in 122 candidate genes with 12 CHD-associated phenotypes in 2775 middle aged men (a genic scan). Of these, 140 SNPs indexed differences in HDL- and LDL-cholesterol, triglycerides, C-reactive protein, fibrinogen, factor VII, apolipoproteins AI and B, lipoprotein-associated phospholipase A2, homocysteine or folate, some with large effect sizes and highly significant P-values (e.g. 2.15 standard deviations at P = 9.2 x 10(-140) for F7 rs6046 and FVII levels). Top ranking SNPs were then tested for association with additional biomarkers correlated with the index phenotype (phenome scan). Several SNPs (e.g. in APOE, CETP, LPL, APOB and LDLR) influenced multiple phenotypes, while others (e.g. in F7, CRP and FBB) showed restricted association to the index marker. SNPs influencing six blood proteins were used to evaluate the nature of the associations between correlated blood proteins utilizing Mendelian randomization. Multiple SNPs were associated with CHD-related quantitative traits, with some associations restricted to a single marker and others exerting a wider genetic 'footprint'. SNPs indexing biomarkers provide new tools for investigating biological relationships and causal links with disease. Broader and deeper integrated analyses, linking genomic with transcriptomic, proteomic and metabolomic analysis, as well as clinical events could, in principle, better delineate CHD causing pathways amenable to treatment.

    Funded by: British Heart Foundation: FS2005/125, PG2005/014, RG/08/008/25291; Wellcome Trust: 082178

    Human molecular genetics 2009;18;12;2305-16

  • Interaction between HSP60 and beta-catenin promotes metastasis.

    Tsai YP, Yang MH, Huang CH, Chang SY, Chen PM, Liu CJ, Teng SC and Wu KJ

    Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

    Heat shock protein 60 (HSP60) plays an essential role in assisting many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in different types of human cancers with metastasis (e.g. pancreatic cancer and large bowel carcinoma). However, the role of HSP60 in metastasis remains little known. Aberrant activation of beta-catenin plays a key role in tumorigenesis and metastasis. Here, we show that overexpression of HSP60 induces metastatic phenotypes in vitro and in vivo. HSP60 interacts with beta-catenin, increases beta-catenin protein levels through the apical domain and enhances its transcriptional activity. Short-interference RNA-mediated repression of beta-catenin reverts metastatic activity caused by HSP60 overexpression. Proteosomal activity is not required for the induction of beta-catenin by HSP60. Coexpression of HSP60 and nuclear beta-catenin predicts a worse prognosis of metastatic head and neck cancer patients. These results implicate a novel role of HSP60 in metastasis.

    Carcinogenesis 2009;30;6;1049-57

  • Mitochondrial DNA variability modulates mRNA and intra-mitochondrial protein levels of HSP60 and HSP75: experimental evidence from cybrid lines.

    Bellizzi D, Taverna D, D'Aquila P, De Blasi S and De Benedictis G

    Department of Cell Biology, University of Calabria, 87036, Rende, Italy. dina.bellizzi@unical.it

    To explore possible relationships between mitochondrial DNA (mtDNA) polymorphism and the expression levels of stress-responder nuclear genes we assembled five cybrid cell lines by repopulating 143B.TK(-) cells, depleted of their own mtDNA (Rho(0) cells), with foreign mitochondria with different mtDNA sequences (lines H, J, T, U, X). We evaluated, at both basal and under heat stress conditions, gene expression (mRNA) and intra-mitochondrial protein levels of HSP60 and HSP75, two key components in cellular stress response. At basal conditions, the levels of HSP60 and HSP75 mRNA were lower in one cybrid (H) than in the others (p = 0.005 and p = 0.001, respectively). Under stress conditions, the H line over-expressed both genes, so that the inter-cybrid difference was abolished. Moreover, the HSP60 intra-mitochondrial protein levels differed among the cybrid lines (p = 0.001), with levels higher in H than in the other cybrid lines. On the whole, our results provide further experimental evidence that mtDNA variability influences the cell response to stressful conditions by modulating components involved in this response. Sentence summary of the article: the results reported in the present study provide important experimental evidence that in human cells mtDNA variability is able to influence the cellular response to heat stress by modulating both the transcription of genes involved in this response and their intra-mitochondrial protein levels.

    Cell stress & chaperones 2009;14;3;265-71

  • A comparative analysis of the products of GROEL-1 gene from Chlamydia trachomatis serovar D and the HSP60 var1 transcript from Homo sapiens suggests a possible autoimmune response.

    Campanella C, Marino Gammazza A, Mularoni L, Cappello F, Zummo G and Di Felice V

    Sezione di Anatomia Umana 'E. Luna', Dipartimento di Medicina Sperimentale, Università di Palermo, Palermo, Italy.

    Chlamydia trachomatis ser 1f40 ovar D produces large quantities of HSP60-1 during infections, which accumulate inside the host cell inducing autoimmunity. We compare the aminoacid sequences of the human HSP60 with the bacterial counterpart to better elucidate how CTHSP60 may simulate HSP60 from human origin during infection and may induce an autoimmune response. As a result of the comparison we suggest several possible epitopes of the CTHSP60, which may induce autoimmunity.

    International journal of immunogenetics 2009;36;1;73-8

  • Correlation of fragile histidine triad (Fhit) protein structural features with effector interactions and biological functions.

    Pichiorri F, Okumura H, Nakamura T, Garrison PN, Gasparini P, Suh SS, Druck T, McCorkell KA, Barnes LD, Croce CM and Huebner K

    Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University and Comprehensive Cancer Center, Columbus, Ohio 43210, USA.

    We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G(2)/M or sub-G(1) fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.

    Funded by: NCI NIH HHS: CA115965, CA132453, CA77738, P01 CA077738, R01 CA115965, R01 CA132453

    The Journal of biological chemistry 2009;284;2;1040-9

  • Is heat shock protein 60 associated with type 2 diabetes mellitus?

    Imatoh T, Sugie T, Miyazaki M, Tanihara S, Baba M, Momose Y, Uryu Y and Une H

    Department of Hygiene and Preventive Medicine, Faculty of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan. imatoh@cis.fukuoka-u.ac.jp

    Aims: HSP60 plays a protective role against heat, oxidative injury and ultraviolet. Recently, animal and clinical studies have suggested that HSP60 plays a role in various diseases. However, few epidemiological studies have demonstrated an association between HSP60 levels and type 2 diabetes mellitus. Therefore, an epidemiological study was conducted to examine the association of HSP60 with type 2 diabetes mellitus.

    Methods: This study included 83 type 2 diabetes mellitus patients and 161 controls that were recruited from male employees who received annual health check-ups between 2005 and 2007. The serum HSP60 levels were measured using the ELISA method.

    Results: Because the HSP60 levels were not detectable (<3.125 ng/mL) in 48.0% of the study subjects, HSP60 levels were divided into two categories (detectable or undetectable). A logistic regression analysis showed that the subjects in the undetectable had a 2.03 times higher risk of diabetes mellitus than those in the detectable after adjustment for age, BMI and rate of hypertension medication.

    Conclusions: This study was the first epidemiological study to demonstrate an association between type 2 diabetes mellitus and HSP60, thus suggesting that HSP60 may play an important role in the type 2 diabetes mellitus pathology.

    Diabetes research and clinical practice 2009;85;2;208-12

  • RNA interference mediated silencing of Hsp60 gene in human monocytic myeloma cell line U937 revealed decreased dengue virus multiplication.

    Padwad YS, Mishra KP, Jain M, Chanda S, Karan D and Ganju L

    Immunomodulation Laboratory, Defence Institute of Physiology and Allied Sciences, Timarpur, Delhi 110054, India.

    Heat shock proteins (Hsps) or stress proteins are highly conserved molecules and expressed in all cell types under stressful conditions like heat, cold, hypoxia and infections. The objective of the present study was to determine the effect of dengue virus infection on relative expression of stress proteins and their role in the progression of the infection. As macrophages are the primary host for dengue, human promonocytic myeloblastoma U937 cells were infected with dengue virus type 2 New Guinea C strain for the evaluation of Hsps expression. A significant expression of Hsp60 was observed in virally infected U937 cells as compared to controls. In order to determine the correlation between Hsp60 expression and viral multiplication in infected cells, expression of Hsp60 was down regulated by RNA interference. Viral multiplication was determined by quantification of viral RNA copy number using Real Time PCR and plaque formation assay in cellular supernatants of Hsp60 silenced cells. Intracellular quantification of viral load was also determined by flow cytometry. It was observed that down regulation of Hsp60 in virally infected cells resulted into decrease in viral RNA copy number, plaque forming units and intracellular viral load. At the same time down regulation also resulted in increased IFN-alpha level. These observations suggest that, elevated levels of Hsp60 expression in virally infected cells may help in viral multiplication and could be possible therapeutic targets for the management of dengue virus infection.

    Immunobiology 2009;214;6;422-9

  • Elevated heat shock protein 60 levels are associated with higher risk of coronary heart disease in Chinese.

    Zhang X, He M, Cheng L, Chen Y, Zhou L, Zeng H, Pockley AG, Hu FB and Wu T

    Institute of Occupational Medicine, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd, Wuhan, Hubei 430030, China.

    Background: Although heat shock protein 60 (Hsp60) is implicated in the pathogenesis of atherosclerosis, its role in coronary heart disease (CHD) is uncertain. This study explored the influence of circulating Hsp60 on CHD in a large case-control study, as well as the impact of acute myocardial infarction on Hsp60 levels in a prospective study.

    Plasma Hsp60 and anti-Hsp60 antibody levels were determined by immunoassay. In the case-control study (1003 patients with CHD, 1003 matched control subjects), Hsp60 levels were higher in patients with CHD and were related to CHD (OR comparing extreme quartiles=4.14, P<0.0001). This association remained after adjustment for traditional risk factors (P for trend <0.0001). Individuals having high levels of Hsp60 (greater than the median of 160.24 ng/mL) and anti-Hsp60 antibody (greater than the median of 38.42 U/mL) were at a greater risk of CHD than those with low levels (OR 2.30, P<0.0001). Stronger additive effects (OR 14.04, P<0.0001) were apparent at higher Hsp60 and anti-Hsp60 antibody levels (>1000 ng/mL and greater than the median of 38.42 U/mL, respectively). The simultaneous presence of high Hsp60 and anti-Hsp60 antibody levels, current smoking, hypertension, and diabetes were cumulatively associated with CHD. Individuals who had any 4 or more of these 5 factors had an OR of 38.61 for CHD (P<0.0001) compared with individuals who had none of these factors. For the prospective study, blood was drawn from 20 patients immediately after admission for acute myocardial infarction and 2, 3, and 7 days thereafter. Hsp60 levels were significantly higher on the day of and the day after arrival than 7 days after an acute myocardial infarction (P=0.011 and P=0.026, respectively).

    Conclusions: Elevated Hsp60 levels are associated with an increased risk for CHD, and Hsp60 and anti-Hsp60 antibody levels combine to increase this risk. In addition, acute myocardial infarction induces Hsp60 release.

    Circulation 2008;118;25;2687-93

  • Direct regulation of HSP60 expression by c-MYC induces transformation.

    Tsai YP, Teng SC and Wu KJ

    Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No. 115, Li-Nong St., Peitou, Taipei 112, Taiwan.

    The c-MYC proto-oncogene encodes a ubiquitous transcription factor involved in cell proliferation and tumorigenesis. Heat shock protein 60 (HSP60) plays an essential role in assisting many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in different types of human cancer. Here we show that c-MYC directly activates HSP60 transcription through an E-box (CACGTG) site located in the proximal promoter of the HSP60 gene. Overexpression of HSP60 induces transformation. Short-interference RNA (siRNA) mediated repression of HSP60 reduces transformation caused by c-MYC overex 1f40 pression. These results indicate that c-MYC may promote transformation through the induction of HSP60 expression.

    FEBS letters 2008;582;29;4083-8

  • The relationship between carotid stiffness and circulating levels of heat shock protein 60 in middle-aged men and women.

    Ellins E, Shamaei-Tousi A, Steptoe A, Donald A, O'Meagher S, Halcox J and Henderson B

    Vascular Physiology Unit, Institute of Child Health, University College London, UK.

    Objective: There is growing evidence that the presence of the cell stress protein heat shock protein (HSP) 60 in the circulation is associated with risk of coronary heart disease. In this study, we measured the association between plasma HSP60 and carotid arterial stiffness in middle-aged men and women.

    Methods: Six hundred and forty-seven men and women aged 50-72 years and free of cardiovascular disease and medication were tested. Carotid artery distensibility coefficient was assessed ultrasonically as a measure of arterial stiffness, and plasma HSP60 was assessed using a sensitive immunoassay.

    Results: We found a significant, independent association between high plasma levels of HSP60 and increased carotid stiffness. Carotid distensibility coefficient was also related to diabetes, adiposity, blood pressure, lipids, plasma interleukin-6 and C-reactive protein. After adjusting for these factors, the odds of HSP60 concentration of at least 1000 ng/ml were 1.79 (95% confidence intervals 1.06-3.04) for participants in the lowest compared with the highest tertile of the distensibility coefficie 1d2f nt.

    Conclusion: HSP60 is a potent activator of vascular endothelial cells and smooth muscle cells. Thus, it is possible that long-term stimulation of these cell populations by blood-borne HSP60 acts to drive blood vessel changes resulting in decreased arterial elasticity.

    Funded by: British Heart Foundation; Medical Research Council

    Journal of hypertension 2008;26;12;2389-92

  • cis-acting sequences and trans-acting factors in the localization of mRNA for mitochondrial ribosomal proteins.

    Russo A, Cirulli C, Amoresano A, Pucci P, Pietropaolo C and Russo G

    Dipartimento di Biochimica e Biotecnologie Mediche, Università Federico II, Via Sergio Pansini 5, Napoli 80131, Italy.

    mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3'-UTR. Here we show by biochemical analysis that these mRNA transcripts are associated with the cytoskeleton through the microtubule network. Cytoskeleton association is functional for their intracellular localization near the mitochondrion, and the 3'-UTR is involved in this cytoskeleton-dependent localization. To identify the minimal elements required for localization, we generated DNA constructs containing, downstream from the GFP gene, deletion mutants of mitochondrial ribosomal protein S12 3'-UTR, and expressed them in HeLa cells. RT-PCR analysis showed that the localization signals responsible for mRNA localization are located in the first 154 nucleotides. RNA pull-down assays, mass spectrometry, and RNP immunoprecipitation assay experiments, demonstrated that mitochondrial ribosomal protein S12 3'-UTR interacts specifically with TRAP1 (tumor necrosis factor receptor-associated protein1), hnRNPM4 (heterogeneous nuclear ribonucleoprotein M4), Hsp70 and Hsp60 (heat shock proteins 70 and 60), and alpha-tubulin in vitro and in vivo.

    Biochimica et biophysica acta 2008;1779;12;820-9

  • Chaperonin chamber accelerates protein folding through passive action of preventing aggregation.

    Apetri AC and Horwich AL

    Howard Hughes Medical Institute and Department of Genetics, Yale School of Medicine, Boyer Center, New Haven, CT 06510, USA.

    The original experiments reconstituting GroEL-GroES-mediated protein folding were carried out under "nonpermissive" conditions, where the chaperonin system was absolutely required and substrate proteins could not achieve the native state if diluted directly from denaturant into solution. Under "permissive" conditions, however, employing lower substrate concentration and lower temperature, some substrate proteins can be refolded both by the chaperonin system and while free in solution. For several of these, the protein refolds more rapidly inside the GroEL-GroES cis chamber than free in solution, suggesting that the chamber may have an active role in assisting protein folding. Here, we observe that the difference is caused by reversible multimolecular association while folding in solution, an avenue of kinetic partitioning that slows the overall rate of renaturation relative to the chaperonin chamber, where such associations cannot occur. For Rubisco, reversible aggregation during folding in solution was observed by gel filtration. For a mutant of maltose-binding protein (DM-MBP), the rate of folding in solution declined with increasing concentration, and the folding reaction produced light scattering. Under solution conditions where chloride was absent, however, light scattering no longer occurred, and DM-MBP folded at the same rate as in the cis cavity. In a further test, dihydrofolate reductase, thermally inactivated in the cis cavity or in solution, was substantially reactivated upon temperature downshift in the cis cavity but not in solution, where aggregation occurred. We conclude that the GroEL-GroES chamber behaves as a passive "Anfinsen cage" whose primary role is to prevent multimolecular association during folding.

    Funded by: Howard Hughes Medical Institute

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;45;17351-5

  • Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells.

    Qiu J, Gao HQ, Liang Y, Yu H and Zhou RH

    Department of Geriatrics, Qilu Hospital of Shandong University, 107 Wenhua Xi Road, Jinan 250012, PR China.

    Although digitalis has been used in clinical treatment extensively, the precise mechanism of its toxic actions on cardiovascular system remained unclear, it would be of interest to study the differential proteomic analysis of vascular endothelial cells in response to toxic concentrations of digitalis thus to provide new agents for treatment of digitalis-induced cytotoxicity. We employed human umbilical vein endothelial cells (HUVEC) as our model system. HUVEC were exposed to increasing concentrations (0.1 nM-10 microM) of digoxin at 12-96 h intervals. Cell viability tests revealed that digoxin played dual effects on cell growth. Apoptosis detection confirmed that apoptosis was primarily responsible for digoxin-induced cell death. Proteomics analysis further revealed that the digoxin-induced apoptosis was accompanied by regulated expression of ATP synthase beta chain, cystatin A, electron transfer flavoprotein, heterogeneous nuclear ribonucleoproteins H3, lamin A, profilin-1, proteasome subunit 5, succinyl-CoA ligase beta chain and heat shock protein 60 (HSP60). Deep study on the overexpression of HSP60 confirmed that HSP60 exerted a protective role in digoxin-induced apoptosis through inhibition of caspase-3 activity in HUVEC. These results provided an impetus for further delineation of mechanism of digoxin-induced cytotoxicity and offered new agents that help attenuate its toxicity.

    Biochimica et biophysica acta 2008;1784;11;1857-64

  • Serum levels of antibodies to Chlamydia pneumoniae and human HSP60 in giant cell arteritis patients.

    López-Hoyos M, Alvarez L, Ruiz Soto M, Blanco R, José Bartolomé M and Martínez-Taboada VM

    Divisions of Immunology, Hospital Universitario Marqués de Valdecilla, Spain. inmlhm@humv.es

    Objective: To measure the serum levels of IgG anti-Chlamydia pneumoniae (C. pneumoniae) and human heat shock protein (hHSP) 60 antibodies in patients with active giant cell arteritis (GCA) and to determine whether such levels decrease with corticosteroid therapy and remission of symptoms.

    Methods: IgG anti-C. pneumoniae and anti-hHSP60 antibodies were quantified by commercial and in-house ELISA tests, respectively, in serum samples from 17 GCA patients, 39 polymyalgia rheumatica (PMR) patients and 23 age-matched healthy subjects.

    Results: Serum IgG anti-hHSP60, but not anti-C. pneumoniae, antibodies were significantly increased in GCA patients in comparison with PMR patients or healthy controls. After steroid therapy, both anti-hHSP60 and -C. p 192d neumoniae antibodies decreased significantly in both groups of patients.

    Conclusions: Although some infectious agents have been suggested to participate in GCA pathogenesis, our data do not suggest that C. pneumoniae might be one of them. The production of anti-hHSP60 antibodies is shared in GCA with other systemic diseases and may be triggered by unknown infectious agents and/or other inflammatory factors.

    Clinical and experimental rheumatology 2008;26;6;1107-10

  • Heat shock proteins 27, 60 and 70 as prognostic markers of prostate cancer.

    Glaessgen A, Jonmarker S, Lindberg A, Nilsson B, Lewensohn R, Ekman P, Valdman A and Egevad L

    Department of Oncology, Karolinska Institutet, Stockholm, Sweden.

    Heat shock proteins (HSPs) protect cells against stress-associated injury and are overexpressed in several malignant tumors. We aimed to investigate their value as prognostic markers in prostate cancer. A tissue microarray (TMA) was constructed of 289 prostate cancers from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. Slides were immunostained for HSP27, HSP60 and HSP70. Intensity and extent of immunoreactivity (IR) and their product (IRp) was evaluated by two observers. The IRp of HSP27 and HSP60, but not of HSP70, significantly predicted biochemical recurrence (p=0.014, 0.034 and 0.160, respectively). Recurrence-free survival in patients with strong HSP27 and HSP60 staining was shorter than in those with weak expression (p=0.019 and 0.001, respectively). IRp of HSP27 and HSP60 correlated with Gleason score (p<0.01). HSP60 was an independent predictor of biochemical recurrence in multivariate analysis, including extraprostatic extension, margin status, seminal vesicle invasion and Gleason score. Weighted kappa for interobserver agreement of HSP27, HSP60 and HSP70 IR was 0.613-0.823 for intensity and 0.584-0.719 for IRp, but only 0.036-0.244 for extent, raising the question whether staining extent should be estimated on TMA. We conclude that HSP27 and HSP60 are predictors of biochemical recurrence after RP.

    APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 2008;116;10;888-95

  • Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells.

    Campanella C, Bucchieri F, Ardizzone NM, Marino Gammazza A, Montalbano A, Ribbene A, Di Felice V, Bellafiore M, David S, Rappa F, Marasà M, Peri G, Farina F, Czarnecka AM, Conway de Macario E, Macario AJ, Zummo G and Cappello F

    Human Anatomy Section, Department of Experimental Medicine, University of Palermo, via del Vespro 129, 90127 Palermo, Italy.

    Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.

    European journal of histochemistry : EJH 2008;52;4;221-8

  • Mitochondrial hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain hypomyelination and leukodystrophy.

    Magen D, Georgopoulos C, Bross P, Ang D, Segev Y, Goldsher D, Nemirovski A, Shahar E, Ravid S, Luder A, Heno B, Gershoni-Baruch R, Skorecki K and Mandel H

    Pediatric Nephrology Unit, Rambam Health Care Campus, Haifa 31096, Israel.

    Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration.

    American journal of human genetics 2008;83;1;30-42

  • The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo.

    Bross P, Naundrup S, Hansen J, Nielsen MN, Christensen JH, Kruhøffer M, Palmfeldt J, Corydon TJ, Gregersen N, Ang D, Georgopoulos C and Nielsen KL

    Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, Arhus 8200, Denmark. peter.bross@Kl.au.dk

    We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harb c8f ors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.

    The Journal of biological chemistry 2008;283;23;15694-700

  • Differential effect of oxidative stress on the apoptosis of early and late passage human diploid fibroblasts: implication of heat shock protein 60.

    Lee YH, Lee JC, Moon HJ, Jung JE, Sharma M, Park BH, Yi HK and Jhee EC

    Department of Oral Biochemistry, School of Dentistry, Chonbuk National University, Korea.

    Since an attenuated response to stress is a characteristic of senescence, a cellular senescence model was used to examine the mechanism of resistance against oxidative stress using human diploid fibroblasts (HDF). With increasing passage, the HDF showed increased production of reactive oxygen species (ROS). Late passage HDF were resistant to the lethal effects of oxidative stress, showing less cleavage of pro-caspase-3 and PARP than those of early ones. Since heat shock proteins (Hsps) are not only cytoprotective but also interfere with the apoptotic cascade, the expression patterns of Hsps during cellular senescence were next examined. Oxidative stress induced a decrease in the mitochondrial Hsp60 levels with a concomitant increase in the cytosolic Hsp60 levels in the early passage HDF, but not in late ones. To show that the resistance to oxidative stress is a specific effect of Hsp60, the levels of Hsp60 were knocked down by siRNA. As expected the Hsp60 knock-down cells were more resistant to oxidative stress. These findings show that Hsp60 is a key player in the resistance mechanism against oxidative stress and aging.

    Cell biochemistry and function 2008;26;4;502-8

  • Fhit interaction with ferredoxin reductase triggers generation of reactive oxygen species and apoptosis of cancer cells.

