G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
heat shock 27kDa protein 1
G00000284 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000023228 (Vega human gene)
ENSG00000106211 (Ensembl human gene)
3315 (Entrez Gene)
617 (G2Cdb plasticity & disease)
HSPB1 (GeneCards)
602195 (OMIM)
Marker Symbol
HGNC:5246 (HGNC)
Protein Expression
2061 (human protein atlas)
Protein Sequence
P04792 (UniProt)

Synonyms (4)

  • HSP27
  • HSP28
  • Hs.76067
  • Hsp25

Literature (166)

Pubmed - other

  • PRAK is essential for ras-induced senescence and tumor suppression.

    Sun P, Yoshizuka N, New L, Moser BA, Li Y, Liao R, Xie C, Chen J, Deng Q, Yamout M, Dong MQ, Frangou CG, Yates JR, Wright PE and Han J

    Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. pqsun@scripps.edu

    Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence upon activation by p38 in response to oncogenic ras. PRAK deficiency in mice enhances DMBA-induced skin carcinogenesis, coinciding with compromised senescence induction. In primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Furthermore, we show that PRAK activates p53 by direct phosphorylation. We propose that phosphorylation of p53 by PRAK following activation of p38 MAPK by ras plays an important role in ras-induced senescence and tumor suppression.

    Funded by: NCI NIH HHS: CA106768, CA91922; NIAID NIH HHS: AI041637, AI054696; NIGMS NIH HHS: GM67101


  • The small heat-shock protein Hsp27 undergoes ERK-dependent phosphorylation and redistribution to the cytoskeleton in response to dual leucine zipper-bearing kinase expression.

    Robitaille H, Simard-Bisson C, Larouche D, Tanguay RM, Blouin R and Germain L

    Laboratoire de Recherche des Grands Brûlés/LOEX, Centre de recherche (FRSQ) du CHA de Québec, Québec, Québec, Canada.

    Hsp27, a small heat-shock protein, has important roles in many cellular processes, including cytoskeleton dynamics, cell differentiation, and apoptosis. Its expression in normal epidermis correlates with differentiation; however, little is known about the regulatory mechanisms involved. In this study, we report that Hsp27 undergoes upregulation, phosphorylation, and redistribution to the cytoskeleton during the late phase of epidermal keratinocyte differentiation. Our results also show that the expression of the dual leucine zipper-bearing kinase (DLK), an upstream activator of the MAP kinase pathways, is sufficient by itself to induce Hsp27 phosphorylation, cell periphery localization, and redistribution to the insoluble protein fraction (cytoskeleton) in poorly differentiated keratinocytes. This redistribution correlates with the insolubilization of cornified envelope-associated proteins such as involucrin. Interestingly, the effects of DLK on Hsp27 were blocked by PD98059, a selective inhibitor of the extracellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of Hsp27 by small interfering RNA in epithelial cells expressing DLK was accompanied by attenuated expression of involucrin in the cytoskeleton. Thus, these observations suggest that the DLK-ERK signaling pathway may act as a regulator of the interaction that occurs between Hsp27 and the cytoskeleton during the formation of the cornified cell envelope, a process conferring to the skin its crucial barrier function.

    Funded by: Canadian Institutes of Health Research

    The Journal of investigative dermatology 2010;130;1;74-85

  • Proteomics of uveal melanomas suggests HSP-27 as a possible surrogate marker of chromosome 3 loss.

    Coupland SE, Vorum H, Mandal N, Kalirai H, Honoré B, Urbak SF, Lake SL, Dopierala J and Damato B

    Department of Pathology, Schoo 12d6 l of Cancer Studies, University of Liverpool, Liverpool, United Kingdom. s.e.coupland@liverpool.ac.uk

    Purpose: To compare the proteomic profiles of primary uveal melanomas, with and without loss of chromosome 3.

    Methods: Frozen specimens from three uveal melanomas with disomy 3 and from four tumors with monosomy 3, according to fluorescence in situ hybridization (FISH) analysis, were subjected to high-resolution, two-dimensional (2-D) gel electrophoresis. The protein expression profiles of the two uveal melanoma cytogenetic groups were compared: Proteins that differed significantly were excised and analyzed by tandem mass spectrometry. Differentially expressed proteins were further analyzed with Western blot analysis. An independent cohort of 41 formalin-fixed, paraffin-embedded (FFPE) uveal melanomas, whose chromosome 3 status had been determined by multiplex ligation-dependent probe amplification (MLPA), was examined for the appropriate antigens by immunohistochemistry.

    Results: Four protein spots were 1.5-fold (Student's t-test, P < 0.05) differentially expressed in the two uveal melanoma types: two spots were overexpressed in the disomy 3 group compared with the monosomy 3 group, whereas two spots were underexpressed. Identification of the four spots yielded nine proteins. Western blot analysis confirmed the results for heat shock protein (HSP)-27, vimentin, and pyruvate dehydrogenase beta (PDHB), with a statistical significance for the first two proteins. HSP-27 was significantly downregulated, whereas vimentin was upregulated in the monosomy 3 tumors (Student's t-test, P = 0.003 and P = 0.005, respectively). Immunohistochemistry confirmed low-to-negative HSP-27 protein expression in monosomy 3 uveal melanomas (Student's t-test; P = 0.011).

    Conclusions: Low-to-negative HSP-27 protein expression in uveal melanoma correlates strongly with monosomy 3. Further validation is necessary to determine whether immunohistochemical assessment of HSP-27 expression correlates with metastatic mortality.