    Trapasso F, Pichiorri F, Gaspari M, Palumbo T, Aqeilan RI, Gaudio E, Okumura H, Iuliano R, Di Leva G, Fabbri M, Birk DE, Raso C, Green-Church K, Spagnoli LG, Venuta S, Huebner K and Croce CM

    Ohio State University, Comprehensive Cancer Center, Columbus, Ohio 43210, USA.

    Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We 1f40 have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

    Funded by: NCI NIH HHS: CA77738, CA78890, P01 CA077738, P01 CA078890

    The Journal of biological chemistry 2008;283;20;13736-44

  • HSP60 is a rare cause of hereditary spastic paraparesis, but may act as a genetic modifier.

    Hewamadduma CA, Kirby J, Kershaw C, Martindale J, Dalton A, McDermott CJ and Shaw PJ

    Academic Neurology Unit, Medical School, Beech Hill Road, University of Sheffield, S10 2RX, UK. channa.hewamadduma@sth.nhs.uk

    Funded by: Department of Health; Wellcome Trust

    Neurology 2008;70;19;1717-8

  • A vicious cycle involving release of heat shock protein 60 from injured cells and activation of toll-like receptor 4 mediates neurodegeneration in the CNS.

    Lehnardt S, Schott E, Trimbuch T, Laubisch D, Krueger C, Wulczyn G, Nitsch R and Weber JR

    Center for Anatomy, Institute for Cell Biology and Neurobiology, Charité-Universitaetsmedizin Berlin, 10117 Berlin, Germany. seija.lehnardt@charite.de

    Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;10;2320-31

  • Hsp60-mediated T cell stimulation is independent of TLR4 and IL-12.

    Osterloh A, Veit A, Gessner A, Fleischer B and Breloer M

    Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany. osterloh@bni-hamburg.de

    Heat shock protein (Hsp) 60 is thought to function as endogenous danger signal by activating professional antigen-presenting cells (APC) through toll-like receptor (TLR) 4 and CD14, a mechanism that is also used by bacterial LPS. We recently showed that Hsp60 binds LPS and enhances LPS-induced immune stimulation. On the other hand, we also observed immune stimulation by Hsp60 independent of LPS which was partially mediated by Hsp60-induced IFN alpha. Here, we study the mechanisms involved in immune stimulation mediated by endotoxin-free Hsp60. We show that T cell co-stimulation induced by LPS-free Hsp60 was independent of TLR4 and the TLR-associated myeloide differentiation factor 88-signaling pathway. LPS-free Hsp60 did not induce IL-6, IL-12 or tumor necrosis factor alpha production in APC nor were these cytokines needed for Hsp60-mediated T cell co-stimulation in the absence of LPS. In contrast to endotoxin-free Hsp60, T cell co-stimulation induced by LPS or Hsp60/LPS complexes strictly depended on IL-12 and functional TLR-4. Furthermore, we show that LPS-free Hsp60 enhances IFN alpha expression in APC and that this cytokine represents one important mediator in immune stimulation by Hsp60 in the absence of LPS. Taken together, we provide evidence that endotoxin-free Hsp60 and LPS or Hsp60/LPS complexes employ different signaling mechanisms to transduce co-stimulatory signals.

    International immunology 2008;20;3;433-43

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Hsp60 regulation of tumor cell apoptosis.

    Ghosh JC, Dohi T, Kang BH and Altieri DC

    Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachustetts 01605, USA.

    Molecular chaperones may promote cell survival, but how this process is regulated, especially in cancer, is not well understood. Using high throughput proteomics screening, we identified the cell cycle regulator and apoptosis inhibitor survivin as a novel protein associated with the molecular chaperone Hsp60. Acute ablation of Hsp60 by small interfering RNA destabilizes the mitochondrial pool of survivin, induces mitochondrial dysfunction, and activates caspase-dependent apoptosis. This response involves disruption of an Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and Bax-dependent apoptosis. In vivo, Hsp60 is abundantly expressed in primary human tumors, as compared with matched normal tissues, and small interfering RNA ablation of Hsp60 in normal cells is well tolerated and does not cause apoptosis. Therefore, Hsp60 orchestrates a broad cell survival program centered on stabilization of mitochondrial survivin and restraining of p53 function, and this process is selectively exploited in cancer. Hsp60 inhibitors may function as attractive anticancer agents by differentially inducing apoptosis in tumor cells.

    Funded by: NCI NIH HHS: CA78810, CA90917, R01 CA090917; NHLBI NIH HHS: HL54131, R37 HL054131

    The Journal of biological chemistry 2008;283;8;5188-94

  • Hsp60 in inflamed muscle tissue is the target of regulatory autoreactive T cells in patients with juvenile dermatomyositis.

    Elst EF, Klein M, de Jager W, Kamphuis S, Wedderburn LR, van der Zee R, Albani S, Kuis W and Prakken BJ

    University Medical Center Utrecht, Utrecht, The Netherlands.

    Objective: Juvenile dermatomyositis (DM) is an autoimmune disease of unknown origin characterized by muscle weakness and skin manifestations. No definite autoantigen has yet been identified. Heat-shock proteins (HSPs) can be up-regulated at sites of inflammation, and immune reactivity to Hsp60 is suggested to play a regulatory role in various chronic inflammatory diseases. The purpose of this study was to determine whether Hsp60 could serve as an autoantigen in juvenile DM.

    Methods: Muscle tissue from 4 patients with juvenile DM and 1 healthy control subject without evidence of muscle disease was stained for Hsp60. Peripheral blood mononuclear cells (PBMCs) from 22 patients and 10 healthy control subjects were tested for T cell proliferation induced by human and microbial Hsp60. Cytokine production in response to Hsp60 was examined in 15 patients and 6 healthy controls. T cell reactivity to Hsp60 was determined in muscle biopsy samples from 2 patients.

    Results: We found significantly increased T cell proliferation to human Hsp60 in PBMCs from juvenile DM patients, which was higher during disease remission. Following in vitro activation with Hsp60, significant amounts of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 were produced. In contrast to muscle biopsy samples from healthy controls, samples from juvenile DM patients showed up-regulation of Hsp60, induction of T cell proliferation, and production of cytokines. Production of proinflammatory cytokines by muscle-derived cells in response to Hsp60 was associated with a poor clinical prognosis, whereas human Hsp60-specific induction of IL-10 was followed by clinical remission.

    Conclusion: These findings suggest that human (self) Hsp60 is a disease-relevant autoantigen in juvenile DM. The difference in T cell response with regard to disease activity indicates an immune regulatory effect of Hsp60-specific T cells, opening up perspectives for antigen-specific immunotherapy.

    Arthritis and rheumatism 2008;58;2;547-55

  • Genetic variation in heat shock protein 60 gene and coronary heart disease in China: tagging-SNP haplotype analysis in a case-control study.

    He MA, Zhang X, Wang J, Cheng L, Zhou L, Zeng H, Wang F, Chen Y, Xu Z, Wei Q, Hu FB and Wu T

    Institute of Occupational Medicine and the Ministry of Education Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

    Background: High levels of circulating heat shock protein 60 (Hsp60) and antibody to human Hsp60 have been associated with greater risk of coronary heart disease (CHD) in several studies, but associations between polymorphisms of the hsp60 gene and CHD risk have not been investigated.

    Methods: By resequencing DNA from 30 unrelated Han Chinese and using HapMap Phase I Chinese data of hsp60 gene, we selected four tagging single nucleotide polymorphisms (tagSNPs) named rs2340690, rs788016, rs2305560, and rs2565163, and determined their frequencies in 1,003 Chinese CHD patients and 1,003 age- and sex-frequency-matched controls. Furthermore, we used PHASE 2.0 software to reconstruct haplotypes and logistic regression to control for potential confounders in multivariate analyses.

    Results: We found 13 SNPs in hsp60 gene (including four novel SNPs) in Han Chinese subjects. Our results showed no significant differences in four selected SNPs in patients with CHD and controls after adjusting for other conventional risk factors and stratifying by age, sex, smoking status, past history of hypertension and DM; however, our results showed that subjects with the GCTC haplotype had about twofold higher risk of CHD than those with the GTTC haplotype (OR = 1.91, 95%CI: 1.26-2.89, P = 0.002).

    Conclusions: Our results suggest that the GCTC haplotype in the hsp60 gene is significantly associated with higher CHD risk in a Chinese population.

    Cell stress & chaperones 2008;13;2;231-8

  • Cytosolic accumulation of HSP60 during apoptosis with or without apparent mitochondrial release: evidence that its pro-apoptotic or pro-survival functions involve differential interactions with caspase-3.

    Chandra D, Choy G and Tang DG

    Department of Carcinogenesis, the University of Texas M.D. Anderson Cancer Center, Science Park--Research Division, Smithville, Texas 78957, USA.

    Most heat shock proteins (HSPs) have pro-survival functions. However, the role of HSP60, a mitochondrial matrix protein, is somewhat controversial with both pro-survival and pro-apoptotic functions reported. Here we show that in numerous apoptotic systems HSP60 protein accumulates in the cytosol. In BMD188-induced cell death, HSP60 accumulates in the cytosol with significant mitochondrial release. In contrast, in apoptosis induced by multiple other inducers, the cytosolic HSP60 accumulates without an apparent mitochondrial release. The short interfering RNA-mediated knockdown experiments revealed that in BMD188-induced apoptosis, HSP60 has a pro-death function and that the pro-death role of HSP60 seems to involve caspase-3 maturation and activation in the cytosol. In contrast, HSP60 appears to play a pro-survival role in other apoptotic systems where there is no apparent mitochondrial release as its knockdown promotes cell death. In these latter apoptotic systems HSP60 does not associate with active caspase-3. In both cases, HSP60 does not appreciably interact with Bax. Taken together, our results suggest the following: 1) cytosolic accumulation of HSP60 represents a common phenomenon during apoptosis induction; 2) cytosolic HSP60 accumulation during apoptosis occurs either with or without apparent mitochondrial release; and 3) the cytosolically accumulated HSP60 possesses either pro-survival or pro-death functions, which involves differential interactions with caspase-3.

    Funded by: NCI NIH HHS: K012 CA123142; NIA NIH HHS: AG023374; NIEHS NIH HHS: ES07784, ES15888

    The Journal of biological chemistry 2007;282;43;31289-301

  • Expression of Hsp60 and Grp78 in the human endometrium and oviduct, and their effect on sperm functions.

    Lachance C, Bailey JL and Leclerc P

    Département d'Obstétrique et de Gynécologie, Centre de Recherche en Biologie de la Reproduction, Université Laval, Unité de Recherche en Ontogénie et Reproduction, Centre de recherche du CHUQ (CHUL), T1-49, 2705 boul. Laurier, Québec, QC, Canada G1V 4G2.

    Background: Within the female genital tract, spermatozoa undergo a series of membranous and intracellular transformations to become competent at fertilizing the oocyte. In the bovine, previous studies have shown that two oviductal proteins, heat shock protein 60 (Hsp60) and glucose regulated protein 78 (Grp78), bind to spermatozoa and may be involved in this acquisition of fertilizing competence.

    Methods: Immunohistochemical studies were performed on human endometrial and oviduct tissues to localize these two chaperones in the female genital tract. Human spermatozoa were incubated under capacitating conditions in the presence or absence of recombinant Hsp60 or Grp78. Following a 4-h incubation, the effects of these proteins were evaluated on sperm acrosomal integrity, motility, protein phosphotyrosine content and free intracellular calcium concentrations.

    Results: Both chaperones were present in the uterus and oviduct epithelial cells and were shown to bind to human spermatozoa. Incubation with either exogenous Hsp60 or Grp78 did not affect sperm viability, motility or acrosomal integrity. Hsp60 partially prevented the increase in p81 phosphotyrosine content induced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and both chaperones significantly increased the sperm intracellular calcium concentration. Moreover, the progesterone-induced increase in intracellular calcium was higher when sperm were pre-treated with either Hsp60 or Grp78.

    Conclusions: Our study suggests that these two proteins may affect human sperm intracellular signalling pathways and capacitation.

    Human reproduction (Oxford, England) 2007;22;10;2606-14

  • HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis.

    Lin L, Kim SC, Wang Y, Gupta S, Davis B, Simon SI, Torre-Amione G and Knowlton AA

    Molecular and Cellular Cardiology, Cardiovascular Division, University of California, Davis, California 95616, USA.

    Heat shock protein (HSP) 60 is a mitochondrial and cytosolic protein. Previously, we reported that HSP60 doubled in end-stage heart failure, even though levels of the protective HSP72 were unchanged. Furthermore, we observed that acute injury in adult cardiac myocytes resulted in movement of HSP60 to the plasma membrane. We hypothesized that the inflammatory state of heart failure would cause translocation of HSP60 to the plasma membrane and that this would provide a pathway for cardiac injury. Two models were used to test this hypothesis: 1) a rat model of heart failure and 2) human explanted failing hearts. We found that HSP60 localized to the plasma membrane and was also found in the plasma early in heart failure. Plasma membrane HSP60 localized to lipid rafts and was detectable on the cell surface with the use of both flow cytometry and confocal microscopy. Localization of HSP60 to the cell surface correlated with increased apoptosis. In heart failure, HSP60 is in the plasma membrane fraction, on the cell surface, and in the plasma. Membrane HSP60 correlated with increased apoptosis. Release of HSP60 may activate the innate immune system, promoting a proinflammatory state, including an increase in TNF-alpha. Thus abnormal trafficking of HSP60 to the cell surface may be an early trigger for myocyte loss and the progression of heart failure.

    Funded by: NHLBI NIH HHS: HL077281, HL079071, R01 HL077281, R01 HL079071; NIAID NIH HHS: AI47294

    American journal of physiology. Heart and circulatory physiology 2007;293;4;H2238-47

  • The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes.

    Xu C, Lu Y, Pan Z, Chu W, Luo X, Lin H, Xiao J, Shan H, Wang Z and Yang B

    Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, Heilongjiang 150086, People's Republic of China.

    The microRNAs miR-1 and miR-133 are preferentially expressed in cardiac and skeletal muscles and have been shown to regulate differentiation and proliferation of these cells. We report here a novel aspect of cellular function of miR-1 and miR-133 regulation of cardiomyocyte apoptosis. miR-1 and miR-133 produced opposing effects on apoptosis, induced by oxidative stress in H9c2 rat ventricular cells, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. miR-1 level was significantly increased in response to oxidative stress. We identified single target sites for miR-1 only, in the 3'-untranslated regions of the HSP60 and HSP70 genes, and multiple putative target sites for miR-133 throughout the sequence of the caspase-9 gene. miR-1 reduced the levels of HSP60 and HSP70 proteins without changing their transcript levels, whereas miR-133 did not affect HSP60 and HSP70 expression at all. By contrast, miR-133 repressed caspase-9 expression at both the protein and mRNA levels. The post-transcriptional repression of HSP60 and HSP70 and caspase-9 was further confirmed by luciferase reporter experiments. Our results indicate that miR-1 and miR-133 are involved in regulating cell fate with increased miR-1 and/or decreased miR-133 levels favoring apoptosis and decreased miR-1 and/or miR-133 levels favoring survival. Post-transcriptional repression of HSP60 and HSP70 by miR-1 and of caspase-9 by miR-133 contributes significantly to their opposing actions.

    Journal of cell science 2007;120;Pt 17;3045-52

  • The role of hereditary spastic paraplegia related genes in multiple sclerosis. A study of disease susceptibility and clinical outcome.

    DeLuca GC, Ramagopalan SV, Cader MZ, Dyment DA, Herrera BM, Orton S, Degenhardt A, Pugliatti M, Sadovnick AD, Sotgiu S and Ebers GC

    University Dept. of Clinical Neurology, University of Oxford, Radcliffe Infirmary, Woodstock Rd, Oxford, OX2 6LE, UK.

    Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system unsurpassed for its variability in disease outcome. It has been observed that axonal loss in MS is significant and that irreversible clinical disability relates to such axonal loss. The clinical similarities between Hereditary Spastic Paraplegia (HSP) and progressive MS, along with their analogous profiles of axonal loss in the long tracts, make the genes known to cause HSP biologically relevant candidates for the study of clinical outcome in MS. A cohort of sporadic MS cases and a set of unaffected controls were used to determine the role of HSP genes on MS susceptibility and disease severity. The MS cases were taken from opposite extremes of the putative distribution of long-term outcome using the most stringent clinical criteria to date. Genotyping the two sets of MS patients and controls could not provide any evidence to suggest that genes involved in the pathogenesis of HSP (Paraplegin, NIPA1, KIF5A, HSPD1, Atlastin, Spartin, Spastin, PLP1, L1CAM, Maspardin and BSCL2) play a role in susceptibility to, or modifying the course of, MS, although small effects of these genes cannot be ruled out.

    Journal of neurology 2007;254;9;1221-6

  • Diverse regulatory activity of human heat shock proteins 60 and 70 on endotoxin-induced inflammation.

    Bangen JM, Schade FU and Flohé SB

    Surgical Research, Department of Trauma Surgery, University Hospital Essen, University Duisburg-Essen, Virchowstr. 171, D-45147 Essen, Germany.

    Tissue injury is often associated with bacterial infection. Intracellular heat shock proteins (HSPs) are released from damaged tissue, come in contact with cells of the immune system, and might affect the immune response against bacteria. In the present study, we investigated the capacity of highly purified human HSP60 and HSP70 to modulate the response of human peripheral blood-derived mononuclear cells (PBMC) to lipopolysaccharide (LPS). HSP70 but not HSP60 decreased the LPS-induced secretion of TNF-alpha when added simultaneously with LPS. In contrast, HSP60 and HSP70 primed PBMC for enhanced secretion of TNF-alpha when added 24h prior to the stimulation with LPS. Neither HSP60 nor HSP70 alone induced the release of TNF-alpha. The capacity of LPS to bind to monocytes was not affected by HSPs, but HSP70 increased the expression of Toll-like receptor 4. Thus, HSP60 and HSP70 released upon tissue damage might play a role in the regulation of bacteria-induced inflammation.

    Biochemical and biophysical research communications 2007;359;3;709-15

  • Ufd1 is a cofactor of gp78 and plays a key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase.

    Cao J, Wang J, Qi W, Miao HH, Wang J, Ge L, DeBose-Boyd RA, Tang JJ, Li BL and Song BL

    State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.

    The membrane-anchored ubiquitin ligase gp78 promotes degradation of misfolded endoplasmic reticulum (ER) proteins and sterol-regulated degradation of HMG-CoA reductase. It was known previously that Ufd1 plays a critical role in ER-associated degradation (ERAD) together with Npl4 and VCP. The VCP-Ufd1-Npl4 complex recognizes polyubiquitin chains and transfers the ubiquitinated proteins to the proteasome. Here we show that Ufd1 directly interacts with gp78 and functions as a cofactor. Ufd1 enhances the E3 activity of gp78, accelerates the ubiquitination and degradation of reductase, and eventually promotes receptor-mediated uptake of low-density lipoprotein. Furthermore, we demonstrate that the monoubiquitin-binding site in Ufd1 is required for the enhancement of gp78 activity and that the polyubiquitin-binding site in Ufd1 is critical for a postubiquitination step in ERAD. In summary, our study identifies Ufd1 as a cofactor of gp78, reveals an unappreciated function of Ufd1 in the ubiquitination reaction during ERAD, and illustrates that Ufd1 plays a critical role in cholesterol metabolism.

    Cell metabolism 2007;6;2;115-28

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • The tumor suppressor PP2A Abeta regulates the RalA GTPase.

    Sablina AA, Chen W, Arroyo JD, Corral L, Hector M, Bulmer SE, DeCaprio JA and Hahn WC

    Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

    The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme family that regulates numerous signaling pathways. Biallelic mutations of the structural PP2A Abeta subunit occur in several types of human tumors; however, the functional consequences of these cancer-associated PP2A Abeta mutations in cell transformation remain undefined. Here we show that suppression of PP2A Abeta expression permits immortalized human cells to achieve a tumorigenic state. Cancer-associated Abeta mutants fail to reverse tumorigenic phenotype induced by PP2A Abeta suppression, indicating that these mutants function as null alleles. Wild-type PP2A Abeta but not cancer-derived Abeta mutants form a complex with the small GTPase RalA. PP2A Abeta-containing complexes dephosphorylate RalA at Ser183 and Ser194, inactivating RalA and abolishing its transforming function. These observations identify PP2A Abeta as a tumor suppressor gene that transforms immortalized human cells by regulating the function of RalA.

    Funded by: NCI NIH HHS: P01 CA050661, P01 CA050661-190009, P01 CA50661

    Cell 2007;129;5;969-82

  • Altered distribution of heat shock protein 60 (Hsp60) with dysregulated expression of DHX32.

    Chen Y, Alli Z, Ackerley C, Al-Saud B and M

    Division of Haematopathology, Department of Paediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Canada.

    DHX32 has an overall similarity to the DHX family of RNA helicases but with a novel helicase domain and nuclear and mitochondrial localizations. The expression of DHX32 is highly regulated during lymphocyte activation and is dysregulated in lymphoid malignancies. In this study, we report our finding of an altered subcellular localization of heat shock protein 60 (Hsp60) in Jurkat-DHX32 cell line in which DHX32 is constitutively expressed. Two-dimensional gel electrophoresis followed by mass spectrometry, electron microscopic immunocytochemistry, and immunoblot analysis showed mainly cytoplasmic localization of Hsp60 in Jurkat-DHX32 cells instead of its mainly mitochondrial localization in control cells. No significant changes were detected in the mitochondrial ultra-structure and the mitochondrial membrane potential activity as a result of dysregulated DHX32 expression. The subcellular distribution of several mitochondrial proteins including cytochrome c, peroxiredoxin 3, manganese superoxide dismutase, Leucine-Rich PentatricoPeptide Repeat Cassette and the 49-kDa subunit of the mitochondrial respiratory Complex I were similar in control and Jurkat-DHX32 cells ruling out non-specific cytoplasmic leakage of mitochondrial proteins. No significant changes in the expression of Hsp60 transcript or total cellular protein were detected. These findings suggest that dysregulated expression of DHX32 might lead to as of yet unknown changes in mitochondrial homeostasis manifested by cytoplasmic redistribution of the molecular chaperon Hsp60.

    Experimental and molecular pathology 2007;82;3;256-61

  • Plasminogen and angiostatin interact with heat shock proteins.

    Dudani AK, Mehic J and Martyres A

    Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Sir Frederick Banting Research Centre, Health Canada, 251 Sir Frederick Banting Way, Tunney's Pasture, Ottawa, Ontario, Canada. anil_dudani@hc-sc.gc.ca

    Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85-90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin's interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15-20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin's binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.

    Molecular and cellular biochemistry 2007;300;1-2;197-205

  • Extracellular heat shock protein 60 (Hsp60) levels in children with septic shock.

    Wheeler DS, Lahni P, Odoms K, Jacobs BR, Carcillo JA, Doughty LA and Wong HR

    Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, 45229-3039, USA. derek.wheeler@cchmc.org

    Recent data suggest that extracellular Hsp60 modulates the host innate immune response. We analyzed plasma Hsp60 levels in children admitted to a level III tertiary care PICU with septic shock.

    Blood samples were obtained from children meeting criteria for septic shock (n = 63), critically ill children without septic shock (n = 10), and healthy controls (n = 24).

    Treatment: Not applicable.

    Methods: Hsp60 levels were measured in the plasma using a commercially available ELISA. Differences between groups were analyzed with a Kruskal-Wallis one way ANOVA due to the non-parametric nature of the data. A p value < or = 0.05 was considered significant.

    Results: Extracellular Hsp60 levels were significantly higher in children with septic shock (median, 16.7 ng/mL) compared to both critically ill children without septic shock (median, 0 ng/mL) and healthy controls (median, 0 ng/mL, p <0.001).

    Conclusions: Extracellular Hsp60 levels are significantly elevated in children with septic shock compared with both healthy controls and critically ill children without sepsis. Extracellular Hsp60 may play a role in the pathogenesis of sepsis in children.

    Funded by: NIGMS NIH HHS: K08-GM077432-01, R01-GM064619, R01-GM61723

    Inflammation research : official journal of the European Histamine Research Society ... [et al.] 2007;56;5;216-9

  • Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.

    Osterloh A, Kalinke U, Weiss S, Fleischer B and Breloer M

    Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany. osterloh@bni.uni-hamburg.de

    Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.

    The Journal of biological chemistry 2007;282;7;4669-80

  • Protective effect of human heat shock protein 60 suggested by its association with decreased seropos 13e7 itivity to pathogens.

    Steptoe A, Shamaei-Tousi A, Gylfe A, Bailey L, Bergström S, Coates AR and Henderson B

    Department of Epidemiology and Public Health, UCL Eastman Dental Institute, University College London, London, United Kingdom.

    The presence of heat shock protein 60 (Hsp60) in human plasma has been linked with cardiovascular disease (CVD). In this study, the examination of the relationship between Hsp60 in plasma and seropositivity for three microbial agents, which are thought to be risk factors for CVD, surprisingly revealed a negative association between Hsp60 and seropositivity, suggesting a protective effect of this circulating stress protein.

    Clinical and vaccine immunology : CVI 2007;14;2;204-7

  • Circulating heat shock proteins and inflammatory markers in patients with idiopathic left ventricular dysfunction: their relationships with myocardial and microvascular impairment.

    Giannessi D, Colotti C, Maltinti M, Del Ry S, Prontera C, Turchi S, Labbate A and Neglia D

    Consiglio Nazionale delle Ricerche Institute of Clinical Physiology, Laboratory of Cardiovascular Biochemistry, Pisa 56100, Italy. danielag@ifc.cnr.it

    Little information is available on peripheral levels of Hsp72, Hsp60, and anti-Hsp60 antibodies in patients with left ventricular (LV) dysfunction due to non-atherosclerotic cardiac disease. In this study, serum Hsp72, Hsp60 and anti-Hsp60 antibodies, IL-6, and C-reactive protein (CRP) were measured in 44 healthy controls and in 82 patients with angiographically normal coronary arteries (LV ejection fraction [EF] > or = 50%, n=22; -35% to <50%, n=32; <35%, n=28). Patients with more severe disease (more depressed myocardial blood flow at rest and during dipyridamole, indicative of coronary microvascular impairment) showed more elevated circulating Hsp60 and auto-antibodies, Hsp72, and CRP levels. IL-6 was increased progressively as a function of severity of LV dysfunction. Anti-Hsp60 antibodies, Hsp72, and IL-6 were significantly correlated with brain natriuretic peptide (BNP) levels and LV end-diastolic dimensions (LVEDD) values. IL-6 tended to be related with Hsp72 in particular in patients with more severe disease (r = 0.45, P = 0.021). Hsp60 and Hsp72 activation and inflammatory markers were correlated with the extent of cardiac and microvascular dysfunction in patients with angiographycally normal coronary arteries. These results suggest a pathogenic role of infective-metabolic insult and inflammatory reaction in the development of vascular and myocardial damage in patients with heart failure even in the absence of overt coronary artery disease.

    Cell stress & chaperones 2007;12;3;265-74

  • HSPD1 is not a major susceptibility gene for rheumatoid arthritis in the French Caucasian population.

    Jacq L, Teixeira VH, Garnier S, Michou L, Dieudé P, Rocha D, Pierlot C, Lemaire I, Quillet P, Hilliquin P, Mbarek H, Petit-Teixeira E and Cornélis F

    GenHotel-EA3886, Evry-Paris VII Universities, 2 rue Gaston Crémieux, 91057 Evry-Genopole cedex, France. Laurent@polyarthrite.net

    The heat shock 60-kDa protein 1 (HSP60) is involved in immune and inflammatory reactions, which are hallmarks of rheumatoid arthritis (RA). HSP60 is encoded by the HSPD1 gene located on 2q33, one of the suggested RA susceptibility loci in the French Caucasian population. Our aim was to test whether HSPD1 is a major susceptibility gene by studing families from the French Caucasian population. Three single nucleotide polymorphisms (SNPs) were studied in 100 RA trio families, and 100 other families were used for replication. Genetic analyses were performed by comparing allelic frequencies, by applying the transmission disequilibrium test, and by assessing the genotype relative risk. We observed a significant RA association for the C/C genotype of rs2340690 in the first sample. However, this association was not confirmed when the second sample was added. The two other SNPs and the haplotype analysis did not give any significant results. We conclude that HSPD1 is not a major RA susceptibility gene in the French Caucasian population.

    Journal of human genetics 2007;52;12;1036-9

  • Plasma heat shock protein 60 and cardiovascular disease risk: the role of psychosocial, genetic, and biological factors.

    Shamaei-Tousi A, Steptoe A, O'Donnell K, Palmen J, Stephens JW, Hurel SJ, Marmot M, Homer K, D'Aiuto F, Coates AR, Humphries SE and Henderson B

    Division of Microbial Diseases, Eastman Dental Institute, University College London, London WC1X 8LD, UK.

    The Whitehall Study is a prospective epidemiological study of cardiovascular risk factors in healthy members of the British Civil Service, which has identified psychological distress as a major risk factor for coronary heart disease. The levels of circulating Hsp60 in 860 participants from the Whitehall cohort and 761 individuals diagnosed with diabetes have been measured and related to psychological, biological, and genetic factors. In the Whitehall participants, concentrations of Hsp60 ranged from undetectable to mg/mL levels. Circulating Hsp60 correlated with total and low-density lipoprotein (LDL) cholesterol and was positively associated with a flattened slope of cortisol decline over the day. Levels of this stress protein also correlated with measures of psychological stress including psychological distress, job demand, and low emotional support. Mass spectrometric analysis of circulating immunoreactive Hsp60 reveal that it is predominantly the intact protein with no mitochondrial import peptide, suggesting that this circulating protein emanates from mitochondria. The Hsp60 is stable when added to plasma and the levels in the circulation of individuals are remarkably constant over a 4-year period, suggesting plasma levels are partly genetically controlled. Sequence analysis of the HSP60-HSP10 intergenic promoter region identified a common variant 3175 C>G where the G allele had a frequency of 0.30 and was associated with higher Hsp60 levels in 761 type 2 diabetic patients. The extended range of plasma Hsp60 concentrations in the general population is genuine and is likely to be related to genetic, biological, and psychosocial risk factors for coronary artery disease.

    Funded by: British Heart Foundation: RG/07/008/23674; Medical Research Council: G0100222, G19/35, G8802774

    Cell stress & chaperones 2007;12;4;384-92

  • Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential.

    Bross P, Li Z, Hansen J, Hansen JJ, Nielsen MN, Corydon TJ, Georgopoulos C, Ang D, Lundemose JB, Niezen-Koning K, Eiberg H, Yang H, Kølvraa S, Bolund L and Gregersen N

    Research Unit for Molecular Medicine, Skejby Sygehus, Aarhus University Hospital and Faculty of Health Sciences, Brendstrupgaardsvej 100, 8200, Arhus N, Denmark. Peter.Bross@ki.au.dk

    Molecular chaperones assist protein folding, and variations in their encoding genes may be disease-causing in themselves or influence the phenotypic expression of disease-associated or susceptibility-conferring variations in many different genes. We have screened three candidate patient groups for variations in the HSPD1 and HSPE1 genes encoding the mitochondrial Hsp60/Hsp10 chaperone complex: two patients with multiple mitochondrial enzyme deficiency, 61 sudden infant death syndrome cases (MIM: #272120), and 60 patients presenting with ethylmalonic aciduria carrying non-synonymous susceptibility variations in the ACADS gene (MIM: *606885 and #201470). Besides previously reported variations we detected six novel variations: two in the bidirectional promoter region, and one synonymous and three non-synonymous variations in the HSPD1 coding region. One of the non-synonymous variations was polymorphic in patient and control samples, and the rare variations were each only found in single patients and absent in 100 control chromosomes. Functional investigation of the effects of the variations in the promoter region and the non-synonymous variations in the coding region indic 1f40 ated that none of them had a significant impact. Taken together, our data argue against the notion that the chaperonin genes play a major role in the investigated diseases. However, the described variations may represent genetic modifiers with subtle effects.

    Journal of human genetics 2007;52;1;56-65

  • Identification of immunodominant CD4+ T cell epitopes in patients with Yersinia-induced reactive arthritis by cytometric cytokine secretion assay.

    Thiel A, Wu P, Lanowska M, Dong J, Radbruch A and Sieper J

    Deutsches Rheuma-Forschungszentrum, Berlin, Germany.

    Objective: In reactive arthritis (ReA), a bacteria-specific T cell response to the triggering microbe is detected in synovial fluid. So far, direct characterization of bacteria-specific T cells and identification of the immunodominant fine specificities remain difficult due to the lack of appropriate techniques. The aim of the present study was to directly determine the fine specificity of CD4+ T cells specific to ReA-associated bacteria-derived protein.

    Methods: In 2 patients with Yersinia-induced ReA, live Yersinia Hsp60-specific CD4+ T cells were directly isolated from synovial fluid after stimulation with Yersinia-derived protein Hsp60 using a cytometric cytokine secretion assay. Generated short-term T cell lines were then tested in vitro for their peptide epitope specificity. Also, direct cross-reactivity of one line with Chlamydia- and human-derived Hsp60 was assessed.

    Results: Generated short-term CD4+ T cell lines were highly antigen-specific and revealed single immunodominant peptide epitopes that were confirmed by direct testing with single peptides in both peripheral blood and synovial fluid cells. Yersinia Hsp60-specific T cells of one patient cross-reacted directly with human Hsp60.

    Conclusion: Our results demonstrate the feasibility of direct assessment of live, potentially pathogenic, antigen-specific interferon-gamma+ CD4+ T cells taken from inflammatory lesions of patients with rheumatic diseases such as ReA. This might have implications not only regarding pathogenesis, but also in the design of new immunotherapies.

    Arthritis and rheumatism 2006;54;11;3583-90

  • Differential effects of mitochondrial heat shock protein 60 and related molecular chaperones to prevent intracellular beta-amyloid-induced inhibition of complex IV and limit apoptosis.

    Veereshwarayya V, Kumar P, Rosen KM, Mestril R and Querfurth HW

    Department of Neurology, Caritas St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

    Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal beta-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular beta-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, beta-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of beta-amyloid-induced neuronal dysfunction and death.

    Funded by: NINDS NIH HHS: NS41373

    The Journal of biological chemistry 2006;281;40;29468-78

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy.

    Johansson B, Pourian MR, Chuan YC, Byman I, Bergh A, Pang ST, Norstedt G, Bergman T and Pousette A

    Department of Clinical Neuroscience, Karolinska Hospital, Karolinska Institutet, Stockholm, Sweden. Bjorn.Johansson@neuro.ki.se

    Background: Androgen-sensitive prostate cancer cell-line LNCaP-FGC and androgen-resistant line LNCaP-r constitute a model for development of androgen resistance in prostate cancer.

    Methods: Proteins differently expressed in the two cell-lines were identified by two-dimensional (2-D) electrophoresis and mass spectrometry. HSP60, more abundant in LNCaP-r, was studied by RT-PCR and immunohistochemistry in specimens of human prostate cancer.

    Results: HSP60 was upregulated in LNCaP-r, nm23 in LNCaP-FGC, and titin (two isoforms) in either LNCaP-r or LNCaP-FGC. In non-malignant prostate, HSP60-staining was in the glandular compartment, particularly basal epithelial cells. In prostate cancer, most epithelial cells showed moderate-strong staining without apparent correlation between staining intensity and Gleason grade.

    Conclusions: The LNCaP-FGC/LNCaP-r model, characterized by 2-D electrophoresis, reveals distinct proteomic alterations. With HSP60, results from cell-lines correlated with clinical results, indicating that this model can be used for dissection of mechanisms involved in transformation to androgen resistance and assignment of protein markers in prostate cancer.

    The Prostate 2006;66;12;1235-44

  • [Polymorphisms of hsp 60 gene in Chinese Han people].

    Wang J, Yang XB, Bai Y, Jiang Q, He YF, Chen YW, He MA and Wu TC

    Department of Labor Health and Environmental 1f40 Health, MOE Key Lab of Environment and Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

    Objective: To identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of heat shock protein 60 gene and search for its genetic makers in Chinese Han people.

    Methods: The 5' flank region, parts of the exons and introns of hsp60 gene were resequenced to ide 662 ntify the SNPs in Chinese Han people, and then the sequenced results to the Japanese, European and African's data in National Center for Biotechnology Information (NCBI) and HapMap databases were compared.

    Results: One novel SNP was identified in exon 2 resulting in synonymous variant and the G allele frequency was 0.025. There were 11 reported SNPs in the sequenced region. The minor allele frequencies of rs1116734, rs3749095, rs1050347, rs8539 were 0.51, 0.30, 0.29, 0.49. The heterozygosity of the other 7 SNPs was 0. The distributions of rs1116734, rs1050347, rs8539, rs3749095 in Chinese Han people were similar to the Japanese's. The hsp60 rs3749095 which was not found in Japanese people was a high-frequency SNP in Chinese Han people; the distribution of rs8539 in Chinese Han people was quite different from European and African's (P < 0.01).

    Conclusion: The SNPs of hsp60 in Chinese Han people are different from the other peoples. The SNPs of hsp60 gene rs1116734, rs3749095, rs1050347, rs8539 are very common in Chinese Han people and might be used for candidate genetic markers of hsp60 gene.

    Zhonghua lao dong wei sheng zhi ye bing za zhi = Zhonghua laodong weisheng zhiyebing zazhi = Chinese journal of industrial hygiene and occupational diseases 2006;24;8;471-4

  • Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling.

    Zanin-Zhorov A, Cahalon L, Tal G, Margalit R, Lider O and Cohen IR

    Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

    CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.

    The Journal of clinical investigation 2006;116;7;2022-32

  • Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4.

    de Graaf R, Kloppenburg G, Kitslaar PJ, Bruggeman CA and Stassen F

    Department of Radiology, Cardiovascular Research Institute Maastricht, University Hospital Maastricht, P. Debyelaan 25, 6202 AZ, Maastricht, The Netherlands. rdgr@rdia.azm.nl

    Heat shock proteins (HSPs) of endogenous and exogenous origin are suspected contributors to the initiation and aggravation of vascular pathologies like atherosclerosis and restenosis. Toll-like receptors (TLRs) 2 and 4 are well-known receptors for exogenous pathogen-associated molecular patterns and have recently been thought to play a role in HSP60-induced cellular activation. We hypothesized that human HSP60 directly stimulates venous smooth muscle cell (VSMC) proliferation through a TLR-dependent mechanism. Localization of HSP60, TLR2 and TLR4 was studied in failed venous grafts and normal venous tissue by double immunostaining. In vitro VSMCs were incubated for 48 h with recombinant human HSP60. In other experiments, VSMCs were pre-incubated for 30 min with specific anti-TLR2 and anti-TLR4 antibodies. VSMC proliferation was determined by Ki67 immunoreactivity, and mean values were compared between experimental and control groups. In addition, human embryonic kidney (HEK) cells transfected with human TLR2 or TLR4/MD-2 were exposed to HSP60 for 48 h, and proliferation was determined by using a hemocytometer. Co-localization of HSP60 and TLRs was detected in all neointimal lesions but was virtually absent in normal veins. Human HSP60 stimulated VSMC proliferation in a concentration-dependent fashion. In addition, TLR2 and TLR4 antibodies attenuated VSMC proliferation. The role of TLR-mediated stimulation of cell proliferation by HSP60 was supported by the significant increase in proliferation of transfected HEK cells. These findings provide supporting evidence for the role of HSP60 and TLR2 and TLR4 in vascular disease. Moreover, our data surpass the infection- and autoimmunity-based hypotheses of cardiovascular disease and suggest an additional HSP60-related autocrine process.

    Microbes and infection 2006;8;7;1859-65

  • Binding specificity of Toll-like receptor cytoplasmic domains.

    Brown V, Brown RA, Ozinsky A, Hesselberth JR and Fields S

    Department of Genome Sciences, University of Washington, Seattle 98195, USA.

    MyD88 participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated Toll-like receptors (TLR). Yeast two-hybrid experiments reveal that the TIR domains of human TLR differ in their ability to associate with MyD88: The TIR of TLR2 binds to MyD88 but the TIR of the closely related TLR1, 6, or 10 do not. Using chimeric TIR domains, we define the critical region responsible for differential MyD88 binding, and use a computational analysis of the critical region to reveal the amino acids that differ between MyD88 binders and non-binders. Remarkably, a single missense mutation created in TLR1 (N672D) confers on it the ability to bind MyD88, without affecting its association with other proteins. Mutations identified as critical for MyD88 binding also affect signaling of TLR pairs in mammalian cells. To investigate the difference between MyD88 binders and non-binders, we identify novel interacting proteins for each cytoplasmic domain of TLR1, 2, 6, and 10. For example, heat shock protein (HSP)60 binds to TLR1 but not to TLR2, and HSP60 and MyD88 appear to bind the same region of the TIR domain. In summary, interactions between the TLR, MyD88, and novel associated proteins have been characterized.

    Funded by: Howard Hughes Medical Institute

    European journal of immunology 2006;36;3;742-53

  • Proteomic analysis of SUMO4 substrates in HEK293 cells under serum starvation-induced stress.

    Guo D, Han J, Adam BL, Colburn NH, Wang MH, Dong Z, Eizirik DL, She JX and Wang CY

    Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, GA 30912, USA.

    The substrates of SUMO4, a novel member for the SUMO gene family, were characterized in HEK293 cells cultured under serum starvation by proteomic analysis. We identified 90 SUMO4 substrates including anti-stress proteins such as antioxidant enzymes and molecular chaperones or co-chaperones. The substrates also include proteins involved in the regulation of DNA repair and synthesis, RNA processing, protein degradation, and glucose metabolism. Several SUMO4-associated transcription factors were characterized by Western blot analyses. AP-1 was selected for in vitro conjugation assays to confirm SUMO4 sumoylation of these transcription factors. Further functional analyses of the transcription factors suggested that SUMO4 sumoylation represses AP-1 and AP-2alpha transcriptional activity, but enhances GR DNA binding capacity. These results demonstrate that SUMO4 sumoylation may play an important role in the regulation of intracellular stress.

    Biochemical and biophysical research communications 2005;337;4;1308-18

  • Association of serum-soluble heat shock protein 60 with carotid atherosclerosis: clinical significance determined in a follow-up study.

    Xiao Q, Mandal K, Schett G, Mayr M, Wick G, Oberhollenzer F, Willeit J, Kiechl S and Xu Q

    Department of Cardiac and Vascular Sciences, St George's University of London, United Kingdom.

    Previous work has shown that soluble heat shock protein 60 (HSP60; sHSP60), present in circulating blood, is associated with carotid atherosclerosis. In the current evaluation, we tested the hypothesis that sHSP60 levels are associated with the progression of carotid arteriosclerosis, prospectively.

    Methods: The association of sHSP60 with early atherogenesis (5-year development and progression of nonstenotic carotid plaques) was investigated as part of the population-based prospective Bruneck Study. The current study focused on the follow-up period between 1995 and 2000 and, thus, included 684 subjects.

    Results: sHSP60 levels measured in 1995 and 2000 were highly correlated (r=0.40; P<0.001), indicating consistency over a 5-year period. Circulating HSP60 levels were significantly correlated with antilipopolysaccharide and anti-HSP60 antibodies. It was also elevated in subjects with chronic infection (top quintile group of HSP60, among subjects with and without chronic infection: 23.8% versus 17.0%; P=0.003 after adjustment for age and sex). HSP60 levels were significantly associated with early atherogenesis, both in the entire population (multivariate odds ratio, for a comparison between quintile group V versus I+II: 2.0 [1.2 to 3.5] and the subgroup free of atherosclerosis at the 1995 baseline: 3.8 [1.6 to 8.9]). The risk of early atherogenesis was additionally amplified when high-sHSP60 and chronic infection were present together.

    Conclusions: Our study provides the first prospective data confirming an association between high levels of sHSP60 and early carotid atherosclerosis. This possibly indicates an involvement of sHSP60 in activating proinflammatory processes associated with early vessel pathology.

    Stroke 2005;36;12;2571-6

  • Induction of endothelial cell apoptosis by the binding of anti-endothelial cell antibodies to Hsp60 in vasculitis-associated systemic autoimmune diseases.

    Jamin C, Dugué C, Alard JE, Jousse S, Saraux A, Guillevin L, Piette JC and Youinou P

    Brest University Medical School, Brest, France.

    Objective: Anti-endothelial cell antibodies (AECAs), which recognize a number of endothelial antigens, are seen in patients with systemic autoimmune diseases, more often in the presence of vasculitis than in its absence. Some AECAs induce apoptosis of endothelial cells (ECs), but their target antigens remain unknown. The aim of this study was to determine whether Hsp60 is a target antigen and whether AECAs induce apoptosis in ECs.

    Methods: Two-dimensional electrophoresis and conventional Western blotting techniques were used to characterize AECA targets. Hsp60 reactivity was determined by enzyme-linked immunosorbent assay.

    Results: Hsp60 was shown to be targeted by a proportion of AECAs. The level of reactivity was higher in patients with systemic autoimmune disease and vasculitis than in those without vasculitis and in patients with systemic lupus erythematosus than in patients with other systemic autoimmune diseases. Hsp60 was expressed on the plasma membrane of heat-stressed ECs, and this followed Hsp60 messenger RNA transcription, confinement of the protein to the cytoplasm, and translocation of the protein to the surface. Shedding of Hsp60 from ECs was induced by stress and resulted in the binding of soluble Hsp60 to the surface of ECs, particularly stressed ECs. Apoptosis of ECs was triggered by anti-Hsp60-containing AECA-positive sera and was inhibited by preincubation of the ECs with recombinant Hsp60.

    Conclusion: Our data support the notion that Hsp60 is an important target for AECAs and that such an interaction contributes to pathogenic effects, especially in vasculitis-associated systemic autoimmune disease.

    Arthritis and rheumatism 2005;52;12;4028-38

  • The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase.

    Cappello F, David S, Rappa F, Bucchieri F, Marasà L, Bartolotta TE, Farina F and Zummo G

    Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, Università degli Studi di Palermo, Italy. francapp@hotmail.com

    Background: The involvement of Heat Shock Proteins (HSP) in cancer development and progression is a widely debated topic. The objective of the present study was to evaluate the presence and expression of HSP60 and HSP10 in a series of large bowel carcinomas and locoregional lymph nodes with and without metastases.

    Methods: 82 Astler and Coller's stage C2 colorectal cancers, of which 48 well-differentiated and 34 poorly-differentiated, were selected along with 661 lymph nodes, including 372 with metastases and 289 with reactive hyperplasia only, from the same tumours. Primitive tumours and both metastatic and reactive lymph nodes were studied; specifically, three different compartments of the lymph nodes, secondary follicle, paracortex and medullary sinus, were also analysed. An immunohistochemical research for HSP60 and HSP10 was performed and the semiquantitative results were analysed by statistical analysis to determine the correlation between HSPs expression and 1) tumour grading; 2) degree of inflammation; 3) number of lymph nodes involved; 4) lymph node compartment hyperplasia. Moreover, western blotting was performed on a smaller group of samples to confirm the immunohistochemical results.

    Results: Our data show that the expression of HSP60, in both primary tumour and lymph node metastasis, is correlated with the tumoral grade, while the HSP10 expression is not. Nevertheless, the levels of HSP10 are commonly higher than the levels of HSP60. In addition, statistical analyses do not show any correlation between the degree of inflammation and the immunopositivity for both HSP60 and HSP10. Moreover, we find a significant correlation between the presence of lymph node metastases and the positivity for both HSP60 and HSP10. In particular, metastatic lymph nodes show a higher percentage of cells positive for both HSP60 and HSP10 in the secondary follicles, and for HSP10 in the medullary sinuses, when compared with hyperplastic lymph nodes.

    Conclusion: HSP60 and HSP10 may have diagnostic and prognostic significance in the management of this tumour and their overexpression in tumoral cells may be functionally related to tumoral progression. We hypothesise that their expression in follicular and medullary cells of lymph nodes may be induced by formation of metastases. Further studies based on these observations could lead to a better understanding of the HSPs involvement in colorectal cancer progression, as well as other neoplasms.

    BMC cancer 2005;5;139

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60.

    Wadhwa R, Takano S, Kaur K, Aida S, Yaguchi T, Kaul Z, Hirano T, Taira K and Kaul SC

    Gene Function Research Center, National Institute of Advanced Industrial Science & Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.

    Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

    The Biochemical journal 2005;391;Pt 2;185-90

  • The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    Satoh J, Onoue H, Arima K and Yamamura T

    Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan. satoj@ncnp.go.jp

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

    Journal of neuropathology and experimental neurology 2005;64;10;858-68

  • Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis.

    Bruneel A, Labas V, Mailloux A, Sharma S, Royer N, Vinh J, Pernet P, Vaubourdolle M and Baudin B

    Service de Biochimie A, Hôpital Saint-Antoine, AP-HP, Paris, France. arnaud.bruneel@sat.ap-hop-paris.fr

    We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

    Proteomics 2005;5;15;3876-84

  • Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway.

    Cohen-Sfady M, Nussbaum G, Pevsner-Fischer M, Mor F, Carmi P, Zanin-Zhorov A, Lider O and Cohen IR

    Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

    We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-gamma by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.

    Journal of immunology (Baltimore, Md. : 1950) 2005;175;6;3594-602

  • Anti-heat shock protein 60 autoantibodies induce atherosclerosis in apolipoprotein E-deficient mice via endothelial damage.

    Foteinos G, Afzal AR, Mandal K, Jahangiri M and Xu Q

    Department of Cardiac and Vascular Sciences, St. George's University of London, Cranmer Terrance, London SW17 0RE, UK.

    Background: Accumulating evidence established a positive association of anti-heat shock protein 60 (HSP60) autoantibodies and the presence of atherosclerosis in humans. However, whether these autoantibodies play a causal role in the development of atherosclerosis is unknown.

    In the present study, anti-HSP60 autoantibodies from blood of patients with coronary heart disease were isolated by affinity chromatography and injected into the tail vein of apolipoprotein E-deficient mice. Atherosclerotic lesions in aortas were significantly increased 8 weeks after injection. Furthermore, administration of a specific mouse monoclonal antibody (II-13) recognizing amino acid residues 288 to 366 of HSP60 effectively induced atherosclerotic lesions in apolipoprotein E-deficient mice. II-13 injection resulted in endothelial cell damage, followed by increased leukocyte attachment and accumulation of macrophages and smooth muscle cells in lesions. Interestingly, II-13-induced atherosclerosis was blocked by pretreatment of animals with F(ab)2 segments derived from the antibody, but not mouse IgG F(ab)2.

    Conclusions: Autoantibodies recognizing amino acid residues 288 to 366 of HSP60 induce atherosclerosis via the mechanisms of autoimmune reactions to HSP60 expressed on arterial endothelial cells, which can be prevented by F(ab)2 segments derived from these antibodies.

    Circulation 2005;112;8;1206-13

  • Human 60-kDa heat shock protein is a target autoantigen of T cells derived from atherosclerotic plaques.

    Benagiano M, D'Elios MM, Amedei A, Azzurri A, van der Zee R, Ciervo A, Rombolà G, Romagnani S, Cassone A and Del Prete G

    Department of Internal Medicine, University of Florence, Florence, Italy.

    Epidemiological studies suggest the potential importance of an inflammatory component in atherosclerosis and support the hypothesis that immune responses to Ags of pathogens cross-react with homologous host proteins due to molecular mimicry. Protein candidates involved may be the stress-induced proteins known as heat shock proteins (HSP). In this study, we report that atherosclerotic plaques harbor in vivo-activated CD4(+) T cells that recognize the human 60-kDa HSP. Such in vivo-activated 60-kDa HSP-specific T cells are not detectable in the peripheral blood. In patients with positive serology and PCR for Chlamydia pneumoniae DNA, but not in patients negative for both, most of plaque-derived T cells specific for human 60-kDa HSP also recognized the C. pneumoniae 60-kDa HSP. We characterized the submolecular specificity of such 60-kDa HSP-specific plaque-derived T cells and identified both the self- and cross-reactive epitopes of that autoantigen. On challenge with human 60-kDa HSP, most of the plaque-derived T cells expressed Th type 1 functions, including cytotoxicity and help for monocyte tissue factor production. We suggest that arterial endothelial cells, undergoing classical atherosclerosis risk factors and conditioned by Th type 1 cytokines, express self 60-kDa HSP, which becomes target for both autoreact 1f40 ive T cells and cross-reactive T cells to microbial 60-kDa HSP via a mechanism of molecular mimicry. This hypothesis is in agreement with the notion that immunization with HSP exacerbates atherosclerosis, whereas immunosuppression and T cell depletion prevent the formation of arteriosclerotic lesions in experimental animals.

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;10;6509-17

  • Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry.

    Ahmed M, Forsberg J and Bergsten P

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. meftun.khandker@drl.ox.ac.uk

    Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.

    Journal of proteome research 2005;4;3;931-40

  • Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

    Pfister G, Stroh, Perschinka H, Kind M, Knoflach M, Hinterdorfer P and Wick G

    Institute for Biophysics, University of Linz, A-4040 Linz, Austria.

    The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 3f9 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

    Journal of cell science 2005;118;Pt 8;1587-94

  • Heat shock protein 10 inhibits lipopolysaccharide-induced inflammatory mediator production.

    Johnson BJ, Le TT, Dobbin CA, Banovic T, Howard CB, Flores Fde M, Vanags D, Naylor DJ, Hill GR and Suhrbier A

    CBio Limited, 17 Wakefield St., Alderley, Queensland 4051, Australia. barbara.johnson@cbio.com.au

    Heat shock protein 10 (Hsp10) and heat shock protein 60 (Hsp60) were originally described as essential mitochondrial proteins involved in protein folding. However, both proteins have also been shown to have a number of extracellular immunomodulatory activities. Here we show that purified recombinant human Hsp10 incubated with cells in vitro reduced lipopolysaccharide (LPS)-induced nuclear factor-kappaB activation and secretion of several inflammatory mediators from RAW264.7 cells, murine macrophages, and human peripheral blood mononuclear cells. Induction of tolerance by contaminating LPS was formally excluded as being responsible for Hsp10 activity. Treatment of mice with Hsp10 before endotoxin challenge resulted in the reduction of serum tumor necrosis factor-alpha and RANTES (regulated upon activation, normal T cell expressed and secreted) levels and an elevation of serum interleukin-10 levels. Hsp10 treatment also delayed mortality in a murine graft-versus-host disease model, where gut-derived LPS contributes to pathology. We were unable to confirm previous reports that Hsp10 has tumor growth factor properties and suggest that Hsp10 exerts anti-inflammatory activity by inhibiting Toll-like receptor signaling possibly by interacting with extracellular Hsp60.

    The Journal of biological chemistry 2005;280;6;4037-47

  • Heat shock protein 60: specific binding of lipopolysaccharide.

    Habich C, Kempe K, van der Zee R, Rümenapf R, Akiyama H, Kolb H and Burkart V

    German Diabetes Clinic, German Diabetes Center, Leibniz Institute, Heinrich Heine University, Dusseldorf, Germany. christiane.habich@ddfi.uni-duesseldorf.de

    Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;3;1298-305

  • Nucleolar proteome dynamics.

    Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI and Mann M

    Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, Denmark.

    The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

    Funded by: Wellcome Trust: 073980

    Nature 2005;433;7021;77-83

  • HIV-1 integration: an interplay between HIV-1 integrase, cellular and viral proteins.

    Van Maele B and Debyser Z

    Laboratory for Molecular Virology and Gene Therapy, KULAK and KULeuven, Flanders, Belgium.

    To achieve a productive infection, the reverse transcribed cDNA of the human immunodeficiency virus type 1 (HIV-1) has to be inserted in the host cell genome. The main protein required to accomplish this reaction is the virally encoded integrase. In vitro, the recombinant integrase is capable of catalyzing the two subsequent reactions of the integration process, namely the 3' processing followed by the strand transfer, without other viral and/or cellular proteins. However, a number of studies indicate that the in vivo integration process also involves cellular proteins, assisting the virus to integrate in the cellular genome. These cellular proteins can play a role during different steps of the integration process, including nuclear import, integrase catalysis, integration site selection and DNA gap repair. In this review we summarize the candidate cellular proteins involved in the HIV-1 integration process identified so far and discuss their potential roles during HIV-1 replication.

    AIDS reviews 2005;7;1;26-43

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • Molecular chaperone, HSP60, and cytochrome P450 2E1 co-expression in dilated cardiomyopathy.

    Sidorik L, Kyyamova R, Bobyk V, Kapustian L, Rozhko O, Vigontina O, Ryabenko D, Danko I, Maksymchuk O, Kovalenko VN, Filonenko VV and Chaschin NA

    Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo street, 03143, Kiev-143, Ukraine. sidorik@imbg.org.ua

    Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins - molecular chaperons (HSPs) - are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation. HS 1f40 Ps are involved in numerous diseases including cardiovascular pathologies, revealing changes of expression and cell localization. We studied the possible changes in expression level of abundant mitochondrial chaperon Hsp60 and main human cytochrome P450 monooxygenase (2E1 isoform) at dilated cardiomyopathy (DCM) progression at the end stage of heart failure using Western blot analysis. The ischemic and normal humans' hearts were studied as control samples. We observed the decrease of Hsp60 level in cytoplasmic fraction of DCM- and ischemia-affected hearts' left ventricular and significant increase of Hsp60 in mitochondrial fractions of all hearts investigated. At the same time we detected the increase of P450 2E1 expression level in ischemic and dilated hearts' cytoplasmic fractions in comparison with normal myocardium and no detectable changes in microsomal fractions of hearts investigated which could be linked with increased level of oxidative injury for DCM heart muscle. In addition, all the changes described are accompanied by significant decrease of ATPase activity of myosin purified from DCM-affected heart in comparison with normal and ischemic myocardia as well. The data obtained allow us to propose a working hypothesis of functional link between antistress (HSPs) and antioxidative (cytochromes) systems at DCM progression.

    Cell biology international 2005;29;1;51-5

  • Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design.

    Karki RG, Tang Y, Burke TR and Nicklaus MC

    Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, DHHS, Frederick, MD 21702, USA. mn1@helix.nih.gov

    We report structural models of the full-length integrase enzyme (IN) of the human immunodeficiency virus type 1 (HIV-1) and its complex with viral and human DNA. These were developed by means of molecular modeling techniques using all available experimental evidence, including X-ray crystallographic and NMR structures of portions of the full-length protein. Special emphasis was placed on obtaining a model of the enzyme's active site with the viral DNA apposed to it, based on the hypothesis that such a model would allow structure-based design of inhibitors that retain activity in vivo. This was because bound DNA might be present in vivo after 3'-processing but before strand transfer. These structural models were used to study the potential binding modes of various diketo-acid HIV-1 IN inhibitors (many of them preferentially inhibiting strand transfer) for which no experimentally derived complexed structures are available. The results indicate that the diketo-acid IN inhibitors probably chelate the metal ion in the catalytic site and also prevent the exposure of the 3'-processed end of the viral DNA to human DNA.

    Journal of computer-aided molecular design 2004;18;12;739-60

  • Lipopolysaccharide-free heat shock protein 60 activates T cells.

    Osterloh A, Meier-Stiegen F, Veit A, Fleischer B, von Bonin A and Breloer M

    Bernhard-Nocht-Institute for Tropical Medicine, 20359 Hamburg, Germany.

    A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past. Hsp60 was shown to induce the secretion of proinflammatory cytokines in professional antigen-presenting cells and to enhance the activation of T cells in primary stimulation. However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations. Here, we employ low endotoxin recombinant human Hsp60 and murine Hsp60 expressed by eukaryotic cell lines to dissect the Hsp60 protein-mediated effects from biologic effects that are mediated by prokaryotic contaminants in the Hsp60 protein preparation. The induction of tumor necrosis factor-alpha secretion in mouse macrophages is lost after endotoxin removal and is not mediated by Hsp60 expressed in eukaryotic systems. In contrast, the Hsp60-mediated enhancement of antigen-specific T cell activation does not correlate with endotoxin contamination. Moreover, Hsp60 that is expressed on the surface of different eukaryotic cell lines increases the activation of T cells in primary stimulation. Taken together, we provide evidence that endogenous Hsp60, which is thought to be released from dying infected cells in vivo, has a biological function that is not due to contaminating pathogen-associated molecules.

    The Journal of biological chemistry 2004;279;46;47906-11

  • Inhibition of adjuvant-induced arthritis by DNA vaccination with the 70-kd or the 90-kd human heat-shock protein: immune cross-regulation with the 60-kd heat-shock protein.

    Quintana FJ, Carmi P, Mor F and Cohen 1f40 IR

    Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.

    Objective: Adjuvant arthritis can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt). The mycobacterial 65-kd heat-shock protein (Hsp65) is targeted by arthritogenic T cells. However, Hsp65 and the mycobacterial 71-kd heat-shock protein are also recognized by T cells that can down-regulate adjuvant-induced arthritis (AIA). We have recently demonstrated that vaccination with human Hsp60 DNA inhibits AIA. The present study was undertaken to analyze the role of the T cell responses to self HSP molecules other than Hsp60 in the control of AIA.

    Methods: Lewis rats were immunized with DNA vaccines coding for human Hsp70 or Hsp90 (Hsp70 plasmid [pHsp70] or pHsp90), and AIA was induced. The T cell response to Mt, Hsp60, Hsp70, and Hsp90 (proliferation and cytokine release) was studied, and the T cell response to Hsp60 was mapped with overlapping peptides.

    Results: The Hsp70 or Hsp90 DNA vaccines shifted the arthritogenic T cell response from a Th1 to a Th2/3 phenotype and inhibited AIA. We detected immune crosstalk between Hsp70/90 and Hsp60: both the Hsp70 and Hsp90 DNA vaccines induced Hsp60-specific T cell responses. Similarly, DNA vaccination with Hsp60 induced Hsp70-specific T cell immunity. Epitope mapping studies revealed that Hsp60-specific T cells induced by pHsp70 vaccination reacted with known regulatory Hsp60 epitopes.

    Conclusion: T cell immunity to Hsp70 and to Hsp90, like Hsp60-specific immunity, can modulate the arthritogenic response in AIA. In addition, our results suggest that the regulatory mechanisms induced by Hsp60, Hsp70, and Hsp90 are reinforced by an immune network that connects their reactivities.

    Arthritis and rheumatism 2004;50;11;3712-20

  • Association of anti-heat shock protein 65 antibodies with development of postoperative atrial fibrillation.

    Mandal K, Jahangiri M, Mukhin M, Poloniecki J, Camm AJ and Xu Q

    Department of Cardiothoracic Surgery, St George's Hospital & Medical School, London, UK.

    Background: Atrial fibrillation (AF) is a frequently encountered arrhythmia after cardiac surgery, but its underlying mechanisms are still unclear. We hypothesize that autoimmune and inflammatory responses against heat shock protein 65 (HSP65) may be involved and hence examined the relationship between HSP65 autoantibodies and occurrence of postoperative AF.

    A prospective study of 329 patients undergoing elective primary CABG was undertaken. Cardiovascular risk factors, ECG characteristics, medications, and intraoperative and postoperative features were documented. Anti-HSP65 antibodies and C-reactive protein levels were measured in all preoperative blood samples with ELISA. Postoperative AF was defined as the characteristic arrhythmia, lasting for at least 15 minutes and confirmed on 12-lead ECG and occurring within the first postoperative week. This occurred in 62 patients (19%). In univariate analysis, HSP65 antibodies were significantly higher in patients with postoperative AF (P=0.02). History of previous myocardial infarction, duration of bypass, number of distal anastomosis, and duration of ventilation were also associated with AF (P<0.05), but C-reactive protein levels were not (P=0.13). Multivariate analysis confirmed the positive association of HSP65 antibodies with postoperative AF (OR, 1.41; P=0.04) independent of age, sex, other cardiovascular risk factors, severity of coronary artery disease, duration of ventilation, duration of bypass, and left ventricular function.

    Conclusions: We report a novel association between anti-HSP65 antibodies and occurrence of postoperative AF, indicating a possible role for antibody-mediated immune response in its pathogenesis.

    Circulation 2004;110;17;2588-90

  • Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.

    Aboulaich N, Vainonen JP, Strålfors P and Vener AV

    Division of Cell Biology and Diabetes Research Centre, Faculty of Health Sciences, Linköping University, SE58185 Linköping, Sweden.

    Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

    The Biochemical journal 2004;383;Pt 2;237-48

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow.

    Cappello F, Tripodo C, Farina F, Franco V and Zummo G

    Human Anatomy Section, Department of Experimental Medicine, University of Palermo, Italy. francapp@hotmail.com

    Heat shock proteins (HSPs) constitute a heterogeneous family of proteins involved in cell homeostasis. During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions. It is known that they regulate gene expression, and cell proliferation, differentiation and death. HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding. Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements. We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM) by the means of immunohistochemistry. NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues. By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages. The positivity was restricted to precursor cells, while mature elements were constantly negative. We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin. The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field.

    European journal of histochemistry : EJH 2004;48;3;261-5

  • Perioperative kinetics of heat shock protein 60 in serum during orthotopic liver transplantation.

    Faybik P, Wachauer D, Hetz H, Bachleitner T, Steltzer H, Schett G and Krenn CG

    University Hospital, Vienna, Department of Anaesthesiology and Intensive Care, Vienna, Austria. zralok@hotmail.com

    Introduction: Heat shock proteins (HSP) play essential roles in the synthesis, transport, and folding of proteins. During ischemia/reperfusion (I/R) injury to orthotopic liver transplants (OLT), disassembly of oligomeric complexes and unfolding of proteins are likely to occur, producing a major burden on HSP to prevent and/or reverse these events. To date, all studies have evaluated HSP expression in tissues after an I/R injury. No data are available on HSP serum levels during I/R injury in liver graft recipients.

    We evaluated the intraoperative and perioperative kinetics of HSP60 in the serum of 25 liver graft recipients.

    Results: We observed a significant increase in serum levels of HSP60 at 4 hours compared with 30 minutes after reperfusion of the graft (P = .028). The perioperative HSP60 kinetics in serum neither correlated with the cold ischemia time nor the indocyanin green clearance. The type of preservation solution had no effect on serum HSP60 levels.

    Conclusion: This first study provides evidence for increased serum levels of HSP60 after reperfusion in OLT. The perioperative kinetics of HSP60 in serum may result from suppressed protein synthesis caused by a reduced energy charge of hepatocytes during early reperfusion, impaired transcription, and/or corticosteroid treatment. Further studies are needed to clarify the role of HSP60 under clinical conditions including immunosuppressive medications in human OLT.

    Transplantation proceedings 2004;36;5;1469-72

  • Serum antibody response to the heat shock protein 60 of Chlamydia trachomatis in women with developing cervical cancer.

    Paavonen J, Karunakaran KP, Noguchi Y, Anttila T, Bloigu A, Dillner J, Hallmans G, Hakulinen T, Jellum E, Koskela P, Lehtinen M, Thoresen S, Lam H, Shen C and Brunham RC

    Department of Obstetrics and Gynecology, University of Helsinki, Haartmaninkatu 2, 00290 Helsinki, Finland. jorma.paavonen@helsinki.fi

    Objective: The purpose of this study was to determine whether serum antibody response to the three versions of chlamydial heat shock protein 60 is associated with an increased risk for cervical cancer.

    Women with cervical carcinoma were identified by linking the data files of three Nordic serum banks with cancer registries. Overall, 178 women with invasive cervical carcinoma were identified. For each case, the earliest prediagnostic serum sample was chosen, and three matched control subjects who were free of cancer at the time of the case diagnosis were selected randomly. Serum antibodies to the chlamydial heat shock protein 60 were measured by enzyme-linked immunosorbent assay and correlated with the risk of the subsequent development of cervical cancer.

    Results: Antibodies to chlamydial heat shock protein 60-1 were associated with cervical squamous cell carcinom a9f a among cases with long lag time (>3.5 years; odds ratio, 2.4; 95% CI, 1.1-5.1). Antibodies to chlamydial heat shock protein 60-2 or chlamydial heat shock protein 60-3 were not associated with cervical cancer risk.

    Conclusions: The finding that chlamydial heat shock protein 60-1 antibodies are associated with an increased cervical cancer risk suggests that persistent Chlamydia trachomatis infection may contribute to cervical neoplasia.

    American journal of obstetrics and gynecology 2003;189;5;1287-92

  • Antisense oligodeoxynucleotides targeted against molecular chaperonin Hsp60 block human hepatitis B virus replication.

    Park SG, Lee SM and Jung G

    School of Biological Sciences, Seoul National University, Seoul, 151-742, Korea.

    The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.

    The Journal of biological chemistry 2003;278;41;39851-7

  • DNA fragments of the human 60-kDa heat shock protein (HSP60) vaccinate against adjuvant arthritis: identification of a regulatory HSP60 peptide.

    Quintana FJ, Carmi P, Mor F and Cohen IR

    Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.

    Adjuvant arthritis (AA) is induced by immunizing Lewis rats with Mycobacterium tuberculosis suspended in adjuvant. The mycobacterial 65-kDa heat shock protein (HSP65) contains at least one epitope associated with the pathogenesis of AA: T cell clones that recognize an epitope formed by aa 180-188 of HSP65 react with self-cartilage and can adoptively transfer AA. Nevertheless, vaccination with HSP65 or some of its T cell epitopes can prevent AA by a mechanism that seems to involve cross-reactivity with the self-60-kDa HSP60. We recently demonstrated that DNA vaccination with the human hsp60 gene can inhibit AA. In the present work, we searched for regulatory epitopes using DNA vaccination with HSP60 gene fragments. We now report that specific HSP60 DNA fragments can serve as effective vaccines. Using overlapping HSP60 peptides, we identified a regulator 1f40 y peptide (Hu3) that was specifically recognized by the T cells of DNA-vaccinated rats. Vaccination with Hu3, or transfer of splenocytes from Hu3-vaccinated rats, inhibited the development of AA. Vaccination with the mycobacterial homologue of Hu3 had no effect. Effective DNA or peptide vaccination was associated with enhanced T cell proliferation to a variety of disease-associated Ags, along with a Th2/3-like shift (down-regulation of IFN-gamma secretion and enhanced secretion of IL-10 and/or tumor growth factor beta1) in response to peptide Mt176-190 (the 180-188 epitope of HSP65). The regulatory response to HSP60 or its Hu3 epitope included both Th1 (IFN-gamma) and Th2/3 (IL-10/tumor growth factor beta1) secretors. These results show that regulatory mechanisms can be activated by immunization with relevant self-HSP60 epitopes.

    Journal of immunology (Baltimore, Md. : 1950) 2003;171;7;3533-41

  • Heat shock proteins HSP27, HSP60, HSP70, and HSP90: expression in bladder carcinoma.

    Lebret T, Watson RW, Molinié V, O'Neill A, Gabriel C, Fitzpatrick JM and Botto H

    Department of Urology and Pathology, Hôpital Foch, Suresnes, France.

    Background: Heat shock proteins (HSPs) are synthesized by cells in response to various stress conditions, including carcinogenesis. The expression of HSPs in neoplasia has been implicated in the regulation of apoptosis, and HSPs also can act by increasing immunity. In the current study, the authors attempted to clarify the significance of HSPs in bladder carcinoma and their effect on tumor behavior.

    Methods: Expression levels of the 27-kilodalton HSP (HSP27), HSP60, HSP70, and HSP90 were studied using immunohistochemistry on tissue sections from 42 transitional cell carcinomas of the bladder (14 Grade 1 tumors; 13 Grade 2 tumors; 15 Grade 3 tumors, inclu 1f40 ding 3 tumors associated with carcinoma in situ; 30 Stage Ta tumors; 7 Stage T1 tumors; and 5 Stage T2 tumors). Bladder specimens from 10 healthy patients were used as controls in the study. The selected patients had a mean follow-up of 52 months (range, 24-78 months). Among the 37 patients with superficial bladder carcinoma, 17 patients did not have any recurrence after undergoing primary resection, and 20 patients developed recurrent disease, including 4 recurrences with muscle invasion. HSP expression was evaluated according to the percentage of positively stained cells, and loss of expression was defined as < 80% of stained cells.

    Results: In normal bladder specimens, all four HSPs (HSP27, HSP60, HSP70, and HSP90) were expressed strongly in the cytoplasm and membrane from the basal cell layer to the superficial cell layer. Loss of expression was detected in tumors: respectively, 45.2%, 38.1%, 69.0% and 23.8% of tumors showed a loss of immunostaining for HSP27, HSP60, HSP70, and HSP90. No correlation between HSP expression and grade was found. Low expression levels of HSP27 and HSP60 were correlated with higher tumor stage (87% vs. 6% [P < 0.001] and 78% vs. 9% [P < 0.01], respectively). HSP60 and HSP90 expression levels were correlated with final outcome for patients with superficial bladder carcinoma: loss of expression was associated with the risk of developing an infiltrating recurrence (97% vs. 6.0% [P < 0.001] and 88.2% vs. 52.5% [P = 0.02] for HSP60 and HSP90, respectively).

    Conclusions: HSPs were expressed in normal urothelium, and the current results indicated that loss of HSP60 and HSP90 expression may have prognostic relevance in patients with bladder carcinoma. The authors believe that HSP60 may be a very useful marker for patients with superficial bladder carcinoma and may be used for predicting disease progression. If these data are confirmed, low HSP60 expression levels may be usable as a prognostic marker to identify patients for whom local treatment would be insufficient.

    Cancer 2003;5;970-7

  • Expression levels of hsc70 and hsp60 are developmentally regulated during B-cell maturation and not associated to childhood c-ALL at presentation or relapse.

    Wehner PS, Nielsen B and Hokland M

    Institute of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark. pedersko@dadlnet.dk

    Heat shock proteins are potent regulators of apoptosis, and so they may also be involved in normal cellular differentiation and cancerogenesis. We used quantitative two-dimensional gel electrophoresis for determining whether either the constitutive chaperonic heat shock cognate protein 70 (hsc70) or heat shock protein 60 (hsp60) contribute to B-cell differentiation and leukemogenesis. We compared the expression of these hsps in normal peripheral blood (PB) CD19+ B-cells, in pediatric bone marrow (BM) CD19+ CD10+ B-cell precursors (BCPs) from normal donors, and in BCPs from common acute lymphoblastic leukemia (c-ALL) patients at diagnosis and at relapse. We found that the mean levels of hsc70 in c-ALL BCPs at initial presentation and at relapse failed to differ significantly. Likewise, they failed to differ significantly from the level in high-expressing normal BM BCPs or from that in low-expressing PB B-cells. Mean levels of hsp60 expression in c-ALL BCPs at initial presentation and at relapse were similar and not distinguishable from that in normal BM BCPs, however, elevated (by a factor of 2-3) compared with that in PB B-cells. Hsc70 and Hsp60 expressions were increased (by a factor of 2 of mean levels) in populations of normal BM BCPs as compared with populations of PB B-cells. Thus, no abnormal levels of hsc70 and hsp60 were detectable in populations of pediatric c-ALL BCPs neither at diagnosis nor at relapse. In contrast, our data were in support of developmentally regulated levels of hsc70 and hsp60 expression during B-cell ontogenesis.

    European journal of haematology 2003;71;2;100-8

  • Increased seroreactivity in tic disorder patients to a 60 kDa protein band from a neuronal cell line.

    Hoekstra PJ, Horst G, Limburg PC, Troost PW, van Lang N, de Bildt A, Korf J, Kallenberg CG and Minderaa RB

    Child and Adolescent Psychiatry Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Pieter.Hoekstra@kjpnn.nl

    In tic disorders, increased seroreactivity against neuronal antigens has been demonstrated, without performing molecular characterization of antigens. Here, unselected patients with a tic disorder were compared with healthy controls, autistic disorder (AD), and obsessive-compulsive disorder (OCD) patients. Seroreactivity against neuroblastoma cells was analyzed by Western blot. Anti-60 kDa binding occurred significantly more frequently in tic disorder patients (67.1%) than in AD (40.0%), OCD (40.0%) and healthy controls (41.9%). Sequence analysis of the 60 kDa protein band identified this as a ubiquitous heat shock protein. However, the involvement of other autoantigens with a molecular weight of 60 kDa cannot be excluded.

    Journal of neuroimmunology 2003;141;1-2;118-24

  • Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor alpha from murine macrophages.

    Gao B and Tsan MF

    Institute for Clinical Research, Washington, D. C. 20422, USA. baochong.gao@med.va.gov

    Recent studies have shown that commercially available recombinant human heat shock protein 60 (rhHSP60) could induce tumor necrosis factor alpha (TNF-alpha) release from macrophages and monocytes in a manner similar to that of lipopolysaccharide (LPS), e.g. via CD14 and Toll-like receptor 4 complex-mediated signal transduction pathway. In this study, we demonstrated that a highly purified rhHSP60 preparation with low endotoxin activity (designated rhHSP60-1) was unable to induce TNF-alpha release from murine macrophages at concentrations of up to 10 microg/ml. In contrast, a less purified rhHSP60 preparation (designated rhHSP60-2) was able to induce a marked TNF-alpha release at concentrations as low as 1 microg/ml. Failure of rhHSP60-1 to induce TNF-alpha release was not due to defective physical properties because rhHSP60-1 and rhHSP60-2 contained a similar amount of HSP60 as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHSP60 antibody. Both rhHSP60 preparations also had similar enzymatic activities as judged by their ability to hydrolyze ATP. Polymyxin B added in the incubation media abolished the endotoxin activity but inhibited only about 50% of the TNF-alpha-inducing activity of rhHSP60-2. However, both the endotoxin activity and the TNF-alpha-inducing activity of rhHSP60-2 were essentially eliminated after passing through a polymyxin B-agarose column that removes LPS and LPS-associated molecules from the rhHSP60 preparation. The TNF-alpha-inducing activities of both rhHSP60-2 and LPS with equivalent endotoxin activity present in rhHSP60-2 were equally sensitive to heat inactivation. These results suggest that rhHSP60 does not induce TNF-alpha release from macrophages. Approximately 50% of the observed TNF-alpha-inducing activity in the rhHSP60-2 preparation is due to LPS contamination, whereas the res 19a7 t of the activity was due to the contamination of LPS-associated molecule(s).

    The Journal of biological chemistry 2003;278;25;22523-9

  • Immunohistochemical study of heat shock proteins 27, 60 and 70 in the normal human adrenal and in adrenal tumors with suppressed ACTH production.

    Pignatelli D, Ferreira J, Soares P, Costa MJ and Magalhães MC

    Institute of Histology and Embryology, Faculty of Medicine, University of Porto, Alameda Hernâni Monteiro, 4200-319 Porto, Portugal. dpignatelli@yahoo.com

    Heat shock proteins (HSPs) are known to protect cells against various aggressions and to assist in the correct folding of nascent proteins as well as in the recovery of denatured ones. HSP70 increases its levels in the cell in response to any stress and is induced by ACTH in the adrenal gland. HSP60 is located in the mitochondria and assists in the folding of mitochondrial peptides. HSP27 is the only small HSP that is stress-induced. HSP27 and HSP70 are known to protect cells against apoptosis while, on the contrary, HSP60 is proapoptotic, increasing caspases maturation. We studied the expression of these HSPs in human adrenal tissue both in the normal glands (12 cases) and in tumoral tissue from cortisol producing adrenal adenomas (6 cases). Besides being neoplastic, these cells live in a particular ambience of lack of ACTH due to the suppression of the hypothalamic-pituitary ACTH secretion induced by the elevated levels of cortisol. HSP27 is highly expressed in the normal adrenal and shows a marked reduction of expression in Cushing's adrenal tissue. Although with overall lower levels of expression in the normal adrenal, HSP70 exhibited a similar pattern of reduction in tumoral tissue. HSP60, on the other hand, increased significantly and consistently in adrenal Cushing tumors. Besides the possible consequences of incorrect folding of nascent peptides, the alterations observed in tumoral tissue seem to act in an apoptotic direction. The only factor that we observed that could be contributing to these changes was the lack of plasma ACTH.

    Microscopy research and technique 2003;61;3;315-23

  • Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria.

    Chandra D and Tang DG

    Department of Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park Research Division, Smithville, Texas 78957, USA.

    Active caspase-9 and caspase-3 have been observed in the mitochondria, but their origins are unclear. Theoretically, procaspase-9 might be activated in the mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase-9 and -3 may translocate to the mitochondria, or the mitochondrially localized procaspases may be activated by the translocated active caspases. Here we present evidence that the mitochondrially localized active caspase-9 and -3 result mostly from translocation from the cytosol (into the intermembrane space) and partly from caspase-mediated activation in the organelle rather than from the Apaf-1-mediated activation. Apaf-1 localizes exclusively in the cytosol and, upon apoptotic stimulation, translocates to the perinuclear area but not to the mitochondria. In most cases, the mitochondrially localized procaspase-9 and -3 are released early during apoptosis and translocate to the cytosol and/or perinuclear area. Cytochrome c and the mitochondrial matrix protein Hsp60 are also rapidly released to the cytosol early during apoptosis. Both the early release of proteins like cytochrome c and Hsp60 from the mitochondria as well as the later translocation of the active caspase-9/-3 are partially inhibited by cyclosporin A, an inhibitor of mitochondrial membrane permeabilization. The mitochondrial active caspases may function as a positive feedback mechanism to further activate other or residual mitochondrial procaspases, degrade mitochondrial constituents, and disintegrate mitochondrial functions.

    Funded by: NCI NIH HHS: CA 90297; NIEHS NIH HHS: ES07784

    The Journal of biological chemistry 2003;278;19;17408-20

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Heat shock protein 60 expression in patients undergoing cardiac operations.

    Schäfler AE, Kirmanoglou K, Gallmeier U, Pecher P, Hannekum A and Schumacher B

    Department of Cardiac Surgery, University of Ulm, Ulm, Germany. aschaefler@gmx.de

    Aim: Cardiomyocytes respond to stress with the expression of different heat shock proteins (HSP). The mitochondrial HSP60 is known to be expressed by various stress factors, including ischemia and reperfusion. The aim of this study was to investigate if HSP60 is increased in human myocardium after cardiac surgery.

    Methods: To determine if heat shock protein 60 accumulated in the myocardium of patients undergoing car-diac operations, right atrial samples before and after extracorporeal circulation were excised and immediately frozen in liquid nitrogen.

    Patients: we obtained 10 sequential right atrial specimens from 5 male patients in sinus rhythm undergoing elective cardiac surgery.

    Measures: the HSP60 protein level was determined by SDS-PAGE, Western blot and quantified by optical densitometry according to the immunoreactive bands of actin.

    Results: The HSP60 concentration was unchanged in hearts after a single episode of hypothermic ischemia and reperfusion. Immunoblot analysis demonstrated HSP60 expression in all hearts. There was no correlation with the endurance of cardiopulmonary bypass or reperfusion time.

    Conclusion: These findings indicate that myocardial HSP60 of patients undergoing cardiac operations is not increased after an obligatory period of ischemia, cardioplegic arrest and reperfusion. This might reflect an effective cardioprotection during ECC.

    The Journal of cardiovascular surgery 2003;44;2;187-90

  • Human heat shock protein 60 induces maturation of dendritic cells versus a Th1-promoting phenotype.

    Flohé SB, Brüggemann J, Lendemans S, Nikulina M, Meierhoff G, Flohé S and Kolb H

    German Diabetes Research Institute, University of Düsseldorf, Düsseldorf, Germany.

    Heat shock protein (HSP) 60 nonspecifically activates cells of the innate immune system. In the present study, we characterized the effects of human HSP60 maturation, cytokine release, and T cell-activating capacity of bone marrow-derived dendritic cells (DC). Furthermore, we analyzed HSP60-induced signal transduction in DC. HSP60 strongly stimulated DC for maturation and release of TNF-alpha, IL-12, and IL-1 beta. However, HSP60 elicited only a weak IL-10 response in DC suggesting a Th1 bias. HSP60-treated DC induced proliferation of allogeneic T cells. Again, a Th1 bias was noted in that cocultures of allogeneic T cells and HSP60-treated DC released IFN-gamma but only small amounts of IL-10 and no detectable IL-4. Signaling via Toll-like receptor 4 was involved in HSP60-induced cytokine release and maturation because DC of C3H/HeJ mice with a mutant Toll-like receptor 4 showed deficient response to HSP60. HSP60 was found to rapidly activate the mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase as well as I kappa B in DC. Phosphorylation of these signaling molecules was also mediated by LPS, but with much slower kinetics. Thus, HSP60 stimulates DC more rapidly than LPS and elicits a Th1-promoting phenotype. These results suggest that DC play a pivotal role in priming for destructive Th1-type responses at sites of local HSP60 release.

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;5;2340-8

  • Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.

    Shin BK, Wang H, Yim AM, Le Naour F, Brichory F, Jang JH, Zhao R, Puravs E, Tra J, Michael CW, Misek DE and Hanash SM

    Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, Michigan 48109-0656, USA.

    There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.

    The Journal of biological chemistry 2003;278;9;7607-16

  • Genomic structure of the human mitochondrial chaperonin genes: HSP60 and HSP10 are localised head to head on chromosome 2 separated by a bidirectional promoter.

    Hansen JJ, Bross P, Westergaard M, Nielsen MN, Eiberg H, Børglum AD, Mogensen J, Kristiansen K, Bolund L and Gregersen N

    Research Unit for Molecular Medicine, Faculty of Health Sciences and Arhus University Hospital, Arhus, Denmark.

    Although the mitochondrial chaperonin Hsp60 and its co-chaperonin Hsp10 have received great attention in the last decade, and it has been proposed that mutations and variations in these genes may be implicated in genetic diseases, the genome structure of the human HSP60 and HSP10 genes (also known as HSPD1 and HSPE1, respectively) has not been firmly established. The picture has been confused by the presence of many pseudogenes of both HSP60 and HSP10 and the long surviving assumption that the HSP60 gene is intron-less. An earlier report on the partial sequence of the human HSP60 gene and the presence of introns has largely been overlooked. We present the full sequence of the human HSP60 and HSP10 genes. The two genes are linked head to head comprising approximately 17 kb and consist of 12 and 4 exons, respectively. The first exon of the human HSP60 gene is non-coding and the first exon of the human HSP10 gene ends with the start codon. Analysis of human and mouse expressed sequence tag sequences in GenBank indicates that alternative splicing occurs resulting in HSP60 gene transcripts with different exon-1 sequences. By sequencing of the exons, the exon/intron boundaries and the region between the two genes in 10 Danish individuals (five couples), nine nucleotide variations and one intronic deletion have been detected that, by subsequent typing of one child from each couple, have been assigned to five haplotypes. The human HSP60 gene has been localised, by radiation hybrid mapping, between markers AFMA121YH1 and WI-10756 on chromosome 2. This location and the position of two homologous fragments in the Human Genome Assembly are consistent with cytogenetic position 2q33.1. Using a luciferase-reporter assay, we demonstrate that the region between the two genes functions as a bi-directional promoter. The transcriptional activity of the promoter fragment in the HSP60 direction is approximately twice that in the HSP10 direction under normal growth conditions and, upon heat-shock, promoter activity in either direction increased by a factor of approximately 12. One of the nucleotide variations detected is localised in a putative SP1-transcription-factor-binding site in the bidirectional promoter region and analysis of the transcriptional activity of the promoter fragment with this variation has shown that it does not affect transcription levels both with and without heat-shock.

    Human genetics 2003;112;1;71-7

  • Hsp70 is present in human saliva.

    Fábián TK, Gáspár J, Fejérdy L, Kaán B, Bálint M, Csermely P and Fejérdy P

    Prosthetic Dentistry Clinic, Faculty of Dentistry, Semmelweis University, Budapest, Hungary.

    Background: There is increasing evidence that chaperones are also present outside the cell, exerting cytokine-like effects and influencing immune recognition. Hsp70 has been found to be present in human blood sera. Chaperonins Cpn10 and Cpn60 are present in pancreatic juice, but Hsp70 is not. These observations raise the possibility that molecular chaperones may be present in other secretory fluids, such as human saliva.

    Human whole saliva was collected from six participants under resting conditions and secretory stimulation. The samples were precleared by centrifugation and sterile filtered. Salivary volume, protein concentration and amylase activity were determined. For detection of Hsp70 saliva proteins were separated on a 12.5% SDS-PAGE. Semi-dry Western blot analysis was used with a primary antibody against the inducible form of Hsp70. Hsp70 bands were detected with a horseradish peroxidase-linked secondary antibody and ECL-Western blotting analysis.

    Results: A single band was recognized around 70 kDa in the saliva of all the participants. There was a significant decrease of Hsp70, and a non-significant decrease of total protein concentration during stimulation, whereas the activity of salivary amylase increased significantly. Stimulation significantly increased the Hsp70, total protein and amylase outputs as well as the amylase/protein ratio, and decreased the Hsp70/amylase and Hsp70/protein ratios.

    Conclusions: Hsp70 is secreted to saliva, but unlike amylase is not transported by the exocytotic secretory mechanisms of acinar cells. Passive transport mechanisms of Hsp70 from blood serum or from salivary gland cells may be major routes of salivary Hsp70 secretion.

    Medical science monitor : international medical journal of experimental and clinical research 2003;9;1;BR62-5

  • A signal peptide derived from hsp60 binds HLA-E and interferes with CD94/NKG2A recognition.

    Michaëlsson J, Teixeira de Matos C, Achour A, Lanier LL, Kärre K and Söderström K

    Microbiology and Tumor Biology Center, Karolinska Institutet, 171 77 Stockholm, Sweden.

    Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule which presents a restricted set of nonameric peptides, derived mainly from the signal sequence of other MHC class I molecules. It interacts with CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and T cell subsets. Here we demonstrate that HLA-E also presents a peptide derived from the leader sequence of human heat shock protein 60 (hsp60). This peptide gains access to HLA-E intracellularly, resulting in up-regulated HLA-E/hsp60 signal peptide cell-surface levels on stressed cells. Notably, HLA-E molecules in complex with the hsp60 signal peptide are no longer recognized by CD94/NKG2A inhibitory receptors. Thus, during cellular stress an increased proportion of HLA-E molecules may bind the nonprotective hsp60 signal peptide, leading to a reduced capacity to inhibit a major NK cell population. Such stress induced peptide interference would gradually uncouple CD94/NKG2A inhibitory recognition and provide a mechanism for NK cells to detect stressed cells in a peptide-dependent manner.

    Funded by: NCI NIH HHS: CA 89294, R01 CA089294

    The Journal of experimental medicine 2002;196;11;1403-14

  • Deficiency of chaperonin 60 in Down's syndrome.

    Bozner P, Wilson GL, Druzhyna NM, Bryant-Thomas TK, LeDoux SP, Wilson GL and Pappolla MA

    Department of Pathology, University of South Alabama, Mobile, AL 36617, USA.

    Patients with Down syndrome (DS) and Alzheimer's disease (AD) share a number of characteristic neuropathologic lesions. Several lines of evidence suggest that mitochondria and the oxidative stress response are involved in the pathogenesis of both conditions. In the process of investigating the stress response in DS, we discovered a defective basal expression of a major mitochondrial heat shock protein, chaperonin 60 (Cpn60) in non-transformed dermal fibroblast cell lines from DS individuals. Such a defect was not present in control cells that had been cultured under identical physiological growth conditions. A quantitative analysis by Western blots showed a marked reduction of Cpn60 per equal amount of total protein in DS cells to an average of 35% of normal. Northern blot studies confirmed the defect and also showed a marked reduction of the mRNA signal for Cpn60 in all the DS cell lines. To gain further information, experiments were conducted to study the rate of de-novo synthesis of Cpn60 at normal and supraoptimal temperatures in DS and controls. Results showed no significant differences between the two study groups. HSP60 is important in mitochondrial function and defects in these organelles have been reported in DS and AD. Thus, the findings may have potential implications in the neuropathology of DS.

    Funded by: NIA NIH HHS: AG 16783

    Journal of Alzheimer's disease : JAD 2002;4;6;479-86

  • Immunohistochemical analysis of inducible nitric oxide synthase (iNOS) and heat shock proteins (HSPs) in ameloblastomas.

    Kumamoto H, Suzuki T and Ooya K

    Division of Oral Pathology, Department of Oral Medicine and Bioregulation, Tohoku University Graduate School of Dentistry, Sendai, Japan.

    Background: To clarify the possible role of nitric oxide (NO) and stress proteins in oncogenesis and cytodifferentiation of odontogenic epithelium. Inducible NO synthase (iNOS) and heat shock proteins (HSPs) were analyzed in ameloblastomas as well as in tooth germs.

    Methods: Specimens of seven tooth germs, 36 benign ameloblastomas and five malignant ameloblastomas were examined by immunohistochemistry using antibodies against iNOS and 27-, 60- and 70-kDa HSPs (HSP27, HSP60 and HSP70).

    Results: Immunoreactivity for iNOS was detected in normal and neoplastic odontogenic epithelial cells and was higher in malignant ameloblastomas than in tooth germs and benign ameloblastomas. HSP27 was expressed constitutively in all odontogenic epithelial cells in tooth germs and benign and malignant ameloblastomas. Expression of HSP60 and HSP70 was detected in normal and neoplastic odontogenic epithelial cells and was prominent in cells neighboring the basement membrane. HSP60 reactivity showed no apparent difference between normal and neoplastic odontogenic epithelium, whereas HSP70 expression was slightly higher in benign and malignant ameloblastomas than in tooth germs.

    Conclusions: Activation of iNOS might be associated with malignant potential of epithelial odontogenic tumors. Elevated expression of HSP70 is considered to be involved in neoplastic transformation of odontogenic epithelial cells.

    Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 2002;31;10;605-11

  • Chaperonin GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli.

    Lee KH, Kim HS, Jeong HS and Lee YS

    Department of Biochemistry, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-Gu, Seoul 133-791, Republic of Korea. leekihwan@gaiagene.com

    An efficient bacterial expression system for the human mitochondrial aldehyde dehydrogenase (ALDH2) was developed using co-overexpression of heat shock chaperone gene GroESL. On the basis of the ALDH2 amino acid sequence and cDNA sequences a full-length cDNA encoding wild-type ALDH2 was cloned from a human liver library. A mutant-type ALDH2 (ALDH2(2)) was developed using site-directed mutagenesis of the ALDH2 cDNA and also cloned. Both types of ALDH2 cDNA were subcloned for expression in Escherichia coli (E. coli), recombinant ALDH2 and ALDH 1f40 2(2) were successfully expressed as soluble active enzymes following co-expression with a second plasmid construct producing GroES and GroEL, E. coli chaperonin proteins. Purified wild-type ALDH2 and mutant ALDH2(2) had a K(m) for acetaldehyde of 0.65 and 25.73 microM, respectively. Co-expression of ALDH2 with ALDH2(2) in the presence of E. coli chaperonins produced a soluble enzyme with a K(m) for acetaldehyde of 8.79 microM, suggesting that the product was a heteromer. Mitochondrial matrix hsp60 and hsp10 chaperonins are then thought to act on imported ALDH2 and are essential for accurate protein folding and multisubunit formation. Protein-protein interactions between ALDH2s and various chaperones were investigated using the yeast two-hybrid system. The wild-type and mutant-type enzymes strongly interacted with each other and GroEL and ALDH2s also interacted but only weakly. Chaperone hsp10 also interacted with hsp60 and ALDH2(1) and ALDH2(2), but again the interactions were weak ones.

    Biochemical and biophysical research communications 2002;298;2;216-24

  • Paeoniae Radix, a Chinese herbal extract, inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway.

    Lee SM, Li ML, Tse YC, Leung SC, Lee MM, Tsui SK, Fung KP, Lee CY and Waye MM

    Department of Biochemistry and The Hong Kong Bioinformatics Center, The Chinese University of Hong Kong, Hong Kong, People's Republic of China.

    Paeoniae Radix (PR) is the root of traditional Chinese Herb named Paeonia lactiflora Pallas, which is commonly used to treat liver diseases in China for centuries. Several earlier studies have indicated that PR has anticancer growth activities, however the mechanism underlying these activities was unclear and remained to be elucidated. In this study, we evaluated the molecular mechanism of the effect of PR on human hepatoma cell lines, HepG2 and Hep3B. Our results showed that the water-extract of Paeoniae Radix (PRE) had inhibitory effect on the growth of both HepG2 and Hep3B cell lines. The induction of internucleosomal DNA fragmentation and chromatin condensation appearance, and accumulation of sub-G1 phase of cell cycle profile in PRE treated hepatoma cells evidenced that the cytotoxicity of PRE to the hepatoma cells is through activation of the cell death program, apoptosis. The activation of apoptosis by PRE is independent of the p53 pathway as Hep3B cell is p53-deficient. In addition, the differential gene expression of PRE treated HepG2 was examined by cDNA microarray technology and RT-PCR analysis. We found that the gene expression of BNIP3 was up-regulated while ZK1, RAD23B, and HSPD1 were down-regulated during early apoptosis of the hepatoma cell mediated by PRE. The elucidation of the drug targets of PR on inhibition of tumor cells growth should enable further development of PR for liver cancer therapy.

    Life sciences 2002;71;19;2267-77

  • Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD-box protein.

    Will CL, Urlaub H, Achsel T, Gentzel M, Wilm M and Lührmann R

    Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen and EMBL, Bioanalytical Research Group, D-69117 Heidelberg, Germany.

    Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.

    The EMBO journal 2002;21;18;4978-88

  • Inhibition of adjuvant arthritis by a DNA vaccine encoding human heat shock protein 60.

    Quintana FJ, Carmi P, Mor F and Cohen IR

    Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

    Adjuvant arthritis (AA) is an autoimmune disease inducible in rats involving T cell reactivity to the mycobacterial 65-kDa heat shock protein (HSP65). HSP65-specific T cells cross-reactive with the mammalian 60-kDa heat shock protein (HSP60) are thought to participate in the modulation of AA. In this work we studied the effects on AA of DNA vaccination using constructs coding for HSP65 (pHSP65) or human HSP60 (pHSP60). We found that both constructs could inhibit AA, but that pHSP60 was more effective than pHSP65. The immune effects associated with specific DNA-induced suppression of AA were complex and included enhanced T cell proliferation to a variety of disease-associated Ags. Effective vaccination with HSP60 or HSP65 DNA led paradoxically to up-regulation of IFN-gamma secretion to HSP60 and, concomitantly, to down-regulation of IFN-gamma secretion to the P180-188 epitope of HSP65. There were also variable changes in the profiles of IL-10 secretion to different Ags. However, vaccination with pHSP60 or pHSP65 enhanced the production of TGFbeta1 to both HSP60 and HSP65 epitopes. Our results support a regulatory role for HSP60 autoreactivity in AA and demonstrate that this control mechanism can be activated by DNA vaccination with both HSP60 or HSP65.

    Journal of immunology (Baltimore, Md. : 1950) 2002;169;6;3422-8

  • Circulating heat shock protein and heat shock protein antibody levels in established hypertension.

    Pockley AG, De Faire U, Kiessling R, Lemne C, Thulin T and Frostegård J

    Division of Clinical Sciences (North), University of Sheffield, Sheffield, UK.

    Objective: Serum Hsp60 and anti-Hsp65 antibody levels are raised in subjects with borderline hypertension, and there is an association between circulating Hsp60 levels and early atherosclerosis. Given the recognized relationship between hypertension and atherosclerosis, this study determined heat shock protein and heat shock protein antibody levels in subjects with established hypertension.

    Methods: Samples from 111 men with hypertension were obtained from the European Lacidipine study on Atherosclerosis and samples from 75 normotensive controls were taken from a population-screening programme (diastolic pressure, 95 and 80 mmHg, respectively). Hsp60, Hsp70 and anti-human Hsp60, anti-human Hsp70 and anti-mycobacterial Hsp65 antibody levels were measured by enzyme immunoassay. Intima-media thickness (I-M) and the presence of carotid atherosclerosis were determined by ultrasonography.

    Results: Hsp60, Hsp70 and anti-Hsp60 antibody levels in hypertension were similar to those in normotensive controls, whereas anti-Hsp70 and anti-Hsp65 antibody levels were elevated ( 0.001). Hsp60 levels and atherosclerosis were not associated. Anti-Hsp70 and anti-Hsp65 antibody levels were both associated with hypertension, independently of age, smoking habits and blood lipids.

    Conclusions: This study demonstrates elevated levels of selected heat shock protein antibodies in subjects with hypertension. Although the association between heat shock protein antibody levels and human cardiovascular stress/disease appears to be robust, the relationship of the latter with heat shock protein levels is more complex. Further studies are required before the factors inducing, and the clinical significance of, circulating heat shock proteins can be evaluated.

    Journal of hypertension 2002;20;9;1815-20

  • Circulating human heat shock protein 60 in the plasma of British civil servants: relationship to physiological and psychosocial stress.

    Lewthwaite J, Owen N, Coates A, Henderson B and Steptoe A

    Cellular Microbiology Research Group, University College London, London, UK.

    Background: The Whitehall cohort studies (I and II) of British civil servants have identified sociodemographic, psychosocial, and biological risk factors for coronary heart disease (CHD). To identify mechanisms responsible for susceptibility to CHD, specific biological markers of stress are increasingly being measured. One marker linked to susceptibility to CHD is heat shock protein (Hsp) 60.

    Blood was taken from 229 civil servants (126 men and 103 women) in the Whitehall II cohort drawn equally from the range of employment grades. Plasma was assayed for levels of Hsp60, tumor necrosis factor alpha (TNFalpha), C-reactive protein, von Willebrand factor, high density lipoprotein (HDL), total cholesterol, and total/HDL ratio. Psychosocial measures included socioeconomic status, psychological distress, and social isolation. The majority of the participants had Hsp60 in their plasma, and approximately 20% had >1000 ng/mL of this protein (a concentration likely to induce biological effects). A positive association between plasma Hsp60 and TNFalpha and a negative association with von Willebrand factor was found. There was also a significant association between elevated Hsp60 levels, low socioeconomic status, and social isolation, together with an association with psychological distress in women.

    Conclusions: The majority of participants exhibited Hsp60 in their plasma, and there was evidence of an association between levels of this stress protein and the proinflammatory cytokine, TNFalpha, and with various psychosocial measures.

    Circulation 2002;106;2;196-201

  • Cytosolic heat shock protein 60, apoptosis, and myocardial injury.

    Kirchhoff SR, Gupta S and Knowlton AA

    Division of Cardiology Research, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas, USA.

    Background: Heat shock proteins (HSPs) are well known for their ability to "protect" the structure and function of native macromolecules, particularly as they traffic across membranes. Considering the role of key mitochondrial proteins in apoptosis and the known antiapoptotic effects of HSP27 and HSP72, we postulated that HSP60, primarily a mitochondrial protein, also exerts an antiapoptotic effect. Methods and Results- To test this hypothesis, we used an antisense phosphorothioate oligonucleotide to effect a 50% reduction in the levels of HSP60 in cardiac myocytes, a cell type that has abundant mitochondria. The induced decrease in HSP60 precipitated apoptosis, as manifested by the release of cytochrome c, activation of caspase 3, and induction of DNA fragmentation. Antisense treatment was associated with an increase in bax and a decrease in bcl-2 secondary to increased synthesis of bax and degradation of bcl-2. A control oligonucleotide had no effect on these measurements. We further demonstrated that cytosolic HSP60 forms a macromolecular complex with bax and bak in vitro suggesting that complex formation with HSP60 may block the ability of bax and bak to effect apoptosis in vivo. Lastly, we show that as cytosolic (nonmitochondrial) HSP60 decreases, a small unbound fraction of bax appears and that the amount of bax associated with the mitochondria and cell membranes increases.

    Conclusions: These results support a key antiapoptotic role for cytosolic HSP60. To our knowledge, this is the first report suggesting that interactions of HSP60 with bax and/or bak regulate apoptosis.

    Funded by: NHLBI NIH HHS: HL58515, R01 HL058515

    Circulation 2002;105;24;2899-904

  • Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60.

    Hansen JJ, Dürr A, Cournu-Rebeix I, Georgopoulos C, Ang D, Nielsen MN, Davoine CS, Brice A, Fontaine B, Gregersen N and Bross P

    Research Unit for Molecular Medicine, Arhus University Hospital and Faculty of Health Sciences, Arhus, Denmark.

    SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.

    American journal of human genetics 2002;70;5;1328-32

  • Specific incorporation of heat shock protein 70 family members into primate lentiviral virions.

    Gurer C, Cimarelli A and Luban J

    Departments of Microbiology and Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

    To determine if any heat shock proteins are incorporated into human immunodeficiency virus type 1 (HIV-1) virions in a manner similar to that of the peptidyl-prolyl isomerase cyclophilin A, we probed purified virions with antibodies against heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70, Hsc70, and Hsp90. Of these proteins, Hsp60, Hsp70, and Hsc70 associated with virions purified based on either particle density or size and were shown to be incorporated within the virion membrane, where they were protected from digestion by exogenous protease. Virion incorporation of Hsp70 was also observed with HIV-2 and with simian immunodeficiency viruses SIV(MAC) and SIV(AGM), but it appears to be specific for primate lentiviruses, since Hsp70 was not detected in association with Moloney murine leukemia virus virions. Of the HIV-1 genes, gag was found to be sufficient for Hsp70 incorporation, though Hsp70 was roughly equimolar with pol-encoded proteins in virions.

    Funded by: NIAID NIH HHS: AI 41857, P30 AI042848, P30 AI42848, R01 AI041857

    Journal of virology 2002;76;9;4666-70

  • Human class I histone deacetylase complexes show enhanced catalytic activity in the presence of ATP and co-immunoprecipitate with the ATP-dependent chaperone protein Hsp70.

    Johnson CA, White DA, Lavender JS, O'Neill LP and Turner BM

    Chromatin and Gene Expression Group, Department of Anatomy, University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom.

    Antibodies to histone deacetylases (HDACs) have been used to immuno-isolate deacetylase complexes from HeLa cell extracts. Complexes shown to contain HDAC1, HDAC3, HDAC6, and HDAC1+2 as their catalytic subunits have been used in an antibody-based assay that detects deacetylation of whole histones at defined lysines. The class II deacetylase HDAC6 was inactive in this assay, but the three class I enzymes deacetylated all histone lysines tested, although with varying efficiency. In comparison to HDAC1, HDAC3 preferentially deacetylated lysines 5 and 12 of H4 and lysine 5 of H2A. H4 tails in purified mononucleosomes were refractory to deacetylation by both HDAC1 and HDAC3, unless ATP was added to the reaction mix. Surprisingly, ATP also consistently enhanced cleavage of free, non-nucleosomal histones, but not small peptides, by both enzyme complexes. We found no evidence that ATP operates by phosphorylation of components of the HDAC complex, but have shown that HDACs 1, 2, and 3 all co-immunoprecipitate with the ATP-dependent chaperone protein Hsp70. Another common ATP-dependent chaperone, Hsp90, was absent from all HDAC complexes tested, whereas Hsp60 associated with HDAC1 only. We suggest that Hsp chaperone proteins enhance the deacetylase activity of HDAC complexes by ATP-dependent manipulation of protein substrates.

    The Journal of biological chemistry 2002;277;11;9590-7

  • Antibodies against different epitopes of heat-shock protein 60 in children with type 1 diabetes mellitus.

    Horváth L, Cervenak L, Oroszlán M, Prohászka Z, Uray K, Hudecz F, Baranyi E, Madácsy L, Singh M, Romics L, Füst G and Pánczél P

    Faculty of Medicine, 3rd Department of Internal Medicine, Semmelweis University, Kútvölgyi út 4., H-1125, Budapest, Hungary

    The aim of this study was to investigate the amounts and epitope specificity of antibodies against heat shock protein 60 (hsp60) in the sera of type 1 diabetic and healthy children. Antibodies specific for peptide p277 of human hsp60 and of M. bovis as well as for human hsp60, M. bovis hsp65 proteins were measured by ELISA. Other autoantibodies (islet cell antibodies, glutamate decarboxylase antibodies and IA-2 antibodies) were also determined. A total number of 83 serum samples from children with type 1 diabetes mellitus and 81 samples of control children were investigated. Epitope scanning of the hsp60 for linear antibody epitopes was carried out using synthetic peptides attached to pins. The antibody levels specific for peptide p277 of human- and of M. bovis origin were significantly (human: P=0.0002, M. bovis: P=0.0044) higher in the diabetic children group than in the healthy children. We could not find significant difference in the antibody levels to whole, recombinant hsp proteins among the examined groups of children. Antibodies to two epitope regions on hsp60 (AA394-413 and AA 1f40 435-454) were detected in high titres in sera of children with diabetes mellitus. The first region similar to the sequence found in glutamate decarboxylase, whereas the second one overlaps with p277 epitope to a large extent. Presence of antibodies to certain epitopes of hsp60 (AA394-413-glutamic acid decarboxylase-like epitope; AA435-454-p277-like epitope) in diabetic children may reflect their possible role in the autoimmune diabetogenic process of the early diabetes.

    Immunology letters 2002;80;3;155-62

  • Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation.

    Barazi HO, Zhou L, Templeton NS, Krutzsch HC and Roberts DD

    Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.

    The alpha 3 beta 1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the alpha 3 beta 1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the alpha 3 beta 1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate alpha 3 beta 1 integrin but not to activate function of alpha 2 beta 1, alpha v beta 3, alpha 4 beta 1, or alpha 5 beta 1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the alpha 3 beta 1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the alpha 3 beta 1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate alpha 3 beta 1 integrin function and suggest that association of endogenous HSP60 with alpha 3 beta 1 integrin is necessary for IGF1-induced activation.

    Cancer research 2002;62;5;1541-8

  • Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells.

    Lipsker D, Ziylan U, Spehner D, Proamer F, Bausinger H, Jeannin P, Salamero J, Bohbot A, Cazenave JP, Drillien R, Delneste Y, Hanau D and de la Salle H

    INSERM, Equipe Propre 99-08, Etablissement Français du Sang-Alsace, 10 rue Spielmann, BP 36, F-67065 Strasbourg Cedex, France. dlipsker@noos.fr

    Priming of CTL by means of heat shock proteins (hsp) is dependent on antigen-presenting cells (APC), which present the hsp-associated peptides, via their cell surface MHC class I molecules, toCD8(+) T cells. It has not yet been established how human (hu) hsp70 interacts with the major (hu)APC, the dendritic cells (DC). Here we show that (hu)hsp70 is specifically internalized intoCD14(-), Toll-like receptor 4(-) monocyte-derived (hu)DC by receptor-mediated endocytosis. We further demonstrate that (hu)hsp70 and (hu)hsp60 share the same receptors on (hu)monocyte-derived DC. Both molecules as well as MHC class I molecules are spontaneously internalized and reach the MHC class II-enriched compartments. Finally, freshly isolated (hu) epidermal Langerhans cells (LC), the DC of the skin, as well as CD34(+)-derived LC do not bind hsp60 or hsp70. Given the likely importance of the internalization of hsp70 by APC in the induction of the immune responses, the finding that hsp60 and hsp70 are internalized through the same receptor(s) may explain why microbial hsp60 represents a major T cell antigen. This may rationalize the use of microbial hsp60 to prime immune responses against microbes. The lack of hsp60/70 receptors on epidermal LC raises the crucial question as to whether absence of priming of the skin and mucosal immune systems by hsp-polypeptide complexes could account for some tissue-specific diseases. This work also points to a potential advantage of using monocyte-derived DC in human immunotherapeutic applications of hsp60/70.

    European journal of immunology 2002;32;2;322-32

  • Directed proteomic analysis of the human nucleolus.

    Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M and Lamond AI

    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.

    Background: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized.

    Results: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions.

    Conclusions: This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

    Current biology : CB 2002;12;1;1-11

  • Functional interactions of human immunodeficiency virus type 1 integrase with human and yeast HSP60.

    Parissi V, Calmels C, De Soultrait VR, Caumont A, Fournier M, Chaignepain S and Litvak S

    REGER, UMR-5097 Centre National de la Recherche Scientifique (CNRS)-Université Victor Segalen Bordeaux 2, Bordeaux, France. vincent.parissi@reger.u-bordeaux2.fr

    Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.

    Journal of virology 2001;75;23;11344-53

  • H9724, a monoclonal antibody to Borrelia burgdorferi's flagellin, binds to heat shock protein 60 (HSP60) within live neuroblastoma cells: a potential role for HSP60 in peptide hormone signaling and in an autoimmune pathogenesis of the neuropathy of Lyme disease.

    Sigal LH, Williams S, Soltys B and Gupta R

    Department of Medicine, Robert Wood Johnson Medical School. University of Medicine and Dentistry of New Jersey, USA. sigallh@umdnj.edu

    Although Borrelia burgdorferi, the causative agent of Lyme disease, is found at the site of many disease manifestations, local infection may not explain all its features. B. burgdorferi's flagellin cross-reacts with a component of human peripheral nerve axon, previously identified as heat shock protein 60 (HSP60). The cross-reacting epitopes are bound by a monoclonal antibody to B. burgdorferi's flagellin, H9724. Addition of H9724 to neuroblastoma cell cultures blocks in vitro spontaneous and peptide growth-factor-stimulated neuritogenesis. Withdrawal of H9724 allows return to normal growth and differentiation. Using electron microscopy, immunoprecipitation and immunoblotting, and FACS analysis we sought to identify the site of binding of H9724, with the starting hypotheses that the binding was intracellular and not identical to the binding site of II-13, a monoclonal anti-HSP60 antibody. The current studies show that H9724 binds to an intracellular target in cultured cells with negligible, if any, surface binding. We previously showed that sera from patients with neurological manifestations of Lyme disease bound to human axons in a pattern identical to H9724's binding; these same sera also bind to an intracellular neuroblastoma cell target. II-13 binds to a different HSP60 epitope than H9724: II-13 does not modify cellular function in vitro. As predicted, II-13 bound to mitochondria, in a pattern of cellular binding very different from H9724, which bound in a scattered cytoplasmic, nonorganelle-related pattern. H9724's effect is the first evidence that HSP60 may play a role in peptide-hormone-receptor function and demonstrates the modulatory potential of a monoclonal antibody on living cells.

    Cellular and molecular neurobiology 2001;21;5;477-95

  • A yeast four-hybrid system identifies Cdk-activating kinase as a regulator of the XPD helicase, a subunit of transcription factor IIH.

    Sandrock B and Egly JM

    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, B. P. 163, 67404 Illkirch Cedex, France.

    To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast four-hybrid system was designed. When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pig 1f40 mentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK.XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.

    The Journal of biological chemistry 2001;276;38;35328-33

  • Autoantibodies to pancreatic hsp60 precede the development of glucose intolerance in patients with cystic fibrosis.

    Jensen P, Johansen HK, Carmi P, Høiby N and Cohen IR

    Department of Clinical Microbiology and The Danish Cystic Fibrosis Centre, Department of Paediatrics, National University Hospital, Copenhagen, Denmark. pej@biobase.dk

    Persons expressing the genetic disease cystic fibrosis (CF) suffer from a high risk of developing impaired glucose tolerance and diabetes. The development of diabetes in CF has been attributed, in the past, to the destruction of pancreatic islets and their resident beta-cells secondary to the destruction of the surrounding tissue by mechanical clogging of the pancreatic exocrine ducts. However, the discovery that autoimmunity to the 60-kDa heat shock protein (hsp60) may cause type I diabetes in NOD mice raises the possibility that hsp60 autoimmunity may be involved in CF diabetes too; could the hyperimmunization to bacterial hsp60 characteristic of CF spread to self-hsp60 and hence to autoimmune diabetes? We now report that rising levels of IgG autoantibodies to hsp60 do indeed precede the appearance of glucose intolerance and diabetes in CF patients. We produced a recombinant human pancreatic hsp60 protein and investigated the IgG antibody response to hsp60 in prediabetic and non-diabetic patients with CF. To detect hsp60 autoantibodies in the presence of high levels of antibodies to bacterial hsp60, we absorbed test sera with the 60-kDa GroEL of Pseudomonas aeruginosa and used an immunostaining technique. Using this technique, 32 prediabetic CF patients were evaluated over a five-year period, three years, on the average, before the onset of glucose intolerance. We found that a significant increase in hsp60 autoantibody preceded impaired glucose tolerance (P=0.042, n=17), diabetes (P=0.011, n=15) and glucose intolerance (P=0.005, n=32). As has been observed in NOD mice and in type I diabetic patients, the hsp60 autoantibodies decline at the outbreak of glucose intolerance in the CF patients. The association of CF diabetes with the rise and fall of hsp60 autoimmunity suggests that the pathogenesis of the diabetes may not be merely mechanical, but arise in the wake of bacterial hyperimmunisation.

    Journal of autoimmunity 2001;17;2;165-72

  • Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

    Liang Y, Li J, Chen J and Wang CC

    National Laboratory of Biomacromolec faa ules, Institute of Biophysics, Academia Sinica, Beijing, China.

    Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.

    European journal of biochemistry 2001;268;15;4183-9

  • Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes.

    Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth M, Alnemri ES and Cohen GM

    Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.

    During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecu 1311 lar weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex.

    Funded by: NIA NIH HHS: AG 13847

    The EMBO journal 2001;20;5;998-1009

  • The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli.

    Richardson A, Schwager F, Landry SJ and Georgopoulos C

    Département de Biochimie Médicale, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva, Switzerland.

    Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.

    The Journal of biological chemistry 2001;276;7;4981-7

  • Changes in skeletal muscle heat shock proteins: pathological significance.

    Liu Y and Steinacker JM

    Department of Sports and Rehabilitation Medicine, University of Ulm, Steinhövelstr. 9, D-89070 Ulm, Germany. yuefei.liu@medizin.uni-ulm.de

    In response to stress, cells rapidly produce a series of new proteins known as heat shock proteins (HSP). HSPs are considered to be molecular chaperones which play a universal role in maintaining cellular homeostasis. It is known that different HSPs are expressed in skeletal muscle, namely, small HSPs (including ubiquitin, alpha B- crystallin, HSP20 and HSP 27), HSP70, HSP60 and HSP90. Skeletal muscle is a complex and heterogeneous system in that its contractile proteins are made of different isoforms to form various muscle fibre types, and each type of muscle fibre has its own histochemical and functional characteristics. It seems that the induction of HSPs differs with muscle fibre type suggesting HSP expression is muscle fibre type specific. HSPs have been shown to respond in muscle diseases and following exercise. However, the molecular mechanisms of HSP induction, regulation and its role in maintaining the muscle function, are not completely understood. Relatively few studies of HSP have been conducted in human skeletal muscles. This review discusses the significance of changes of HSPs in skeletal muscle in both physiological and pathological conditions.

    Frontiers in bioscience : a journal and virtual library 2001;6;D12-25

  • Identification and characterization of the potential promoter regions of 1031 kinds of human genes.

    Suzuki Y, Tsunoda T, Sese J, Taira H, Mizushima-Sugano J, Hata H, Ota T, Isogai T, Tanaka T, Nakamura Y, Suyama A, Sakaki Y, Morishita S, Okubo K and Sugano S

    Department of Virology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the "oligo-capping" method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA(+)/Inr(+) PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and the TM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.

    Genome research 2001;11;5;677-84

  • Induction and membrane expression of heat shock proteins in heat-treated HPC-4 cells is correlated with increased resistance to LAK-mediated lysis.

    No authors listed

    Dept. of Biology, University of Tor Vergata, Rome, Italy.

    In human pancreatic carcinoma cells (HPC-4), a hyperthermic treatment at 43 degrees C for 30 min resulted in the vigorous induction of Hsp72, along with a less pronounced increase in the rate of synthesis of Hsp90, Hsp60 and Hsp 27. Biotinylation of surface-exposed proteins, followed by isolation of biotin-tagged proteins by affinity chromatography, demonstrated that both Hsp72 and Hsp60 are expressed on plasma membrane. Membrane expression of these two Hsps was confirmed by immunoprecipitation of surface biotinylated proteins with anti-Hsp72 and anti-Hsp60 specific antibodies. Cytotoxic assays showed that untreated HPC-4 cells are intrinsically resistant to NK-mediated lysis, while they were efficiently killed by LAK lymphocytes, as well as by exposure to TNFalpha. Following heat-treatment, cells became much more resistant to LAK-mediated lysis, while their sensitivity to NK-mediated lysis and to TNFalpha cytotoxicity remained unmodified.

    Journal of experimental & clinical cancer research : CR 2000;19;3;329-34

  • Protein substrate binding induces conformational changes in the chaperonin GroEL. A suggested mechanism for unfoldase activity.

    Hammarström P, Persson M, Owenius R, Lindgren M and Carlsson U

    IFM Department of Chemistry and Chemical Physics, Linköping University, S-581 83 Linköping, Sweden.

    Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.

    The Journal of biological chemistry 2000;275;30;22832-8

  • Stress proteins and the immune response.

    Moseley P

    Departments of Medicine and Biochemistry and Molecular Biology, University of New Mexico School of Medicine, ACC 5th Floor, Albuquerque, NM 87131, USA. pmoseley@unm.edu

    The heat shock or stress response is one of the most highly conserved adaptive responses in nature. In single cell organisms, the stress response confers tolerance to a variety of stresses including hyperthermia, hyperoxia, hypoxia, and other perturbations, which alter protein synthesis. This tolerance phenomeno 1409 n is also extremely important in the multicellular organism, resulting in not only thermal tolerance, but also resistance to stresses of the whole organism such as ischemia-reperfusion injury. Moreover, recent data indicates that these stress proteins have the ability to modulate the cellular immune response. Although the terms heat shock proteins (HSPs) and stress proteins are often used interchangeably, the term stress proteins includes the HSPs, the glucose-regulated proteins (GRPs) and ubiquitin. The stress proteins may be grouped by molecular weight ranging from the large 110 kDa HSP110 to ubiquitin at 8 kDa. These proteins serve as cellular chaperones, participating in protein synthesis and transport through the various cellular compartments. Because these proteins have unique cellular localizations, the chaperone function of the stress proteins often involves a transfer of peptides between stress proteins as the peptide is moved between cellular compartments. For example, HSP70 is a cytosolic and nuclear chaperone, which is critical for the transfer of cellular peptides in the mitochondrion through a hand-off that involves mitochondrial HSP60 at the inner mitochondrial membrane. Similarly, cytosolic proteins are transferred from HSP70 to gp96 as they move into the endoplasmic reticulum. The central role of the stress proteins in the transfer of peptides through the cell may be responsible for the recently recognized importance of the stress proteins in the modulation of the immune system [Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243-282.]. This importance in immune regulation is best addressed using Matzinger's model of the immune response - The Danger Theory of Immunity [Matzinger, P., Fuchs, E.J., 1996. Beyond self and non-self: immunity is a conversation, not a war. J. NIH Res. 8, 35-39.]. Matzinger suggests that an immune system model based on the differentiation between "self and non-self" does not easily account for the changes that occur in the organism with growth and development. Why, for example does an organism not self-destruct when the immune system encounters the myriad of new peptides generated at puberty? Instead, she proposes a model of immune function based on the ability to detect and address dangers. This model states that the basic function of all cells of the organism is appropriately timed death "from natural causes". This type of cell death, or apoptosis, generates no stress signals. If, on the other hand, a cell is "murdered" by an infectious agent or dies an untimely death due to necrosis or ischemia, the cell undergoes a stress response with the liberation of stress protein-peptide complexes into the extracellular environment upon cell lysis. Not only do they serve as a "danger signal" to alert the immune system to the death of a cell under stress, but their role as protein carriers allows the immune effector cells to survey the peptides released by this stressed cell and to activate against new or unrecognized peptides carried by the stress protein. Matzinger bases the Danger Theory of Immunity on three "Laws of Lymphotics". These laws state that: (1) resting T lymphocytes require both antigen stimulation by an antigen-presenting cell (APC) and co-stimulation with a danger signal to become activated; (2) the co-stimulatory signal must be received through the APC; and (3) T cells receiving only antigen stimulation without the co-stimulatory signal undergo apoptosis. The Danger Theory gives a simple model for both tolerance and activation. (ABSTRACT TRUNCATED)

    Immunopharmacology 2000;48;3;299-302

  • HSP expression in human leukocytes is modulated by endurance exercise.

    Fehrenbach E, Passek F, Niess AM, Pohla H, Weinstock C, Dickhuth HH and Northoff H

    Department of Transfusion Medicine, University of Tuebingen, Germany. elvira.fehrenbach@med.uni-tuebingen.de

    Purpose: Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression.

    Methods: The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control.

    Results: After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01).

    Conclusions: Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training.

    Medicine and science in sports and exercise 2000;32;3;592-600

  • A new locus for autosomal dominant pure spastic paraplegia, on chromosome 2q24-q34.

    Fontaine B, Davoine CS, Dürr A, Paternotte C, Feki I, Weissenbach J, Hazan J and Brice A

    INSERM CJF9 27e 711, Faculté de Médecine Pitié-Salpêtrière, 105 boulevard de l'hôpital, 75013 Paris, France. fontaine@infobiogen.fr

    Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous disorders causing progressive spasticity and weakness of the lower limbs. We report a large family of French descent with autosomal dominant pure HSP. We excluded genetic linkage to the known loci causing HSP and performed a genomewide search. We found evidence for linkage of the disorder to polymorphic markers on chromosome 2q24-q34: a maximum LOD score of 3. 03 was obtained for marker D2S2318. By comparison with families having linkage to the major locus of pure autosomal dominant HSP (SPG4 on chromosome 2p), there were significantly more patients without Babinski signs, with increased reflexes in the upper limbs, and with severe functional handicaps.

    American journal of human genetics 2000;66;2;702-7

  • gammadelta T cells lyse autologous and allogenic oesophageal tumours: involvement of heat-shock proteins in the tumour cell lysis.

    Thomas ML, Samant UC, Deshpande RK and Chiplunkar SV

    Cellular Immunology Unit, Cancer Research Institute, Tata Memorial Centre, Parel, Mumbai-400012, India.

    T cells expressing gammadelta receptors were isolated from the peripheral blood of oesophageal cancer patients and analysed for their potential to lyse tumour targets. Immunophenotyping by flow cytometry showed that the dominant population of gammadelta T cells expressed the Vgamma9 and the Vdelta2 T cell receptor, and a minor population expressed the Vdelta1 receptor. Cytotoxicity assays revealed that activated gammadelta T cells lysed Daudi Burkitt's lymphoma and K562 cells. Lysis of autologous oesophageal tumours was higher than of allogenic tumours. Anti-hsp60 and anti-hsp70 mAb significantly inhibited the cytotoxicity of gammadelta T cells to both autologous and allogenic oesophageal tumours. Surface expression of hsp60 and hsp70 on oesophageal tumours and Daudi cells was demonstrated by flow cytometry. In conclusion, gammadelta T cells isolated from the peripheral blood of oesophageal cancer patients have the ability of kill oesophageal tumour cells. The lysis of tumour targets by the gammadelta T cells is brought about via recognition of heat-shock proteins expressed on the surface of tumour cells. gammadelta T cells isolated from the peripheral blood may have applications in adoptive immunotherapy of oesophageal cancer.

    Cancer immunology, immunotherapy : CII 2000;48;11;653-9

  • Stress kinases and heat shock proteins in the pancreas: possible roles in normal function and disease.

    Schäfer C and Williams JA

    Department of Physiology 7737 Medical Science II, University of Michigan, Ann Arbor, 48109-0622, USA.

    Mitogen-activated protein kinases (MAPKs) and heat shock proteins (Hsps) are ubiquitous proteins that function in both normal and stress-related pathophysiological states of the cell. Recent advances 489 in the study of such Hsps and their interaction with signaling kinase cascades, including the cloning of new members, has helped to define their physiological roles in various tissues. In the pancreas, three major MAPKs, ERKs, JNKs, and p38, have been demonstrated. While intracellular signals involved in the ERK cascade have been most extensively investigated, only a few upstream regulators and downstream effector proteins of the JNKs and p38 MAPK are known in the pancreas. Similarly, a number of Hsps have been identified in pancreas, including Hsp27, Hsp60, and Hsp70. Although the activation of various MAPKs and the induction of Hsp expression have clearly been demonstrated following experimental exposure of rodent pancreas to stress conditions, it remains to be determined whether Hsps have a protective or detrimental effect during acute pancreatitis or at the onset of pancreatic carcinoma. This review will summarize current knowledge of the regulation and function of stress-activated kinases and stress proteins in the pancreas.

    Funded by: NIDDK NIH HHS: DK 52860

    Journal of gastroenterology 2000;35;1;1-9

  • Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins.

    Hittelman AB, Burakov D, Iñiguez-Lluhí JA, Freedman LP and Garabedian MJ

    Department of Microbiology and the Kaplan Comprehensive Cancer Center, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA.

    The hormone-activated glucocorticoid receptor (GR), through its N- and C-terminal transcriptional activation functions AF-1 and AF-2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two-hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor-interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF-1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF-1-mediated transactivation. Thus, GR AF-1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone-dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF-1 and AF-2, and represent important mediators of GR transcriptional enhancement.

    Funded by: NIDDK NIH HHS: 1R01 DK54836, DK45460; NIGMS NIH HHS: 2T32 GM07308

    The EMBO journal 1999;18;19;5380-8

  • A 60 kD heat-shock protein-like molecule interacts with the HIV transmembrane glycoprotein gp41.

    Speth C, Prohászka Z, Mair M, Stöckl G, Zhu X, Jöbstl B, Füst G and Dierich MP

    Institute for Hygiene and Ludwig Boltzmann-Institute for AIDS Research, Innsbruck, Austria. cornelia.speth@u 1a ibk.ac.at

    The heat-shock protein hsp60 is typically found in mitochondria, but, in smaller amounts, also in the cell cytoplasm and associated with the cell membrane. Since heat-shock proteins are known to interact with a variety of molecules and since purified HIV-1 particles were described to contain hsp60 molecules, we tested the possibility that a previously described putative receptor for HIV transmembrane protein gp41 is identical to hsp60. The gp41-binding human protein P62 was purified from H9 and Raji cell lysates by a gp41-coupled affinity column. We could show crossreactivity of both polyclonal and monoclonal anti-hsp60 antibodies with the purified P62. In addition we analyzed binding of P18, a soluble gp41 fragment harboring the extracellular domain (Env aa539-684), to recombinant hsp60. Hsp60 bound well to P18-coated ELISA plates whereas HIV-1 surface protein gp120 induced no binding of hsp60. Preincubation of hsp60 with gp41 abolished the binding. The possible role of this molecule as a cofactor in the pathogenesis of HIV disease is discussed.

    Molecular immunology 1999;36;9;619-28

  • Overexpression of the human 72 kDa heat shock protein in renal tubular cells confers resistance against oxidative injury and cisplatin toxicity.

    Komatsuda A, Wakui H, Oyama Y, Imai H, Miura AB, Itoh H and Tashima Y

    Third Department of Internal Medicine, Akita University School of Medicine, Japan.

    Background: Recent studies have shown that the 72-kDa heat shock protein (HSP72) can be induced in renal tubular cells by a variety of stress conditions, and suggested its cytoprotective function. We have tested this hypothesis directly by transfection studies.

    Methods: LLC-PK1 cells (porcine renal tubular epithelial cells) were stably transfected with pBK-CMV or pBK-CMV containing the human HSP72 gene (pBK-CMV-HSP72). These cells were then treated with various concentrations of hydrogen peroxide or cisplatin. The cell viability and lytic cell damage were determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release assay.

    Results: Immunoblot and immunocytochemical analyses showed the high level expression of HSP72 in LLC-PK1 cells transfected with pBK-CMV-HSP72. In addition, the expression of other major HSPs (HSP90, HSP73, HSP60 and HSP27) was not affected by transfection. LLC-PK1 cells overexpressing HSP72 were significantly more resistant to hydrogen peroxide and cisplatin treatments than control cells.

    Conclusion: These results indicate that overexpressed HSP72 plays a direct role in protecting renal tubular cells against oxidative injury and cisplatin toxicity.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 1999;14;6;1385-90

  • Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis.

    Xanthoudakis S, Roy S, Rasper D, Hennessey T, Aubin Y, Cassady R, Tawa P, Ruel R, Rosen A and Nicholson DW

    Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada H9H 3L1. steven_xanthoudakis@merck.com

    The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.

    The EMBO journal 1999;18;8;2049-56

  • Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells.

    Samali A, Cai J, Zhivotovsky B, Jones DP and Orrenius S

    Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, S-171 77, Stockholm, Sweden. afshin.samali@imm.ki.se

    Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Recently, mitochondria were found to play a central role in apoptosis through release of cytochrome c and activation of caspases. Moreover, a sub-population of pro-caspase-3 has been found to be localized to this organelle. In the present study, we demonstrate that pro-caspase-3 is present in the mitochondrial fraction of Jurkat T cells in a complex with the chaperone proteins Hsp60 and Hsp10. Induction of apoptosis with staurosporine led to the activation of mitochondrial pro-caspase-3 and its dissociation from the Hsps which were released from mitochondria. The release of Hsps occurred simultaneously with the release of other mitochondrial intermembrane space proteins including cytochrome c and adenylate kinase, prior to a loss of mitochondrial transmembrane potential. In in vitro systems, recombinant Hsp60 and Hsp10 accelerated the activation of pro-caspase-3 by cytochrome c and dATP in an ATP-dependent manner, consistent with their function as chaperones. This finding suggests that the release of mitochondrial Hsps may also accelerate caspase activation in the cytoplasm of intact cells.

    Funded by: NIEHS NIH HHS: ES09047

    The EMBO journal 1999;18;8;2040-8

  • The expression of heat shock protein 60 in patients with dilated cardiomyopathy.

    Latif N, Taylor PM, Khan MA, Yacoub MH and Dunn MJ

    Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, Harefield Hospital, Middx., United Kingdom. Najma.Latif@harefield.nthames.nhs.uk

    Objective: Heat shock proteins (hsps) have been shown to be important antigens in a number of autoimmune diseases. We have previously shown the presence of autoantibodies against hsp60 in a high proportion of patients with dilated cardiomyopathy (DCM). This study set out to investigate the expression of hsp60 in the myocardium of 30 patients with DCM and a control group of 30 normal donors.

    Design: The expression of hsp60 was quantitated at the protein and mRNA level by Western blotting and RT-PCR, respectively, and its distribution was investigated by immunocytochemistry.

    Results: Quantitation of hsp60 showed a 5-fold increase in the DCM hearts over that in the donor hearts. By immunocytochemistry 13 patients with DCM showed increased positive staining localised to the connective tissue. Semi-quantitative RT-PCR analysis of hsp60 showed a significantly increased amount of hsp60 at the mRNA level in the DCM hearts than in the controls.

    Conclusions: These results unequivocally demonstrate a raised level of expression of endogenous hsp60 in the myocardium of patients with DCM. The increased expression of hsp60 in the myocardium of patients with DCM may render such cells susceptible to react with circulating autoantibodies to hsp60 and hsp65 found in a high proportion of patients with this disease.

    Basic research in cardiology 1999;94;2;112-9

  • Identification of human heat shock protein 60 (Hsp60) and anti-Hsp60 antibodies in the peripheral circulation of normal individuals.

    Pockley AG, Bulmer J, Hanks BM and Wright BH

    Division of Clinical Sciences (NGHT), Clinical Sciences Centre, Herries Road, Sheffield, UK. g.pockley@sheffield.ac.uk

    Although primarily regarded as being intracellular, this study has identified the presence of heat shock protein 60 (Hsp60) in the peripheral circulation of normal individuals. The median Hsp60 concentration was approximately 3.5-fold higher in females than in males and significantly higher levels of anti-human Hsp60 antibodies were also detected in females. There were no differences in the levels of antibodies to mycobacterial Hsp60 between males and females, nor did antibody levels correlate with Hsp60 concentrations. Hsp60 was not released from mitogenically stimulated peripheral blood mononuclear cells. The potential physiological roles for circulating Hsp60 are unknown. Given the emerging evidence that inappropriate reactivity to heat shock proteins is involved in autoimmune disease and that T cells responsive to self Hsp60 appear to be protective, these findings suggest that circulating Hsp60 may be involved in the regulation of tolerance to self and immunity to bacterial forms of these widely expressed and structurally conserved proteins.

    Cell stress & chaperones 1999;4;1;29-35

  • EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction.

    Persson M, Hammarström P, Lindgren M, Jonsson BH, Svensson M and Carlsson U

    IFM-Department of Chemistry, Linköping University, Sweden.

    Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature. To further investigate this interaction we used electron paramagnetic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-labeled at selected positions. From our results it is evident that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements. HCA II needs to be unfolded to about the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL. The interaction with GroEL includes interactions with outer parts of the HCA II molecule, such as peripheral beta-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core exhibits higher flexibility than in the molten globule, which is likely to facilitate rearrangements of misfolded structure during the folding process. The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactions appear to be most important in stabilizing the GroEL-substrate complex.

    Biochemistry 1999;38;1;432-41

  • Detection of the human 70-kD and 60-kD heat shock proteins in the vagina: relation to microbial flora, vaginal pH, and method of contraception.

    Giraldo P, Neuer A, Ribeiro-Filho A, Linhares I and Witkin SS

    Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, NY 10021, USA.

    The expression of the 60-kD and 70-kD heat shock proteins (hsp60 and hsp70) in the vaginas of 43 asymptomatic women of reproductive age with or without a history of recurrent vulvovaginitis (RVV) were compared. Vaginal wash samples were obtained and assayed by enzyme-linked immunosorbent assay (ELISA) for human hsp60 and hsp70. Heat shock protein 70 was not detected in any of the 19 women with no history of RVV, and hsp60 was present in only one woman in this group. In contrast, in the RVV group, 11 (45.8%) were hsp60-positive and eight (33.3%) were hsp70-positive. The presence of either heat shock protein in the vagina was associated with an elevated vaginal pH (>4.5). Bacterial vaginosis or Candida was identified in some of the asymptomatic subjects; their occurrence was significantly higher in women with vaginal hsp70 than in women with no heat shock proteins. Oral contraceptives were used by 35.7% of subjects who were negative for vaginal heat shock proteins, as opposed to only 12.5% of women who were positive for hsp70 and 8.3% who were positive for hsp60. Expression of heat shock proteins in the vagina may indicate an altered vaginal environment and a susceptibility to vulvovaginal symptoms.

    Infectious diseases in obstetrics and gynecology 1999;7;1-2;23-5

  • Heat shock proteins and heat shock protein-antibody complexes in placental tissues.

    Ziegert M, Witkin SS, Sziller I, Alexander H, Brylla E and Härtig W

    Department of Obstetrics and Gynecology, University of Leipzig, Germany.

    Objective: The relationship between pregnancy outcome and expression of the heat shock proteins (hsps) or hsp-antibody complexes of 60kD (hsp60), 70kD (hsp70), and 90kD (hsp90) in placental tissue and circulating antibodies to hsps was evaluated.

    Method: Expression of hsp60, hsp70, and hsp90 in placentae from 12 women with preterm birth, eight with intrauterine growth restriction (IUGR), and 10 with term birth, as well as the presence of the corresponding antibodies, was investigated by a new carbocyanine double fluorescence technique. Results were compared with microbiological findings and circulating antibodies to hsps in sera.

    Results: In each placental specimen examined, hsp60, hsp70, and hsp90 were identified. However, hsp70-antibody complexes were detected in only four of the preterm labor cases. Similarly, hsp60-antibody complexes were detected in only five preterm labor patients and in one patient with IUGR. None of the placentae contained hsp90-antibody complexes. In the preterm birth group, all patients with hsp60-antibody complexes were also positive for circulating antibodies to hsp60. The presence of hsp70-antibody complexes also correlated with hsp70 antibody in sera.

    Conclusions: Formation of hsp60- and hsp70-antibody complexes in the placenta may contribute to the induction of preterm birth. Women sensitized to these antibodies may be at increased risk for adverse pregnancy outcome.

    Infectious diseases in obstetrics and gynecology 1999;7;4;180-5

  • Heat shock proteins in human endometrium throughout the menstrual cycle.

    Tabibzadeh S and Broome J

    Department of Pathology, North Shore University Hospital, Biomedical Research Center, Manhasset, NY 11030, USA. tabibzadeh@bioscience.org

    Human endometrium, in response to steroid hormones, undergoes characteristic cycles of proliferation, secretory changes, and tissue shedding. Human endometrium expresses a molecular repertoire which includes the heat shock proteins (Hsps) Hsp27, Hsp60, Hsp70, Hsp90, and alpha crystallin B chain. The expression of Hsp27, Hsp60, and the constitutive form of Hsp70 (Hsc70) shows a sharp increase in human endometrium after ovulation. The maximal expression of the molecular chaperone, alpha crystallin B chain, occurs during the secretory phase. In view of known functions of the Hsps, it is likely that these proteins are involved in protection of the endometrial proteins against factors with the potential to lead to protein denaturation. Tumor necrosis factor-alpha (TNF-alpha) is a cytotoxic cytokine that is produced in progressive amounts during the secretory phase. The function of the Hsps may be to protect cells against the cytotoxic damage of TNF-alpha, particularly during the critical period of "implantation window."

    Funded by: NCI NIH HHS: CA46866

    Infectious diseases in obstetrics and gynecology 1999;7;1-2;5-9

  • Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane.

    Khan IU, Wallin R, Gupta RS and GM

    Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, N. C. 27157, Canada.

    Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4(+) CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

    Funded by: NCI NIH HHS: 5 P30 CA12197-21N,21S1, CA-12197-22, P30 CA012197; NIAMS NIH HHS: AR-39501, R01 AR039501

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;18;10425-30

  • Relationship between heat shock protein 60 (HSP60) mRNA expression and resistance to platinum analogues in human ovarian and bladder carcinoma cell lines.

    Abu-Hadid M, Wilkes JD, Elakawi Z, Pendyala L and Perez RP

    Departments of Solid Tumor Oncology and Investigational Therapeutics, Division of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.

    Heat shock protein 60 (HSP60) participates in protein assembly, folding and transport. Increased HSP60 mRNA expression is associated with cisplatin resistance in some in vitro models and with shorter survival among ovarian cancer patients. HSP60 mRNA expression was quantitated in three independent model systems by reverse-transcription PCR (A2780 human ovarian carcinoma cell line and sublines selected for cisplatin or oxaliplatin resistance in vitro and also in the UCRU-BL13 human bladder carcinoma cell line and a cisplatin-resistant subline). Increased HSP60 mRNA expression was observed in all resistant sublines (range 2.5-15 fold), correlated with relative resistance to cisplatin and oxaliplatin. No differences in HSP60 gene copy number were apparent in resistant sublines. These data provide further evidence of a strong association between in vitro resistance to platinum compounds and increased HSP60 mRNA expression.

    Cancer letters 1997;119;1;63-70

  • Cell surface localization of the 60 kDa heat shock chaperonin protein (hsp60) in mammalian cells.

    Soltys BJ and Gupta RS

    Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.

    To investigate whether the 60-kDa heat shock chaperonin protein (hsp60) is present on the surface of mammalian cells, we used immunogold labeling of intact cells and backscattered electron imaging to image gold particles. Chinese hamster ovary cells and the human leukemic CD4-positive T-cell line CEM-SS on glass coverslips were labeled using affinity-purified monoclonal and polyclonal antibodies specific for hsp60 and 30 nm gold markers. Cells were imaged using the scanning mode of the conventional transmission electron microscope. Backscattered electron imaging provided definitive identification of the gold markers while secondary electron imaging gave information on surface architecture. Labeling intensity was 250-800 gold particles per cell in Chinese hamster ovary cells and 600-2000 in CEM-SS human lymphoblasts. The finding of hsp60 o 1aaf n the cell surface of mammalian cells may signify chaperone involvement in surface functions.

    Cell biology international 1997;21;5;315-20

  • A two-dimensional gel database of human colon carcinoma proteins.

    Ji H, Reid GE, Moritz RL, Eddes JS, Burgess AW and Simpson RJ

    Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Victoria, Australia.

    The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M(r)/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http:(/)/www.ludwig.edu.au.

    Electrophoresis 1997;18;3-4;605-13

  • Two-dimensional electrophoretic analysis of human breast carcinoma proteins: mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2.

    Rasmussen RK, Ji H, Eddes JS, Moritz RL, Reid GE, Simpson RJ and Dorow DS

    Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.

    MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein. To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: http:(/)/ www.ludwig.edu.au/www/jpsl/jpslhome.htm l).

    Electrophoresis 1997;18;3-4;588-98

  • Macrophage-lysis mediated by autoantibodies to heat shock protein 65/60.

    Schett G, Metzler B, Mayr M, Amberger A, Niederwieser D, Gupta RS, Mizzen L, Xu Q and Wick G

    Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria.

    Macrophages in atherosclerotic lesions have been shown to express high amounts of heat shock protein 60 (hsp60), a highly conserved protein. Patients with atherosclerosis have high titers of anti-hsp65/60 antibodies (Ab) recognizing macrophages in the lesions. To elucidate the role of anti-hsp65/60 Ab in macrophage cytotoxicity, human high titer serum and purified anti-hsp65/60 Ab were tested on in vitro heat-stressed cells of a human macrophage cell line (U937) and macrophages derived from peripheral blood. Application of heat stress at 42 degrees C for 30 min resulted in marked upregulation of hsp60 mRNA, followed by increased protein expression as determined by Northern blot and FACS-analysis, respectively. Compared to unstressed cells, high titer serum and anti-hsp65/60 Ab preferentially bound to the surface of stressed U937 macrophages, but not control antibodies. Furthermore, high titer serum and anti-hsp65/60 Ab exerted significant (P < 0.01) complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) on stressed 51Cr-labelled U937 and peripheral blood derived macrophages. Thus, macrophages expressing hsp60 can be lysed by autoantibodies against hsp65/60, which may contribute to cell death in atherosclerotic plaques in vivo.

    Atherosclerosis 1997;128;1;27-38

  • Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

    Gross M, Robinson CV, Mayhew M, Hartl FU and Radford SE

    Oxford Centre for Molecular Sciences, New Chemistry Laboratory, United Kingdom.

    An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.

    Protein science : a publication of the Protein Society 1996;5;12;2506-13

  • Prion protein PrPc interacts with molecular chaperones of the Hsp60 family.

    Edenhofer F, Rieger R, Famulok M, Wendler W, Weiss S and Winnacker EL

    Laboratorium Für Molekulare Biologie-Genzentrum-Institute Für Biochemie der Ludwig-Maximilians-Universität Munchen, Germany.

    Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans. The prion particle consists mainly, if not entirely, of PrPSc, a posttranslationally modified isoform of the cellular host-encoded prion protein (PrPc). It has been suggested that additional cellular factors might be involved in the physiological function of PrPc and in the propagation of PrPSc. Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrPc. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.

    Journal of virology 1996;70;7;4724-8

  • Heat shock proteins in human endometrium throughout the menstrual cycle.

    Tabibzadeh S, Kong QF, Satyaswaroop PG and Babaknia A

    Department of Pathology, University of South Florida, Tampa, USA.

    Human endometrium is a steroid-sensitive tissue and there is evidence that supports the viewpoint that heat shock proteins (HSP) are implicated in the regulation of steroid function. Therefore, in this study we examined the expression of various members of the heat shock family of proteins in the steroid-responsive human endometrium. Western blot analysis revealed that the expression of HSP90 showed minimal changes throughout the menstrual cycle. When normalized to the amount of HSP90, the expression of HSP27, HSP60 and the constitutive form of heat shock protein 70 (HSC70) increased progressively during the late proliferative and early secretory phases, and diminished in the mid- to late secretory and menstrual phases. In contrast, the inducible form of heat shock protein 70 (HSP70) did not undergo these changes. The cellular and subcellular localizations of these proteins were examined in human endometria by immunohistochemical staining. With the exception of HSP70, which was found primarily in the epithelial cells, the immunoreactivity for other heat shock proteins was found in both the stroma and the epithelium. Immunoreactivity for HSP27 was found in the lymphoid aggregates within endometrial stroma, and both HSP27 and HSP90 were found in endothelial cells. The immunoreactive heat shock proteins were found in the nuclei and/or cytoplasm of cells. However, no consistent nuclear versus cytoplasmic staining emerged, and such localization was irrespective of the site, the cell type or the phase of the menstrual cycle. Our findings show that endometrium has a full complement of heat shock proteins. The menstrual cycle-dependent changes in the amounts of heat shock protein suggest regulation by steroid hormones.

    Funded by: NCI NIH HHS: CA46866

    Human reproduction (Oxford, England) 1996;11;3;633-40

  • Induction of heat shock protein in monocytic cells by oxidized low density lipoprotein.

    Frostegård J, Kjellman B, Gidlund M, Andersson B, Jindal S and Kiessling R

    Department of Medicine, Karolinska Hospital, Karolinska Institutet, Stockholm, Sweden.

    The atherosclerotic lesion may be characterized as a chronic inflammatory process, and oxidized LDL is believed to be a key event in the development of atherosclerosis, though the mechanisms by which oxidized LDL exerts its proatherogenic properties are largely unknown. Heat shock proteins (hsp) are a group of proteins with a highly conserved structure and of these, hsp60 has been suggested to play a role in autoimmunity due to T lymphocyte crossreactivity between bacterial and human hsp60. The present study was designed to investigate the effects of oxidized LDL on the expression of hsp60 using the monocytic cell lines U937 and HL60 as models. The expression of hsp60 was determined by using monoclonal antibodies to hsp60 in FACScan, Western blot, and a sandwich ELISA. The results show that hsp60 is induced in both cell types after 2 h exposure to oxidized LDL, with a maximal effect at 20 micrograms/ml for U937 cells and 5 micrograms/ml for HL60 cells. A close to 3-fold increase in the expression of hsp60 was seen after culturing oxidized LDL (20 micrograms/ml) treated U937 cells for a period of 24 h. Interleukin 1-beta had similar effects on da1 hsp60 expression to oxidized LDL. The results indicate that expression of hsp60 by monocytes in the vascular wall may be enhanced by oxidized LDL. It is thus possible that the chronic inflammatory process characterizing atherosclerosis is perpetuated by autoreactive T cells, which recognize hsp60 expressed by monocytes, induced by oxidized LDL.

    Atherosclerosis 1996;121;1;93-103

  • Protein folding in the central cavity of the GroEL-GroES chaperonin complex.

    Mayhew M, da Silva AC, Martin J, Erdjument-Bromage H, Tempst P and Hartl FU

    Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

    The chaperonin GroEL is able to mediate protein folding in its central cavity. GroEL-bound dihydrofolate reductase assumes its native conformation when the GroES cofactor caps one end of the GroEL cylinder, thereby discharging the unfolded polypeptide into an enclosed cage. Folded dihydrofolate reductase emerges upon ATP-dependent GroES release. Other proteins, such as rhodanese, may leave GroEL after having attained a conformation that is committed to fold. Incompletely folded polypeptide rebinds to GroEL, resulting in structural rearrangement for another folding trial in the chaperonin cavity.

    Nature 1996;379;6564;420-6

  • Expression of the human heat shock protein 60 in thyroid, pancreatic, hepatic and adrenal autoimmunity.

    Mallard K, Jones DB, Richmond J, McGill M and Foulis AK

    Department of Pathology, Royal Infirmary, Glasgow, UK.

    Heat shock proteins (hsps) have been proposed to play a role in autoimmune disease. Their highly conserved nature and role as a common antigenic determinant throughout phylogeny has raised the possibility that they may act as cellular targets of an autoimmune response when their expression is altered in stressed tissue cells. Using an antibody to human hsp60 we have demonstrated a wide tissue distribution in normal tissues, including thymus, the degree of staining reflecting the content of mitochondria in the cells, consistent with the known mitochondrial location of this protein. Enhanced staining was also demonstrated in oncocytes (deeply eosinophilic cells which have greatly increased numbers of mitochondria) in both thyroid and adrenal autoimmune disease and also in unrelated conditions where oncocytic change was identified. No enhancement was demonstrated in target cells in organ specific autoimmune diseases where oncocytic change was not seen, for example islet cells in diabetes and bile duct cells in primary biliary cirrhosis. Thus, no alteration of hsp60 expression was demonstrated which was specific to the autoimmune diseases studied.

    Journal of autoimmunity 1996;9;1;89-96

  • [Immunostaining of mitochondrial heat shock proteins (mtHSPs) in skeletal muscle fibers of mitochondrial cytopathy].

    Ibi T, Sahashi K, Ling J, Marui K and Mitsuma T

    Fourth Department of Medicine, Aichi Medical University.

    Expression of the mtHSPs (HSP60 and mtHSP70) was immunohistochemicall observed in biopsied limb muscles of genetically determined mitochondrial cytopathies (chronic progressive ophthalmoplegia 14, MELAS 4, limb girdle syndrome with the A-to-G transition at nt.3243 of tRNALeu(UUR), exertional myoglobinuria with multiple deletions of mtDNA 2, and Leber's hereditary optic neuropathy 2). mtHSP 70 and HSP 60 were strongly localized at ragged-red fibers. In strongly succinate dehydrogenase-reactive vessels of MELAS, mtHSP70 was expressed. GRP78 was expressed in the cytoplasmic body, which is often observed in this disorder. The present data suggest that expression of mtHSPs may reflect increased numbers of mitochondria, an impairment of assembly of mitochondrial proteins encoded by the genomic DNA and abnormal mitochondrial DNA, and/or an impaired mitochondrial function due to recurrent oxygen radical attacks against mitochondria.

    Rinsho shinkeigaku = Clinical neurology 1996;36;1;61-4

  • Common antigenicity between Yersinia enterocolitica-derived heat-shock protein and the retina, and its role in uveitis.

    Tanaka T, Yamakawa N, Yamaguchi H, Okada AA, Konoeda Y, Ogawa T, Kamiya S and Usui M

    Department of Ophthalmology, Tokyo Medical College Hospital, Japan.

    Yersinia enterocolitica-derived heat-shock protein (HSP60) was recently demonstrated to be associated with certain systemic autoimmune diseases. A role for HSP60 is also suspected in the pathogenesis of some types of uveitis believed to involve autoimmune mechanisms, such as Behçet's disease. We report our results on the role of HSP60 in patients with uveitis. HSP60 was subjected to electrophoresis in immunoblot analysis, and then allowed to react with sera from patients with uveitis in order to detect the presence of anti-HSP60 antibody. Tissue extracts from human and bovine retina were also electrophoresed, and then treated with anti-HSP60 monoclonal antibodies to determine whether or not the antibodies recognized ocular tissues. Immunoblot analysis revealed anti-HSP60 antibodies in patient sera. Furthermore, anti-HSP60 monoclonal antibodies reacted against the 60-kD protein derived from human and bovine retinal extracts. These immunological cross-reactions between HSP60 and the retina demonstrate a common antigenicity. Furthermore, detection of specific antibody against HSP60 in patient sera suggests that this common antigenicity between HSP60 and the retina may be related to the pathogenesis of uveoretinitis in some cases.

    Ophthalmic research 1996;28;5;284-8

  • Presence of Chromatium vinosum chaperonins 10 and 60 in mitochondria and peroxisomes of rat hepatocytes.

    Vélez-Granell CS, Arias AE, Torres-Ruíz JA and Bendayan M

    Department of Anatomy, Université de Montréal, Quebec, Canada.

    In the present study we report the occurrence of chaperonins, cpn10 and cpn60, in Chromatium vinosum and rat hepatocytes, using specific polyclonal antibodies in conjunction with the protein A-gold immunocytochemical technique. As demonstrated by quantitative evaluations, the immunolabeling for cpn10 and cpn60 in C vinosum cells was associated primarily with the bacterial cell envelope. In rat liver homogenates, Western immunoblotting analysis has shown that antibodies to cpn10 from C vinosum recognize an unique 25-kDa protein that remains to be further characterized. On the other hand, the antibody to cpn60 from C vinosum revealed the presence of a 60-kDa protein in the rat liver homogenates. Immunofluorescence on rat liver tissue revealed an intracellular granular labeling for both chaperonins. On the other hand, using the post-embedding immunoelectron microscopy technique cpn10 and cpn60 were localized specifically in liver mitochondria and peroxisomes. Interestingly, further analysis of the labeling distribution confirmed the association of both proteins with the mitochondrial inner membrane whereas in the peroxisomes the chaperonins appeared to be located in the matrix, away from the limiting peroxisomal membrane. The colocalization of both chaperonins suggests that, as in other bacteria as well as eukaryotic cells, they may act in tandem for the proper folding of particular proteins.

    Funded by: NIGMS NIH HHS: SO6 GM-68239

    Biology of the cell 1995;85;1;67-75

  • The human myocardial two-dimensional gel protein database: update 1994.

    Corbett JM, Wheeler CH, Baker CS, Yacoub MH and Dunn MJ

    Department of Cardiothoracic Surgery, National Heart and Lung Institute, Heart Science Centre, Middlesex, UK.

    An updated human heart protein two-dimensional electrophoresis (2-DE) database is presented. The database, which contains some 1388 protein spots characterised in terms of M(r) and pI, has been analysed further by Western immunoblotting and protein sequencing. From a total of 103 protein spots analysed, 49 have been identified by immunoblotting and 32 have been identified by protein sequencing. A further six proteins have tentatively been assigned by comparison with the human heart 2-DE protein database of Jungblut et al. (Electrophoresis) 1994, 15, 685-607). This database is being used in studies of alterations in protein expression in the diseased and transplanted human heart.

    Electrophoresis 1994;15;11;1459-65

  • Immuno-gold electron microscopical detection of heat shock protein 60 (hsp60) in mitochondria of rat hepatocytes and myocardiocytes.

    Kreisel W, Hildebrandt H, Schiltz E, Köhler G, Spamer C, Dietz C, Mössner W and Heilmann C

    Medizinische Universitäts-Klinik, Abteilung Gastroenterologie und Hepatologie, Freiburg, Germany.

    We characterize the specificity of a polyclonal antibody against heat shock protein 60 (hsp60) and present an application for ultrastructural localization studies of this protein. The antibody was obtained from an IgG fraction (AB 121) originally raised against the calcium binding protein calsequestrin by immunoabsorption on isolated rat liver hsp60. As shown by partial N-terminal amino acid sequence analysis of immunoprecipitated proteins AB 121 contained reactivities against hsp60, calsequestrin and the glycoprotein fetuin. In rat heart AB 121 recognized calsequestrin and hsp60. In human and rat liver the only reacting protein was hsp60. In rat erythrocytes the antibody bound to 61 kDa and 58 kDa isoforms of fetuin. According to published data no amino acid sequence homologies nor common motifs are found between calsequestrin, hsp60 and fetuin. As the first application the anti-hsp60 antibody was used for immuno-gold electron microscopical localization of hsp60: in myocardiocytes and hepatocytes of the rat strong labelling was obtained exclusively in mitochondria. No extramitochondrial structures were labelled. The specificity of the antibody and its ability to be visualized by immuno-gold electron microscopy offers the possibility to study the expression of this protein in the liver and in other organs. Possible clinical applications of these studies are discussed, since hsp60 could be a target antigen of autoantibodies in diseases such as autoimmune hepatitis, primary sclerosing cholangitis or primary biliary cirrhosis.

    Acta histochemica 1994;96;1;51-62

  • Intestinal expression and cellular immune responses to human heat-shock protein 60 in Crohn's disease.

    Baca-Estrada ME, Gupta RS, Stead RH and Croitoru K

    20f Department of Medicine, McMaster University, Hamilton, Ontario, Canada.

    Changes in the intestinal expression of the endogenous human 60-kDa heat-shock protein (HSP60) were investigated in patients with Crohn's disease. HSP60 immunoreactivity was detected in epithelial cells, vascular smooth muscle, and nerve cell bodies of both small and large bowel from patients with Crohn's disease. However, control tissue showed a similar pattern of HSP60 expression. Western blot analysis confirmed that the HSP60 immunoreactivity detected in the intestine corresponded to the 60-kDa HSP. The proliferative response of peripheral blood lymphocytes (PBL) and intestinal intraepithelial lymphocytes (IEL) to recombinant human HSP60 was examined. The results indicate that there was no significant difference in responses between patients with Crohn's disease and controls. Furthermore, there was no increase in the proportion of gamma/delta T cell receptor-bearing T cells in PBL from patients with Crohn's disease cultured for six days in the presence of human HSP60 as compared to control patients. These results suggest that endogenous human HSP60 is unlikely to be a target for an autoimmune response in patients with Crohn's disease.

    Digestive diseases and sciences 1994;39;3;498-506

  • Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells.

    Vélez-Granell CS, Arias AE, Torres-Ruíz JA and Bendayan M

    Department of Anatomy, Faculty of Medicine, Université de Montréal, Quebec, Canada.

    Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

    Funded by: NIGMS NIH HHS: 506 GM-8239

    Journal of cell science 1994;107 ( Pt 3);539-49

  • Human liver protein map: a reference database established by microsequencing and gel comparison.

    Hochstrasser DF, Frutiger S, Paquet N, Bairoch A, Ravier F, Pasquali C, Sanchez JC, Tissot JD, Bjellqvist B, Vargas R et al.

    Medicine Department, Geneva University, Switzerland.

    This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

    Electrophoresis 1992;13;12;992-1001

  • Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin.

    Dawson SJ and White LA

    Department of Microbiology, Southampton General Hospital, U.K.

    A patient with Haemophilus aphrophilus endocarditis was successfully treated with ciprofloxacin. The response to treatment with cefotaxime and netilmicin for 12 days was poor but was satisfactory to a 6 weeks' course of ciprofloxacin.

    The Journal of infection 1992;24;3;317-20

  • Immunocytochemical localization of heat-shock protein 60-related protein in beta-cell secretory granules and its altered distribution in non-obese diabetic mice.

    Brudzynski K, Martinez V and Gupta RS

    Department of Medicine, University of Western Ontario, London, Canada.

    Immuno-electron microscopy technique was employed to investigate the cellular distribution of 60 kDa heat-shock protein (HSP60) in pancreatic Beta cells of control and non-obese diabetic mice. In thin sections prepared from control mice, antibody to mammalian HSP60 cross-reacted with protein(s) located to mitochondria and secretory granules. In particular, prominent binding of the antibody was seen to the insulin core of the mature insulin-secreting granules. In comparison, very little immunoreactivity was observed with immature secretory granules or with the Golgi apparatus. No binding to secretory granules or mitochondria was observed with normal mouse serum or with unrelated sera. On Western blots, HSP60 antibody specifically interacted with a single 62 kDa islet cell protein. These results suggest the existence of an HSP60-related protein with a novel location in mature secretory granules of Beta cells. The preferential association of the HSP60-related protein with the insulin core was gradually lost in Beta cells of pre-diabetic non-obese diabetic mice, and correlated with the progression of insulitis. The decrease in the granular binding of the HSP60 antibody was accompanied by an increase in cytoplasm staining, and was concomitant with a significant expansion of the insulin core diameter. The altered distribution of the HSP60-related protein in prediabetic mice, together with our observation that immature secretory granules accumulate in these animals indicate that the presence of HSP60-related protein in secretory granules might be associated with the secretory function of Beta cells.

    Diabetologia 1992;35;4;316-24

  • An interaction between p21ras and heat shock protein hsp60, a chaperonin.

    Ikawa S and Weinberg RA

    Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

    Ras proteins play a crucial role in the development of neoplasia and in signal transduction in normal cells. In a search for proteins interacting with p21ras, we previously identified a protein of 60 kDa (p60) through use of a chemical cross-linker. Using information from partial amino acid sequencing of the purified protein, we isolated full-length cDNA clones encoding this 60-kDa protein. Nucleotide sequence analysis revealed that p60 is the murine heat shock protein hsp60, a chaperonin. Association of hsp60 with p21ras appears physiological, as the amount of hsp60 complexed to p21ras was similar even in cells over-expressing p21ras, and reversing the order of cross-linking and lysis of the cells, which releases large amounts of hsp60 from mitochondria, did not alter the amount of hsp60 cross-linked to p21ras.

    Funded by: NCI NIH HHS: 5 R35 CA39826

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;6;2012-6

  • Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.

    Ward LD, Hong J, Whitehead RH and Simpson RJ

    Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Parkville, Australia.

    The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.

    Electrophoresis 1990;11;10;883-91

  • Nucleotide sequences and novel structural features of human and Chinese hamster hsp60 (chaperonin) gene families.

    Venner TJ, Singh B and Gupta RS

    Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.

    A number of clones that specifically hybridize to the human hsp60 cDNA (chaperonin protein; GroEL homolog) were isolated from human and Chinese hamster ovary cell genomic libraries. DNA sequence analysis shows that one of these clones, pGem-10, is completely homologous to the human hsp60 cDNA (in both coding and noncoding regions) with no intervening sequences. The other human clones analyzed were all nonfunctional pseudogenes containing numerous small additions, deletions, and base substitutions, but no introns. On the basis of sequence data, six different hsp60 pseudogenes were identified in human cells. In addition, we also cloned and completely sequenced a genomic clone from CHO cells. This clone, which was also a pseudogene, contained a small 87-nucleotide intron near the 3' end. Southern blot analysis of human, mouse, and Chinese hamster DNA, digested with unique restriction enzymes (no sites in cDNA), indicates the presence of about 8-12 genes for hsp60 in the vertebrate genomes. The sequence data, however, suggest that most of these genes, except one (per haploid genome), are likely to be nonfunctional pseudogenes.

    DNA and cell biology 1990;9;8;545-52

  • Mitochondrial import of the human chaperonin (HSP60) protein.

    Singh B, Patel HV, Ridley RG, Freeman KB and Gupta RS

    Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.

    The mitochondrial import of a member of the "chaperonin" group of proteins which play an essential role in the import of protein into organelles and their subsequent proper folding has been examined. The cDNA for human hsp60 (synonyms: GroEL homolog, P1) was transcribed and translated in vitro and its import into isolated rat heart mitochondria examined. The protein was converted into a mature form of lower molecular mass (= 58 kDa) which was resistant to trypsin treatment. The import of human hsp60 into mitochondria was inhibited in the presence of an uncoupler and also no import occurred when the N-terminal presequence was lacking. These results indicate that the chaperonin protein(s) are transported into mitochondria by a process similar to other imported mitochondrial proteins. Our results also indicate that although the P1 protein precursor was efficiently imported into mitochondria, in comparison to precursors of other mitochondrial proteins (viz. ornithine carbamoyltransferase and uncoupling protein) much less binding of pre P1 to mitochondria was observed. The significance of this latter observation at present is unclear.

    Biochemical and biophysical research communications 1990;169;2;391-6

  • Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen.

    Jindal S, Dudani AK, Singh B, Harley CB and Gupta RS

    Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.

    The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.

    Molecular and cellular biology 1989;9;5;2279-83

  • Amino-acid sequence homology of a polymorphic cellular protein from human lymphocytes and the chaperonins from Escherichia coli (groEL) and chloroplasts (Rubisco-binding protein).

    Waldinger D, Eckerskorn C, Lottspeich F and Cleve H

    Institut für Anthropologie und Humangenetik der Universität München.

    The human p60 (Mr 60,000) is an abundant protein in the two-dimensional electrophoresis pattern of the cellular proteins of human mitogen-stimulated lymphocytes. The p60 shows as remarkable characteristic a genetic polymorphism with two different alleles. Electrotransfer of this protein from two-dimensional gels onto siliconized glass fiber sheets and subsequent amino-acid sequence analysis has revealed a striking homology to the known bacteria and plant chaperonins, the groEL and the Rubisco-subunit-binding protein. From this sequence homology we conclude that we have identified the human chaperonin homologue.

    Biological chemistry Hoppe-Seyler 1988;369;10;1185-9

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000010 G2C Homo sapiens Human mitochondria Human orthologues of mouse mitochondria adapted from Collins et al (2006) 91
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

Cookies Policy | Terms and Conditions. This site is hosted by Edinburgh University and the Genes to Cognition Programme.