    Investigative ophthalmology & visual science 2010;51;1;12-20

  • Heat shock protein 27 mediates repression of androgen receptor function by protein kinase D1 in prostate cancer cells.

    Hassan S, Biswas MH, Zhang C, Du C and Balaji KC

    Division of Urology, Department of Surgery, University of Massachusetts Medical School, Worcester, MA, USA.

    We have previously shown that protein kinase D1 (PKD1), charter member of PKD protein family, is downregulated in advanced prostate cancer (PC) and influences androgen receptor (AR) function in PC cells. Other independent studies showed that serine 82 residue in heat shock protein 27 (Hsp27) undergoes substrate phosphorylation by PKD1 and is associated with nuclear transport of AR resulting in increased AR transcriptional activity. In this study, we show that PKD1 interacts and phosphorylates Hsp27 at Ser82 in PC cells, which is mediated by p38-dependent mitogen-activated protein kinase pathway and is necessary for PKD1 repression of AR transcriptional activity and androgen-dependent proliferation of PC cells. The study provides first in vivo evidence that Hsp27 is a mediator of repression of AR function by PKD1 in PC cells, thereby linking the data in the published literature.

    Oncogene 2009;28;49;4386-96

  • The taming of small heat-shock proteins: crystallization of the alpha-crystallin domain from huma a6d n Hsp27.

    Baranova EV, Beelen S, Gusev NB and Strelkov SV

    Laboratory for Biocrystallography, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium. ebaranov@vub.ac.be

    Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones. sHsps function as homooligomers or heterooligomers that are prone to subunit exchange and structural plasticity. Here, a procedure for obtaining diffraction-quality crystals of the alpha-crystallin domain of human Hsp27 is presented. Initially, limited proteolysis was used to delineate the corresponding stable fragment (residues 90-171). This fragment could be crystallized, but examination of the crystals using X-rays indicated partial disorder. The surface-entropy reduction approach was applied to ameliorate the crystal quality. Consequently, a double mutant E125A/E126A of the 90-171 fragment yielded well ordered crystals that diffracted to 2.0 A resolution.

    Acta crystallographica. Section F, Structural biology and crystallization communications 2009;65;Pt 12;1277-81

  • Concentrations of antibodies against heat shock protein 27 in the sera of women with ovarian carcinoma.

    Olejek A, Damasiewicz-Bodzek A, Bodzek P, Wielkoszyński T, Zamłyński J, Stołtny P and Skutil M

    Department of Obstetrics, Gynaecology and Oncological Gynaecology, Medical University of Silesia, Bytom, Poland.

    Introduction: Heat shock proteins (Hsp) are highly expressed in many malignant human tumors, including tumors of the genital tract. One of the Hsps strongly associated with the process of cancerogenesis is Hsp27. The presence of Hsp27 and anti-Hsp27 in the sera of patients with ovarian carcinoma is still a new research field, and papers contain contradictory results. The aim of this work was to study the concentrations of anti-Hsp27 immunoglobulin G antibodies in the sera of women with ovarian cancer at different clinical stages and with different histopathological types of this cancer.

    Methods: Serum samples from 158 patients with ovarian carcinoma and 80 healthy women were investigated. The concentrations of anti-Hsp27 antibodies were determined by the enzyme-linked immunosorbent assay technique.

    Results: The mean concentration of anti-Hsp27 antibodies in the patients with ovarian carcinoma was significantly higher than that in the control group. Analysis in relation to the stage of clinical progression showed that the less advanced the cancerogenesis process, the higher the concentration of the anti-Hsp27 antibodies is. The mean concentrations of the anti-Hsp27 antibodies in the patients with ovarian carcinoma were not significantly different in relation to the histological type of the cancer. The use of chemotherapy as a primary anticancer treatment in ovarian carcinoma did not cause a significant decrease in the concentration of anti-Hsp27 antibodies.

    Conclusions: An immunological response to Hsp27 is increased in women with ovarian carcinoma. Although the diagnostic concentrations of anti-Hsp27 antibodies have not been precisely defined yet, we believe that this may be a helpful diagnostic parameter particularly to detect early stages of clinical advancement of the disease.

    International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2009;19;9;1516-20

  • Activation of gene transcription by heat shock protein 27 may contribute to its neuronal protection.

    Friedman MJ, Li S and Li XJ

    Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Heat shock proteins are up-regulated as a physiological response to stressful stimuli and generally function as molecular chaperones for improperly folded protein substrates. The small heat shock protein HSP27 (or HSPB1) has multiple cytoplasmic roles. HSP27 also can translocate to the nucleus in response to stress, but the functional significance of this nuclear distribution has not been elucidated. We have previously implicated HSP27 as a genetic modifier of spinocerebellar ataxia 17 (SCA17), a neurological disease caused by a polyglutamine expansion in the TATA-binding protein (TBP). Altered expression of HSP27 is also found in cell models of other polyglutamine diseases, including Huntington disease as well as SCA3 and SCA7. Here, we show that Hsp27, unlike Hsp70, is not detected in mutant TBP aggregates in primary cerebellar granule neurons from transgenic SCA17 mice. Although HSP27 overexpression does not reduce the aggregation of cotransfected mutant TBP containing 105 glutamines, it potentiates activated transcription from both TATA-containing and TATA-lacking promoters. Neither HSP40 nor HSP70 elicits the same transcriptional effect. Moreover, HSP27 interacts with the transcription factor SP1, and coexpression of SP1 and nuclear localization signal-tagged HSP27 synergistically activates reporter constructs for the SP1-responsive neurotrophic receptor genes Ngfr(p75) and TRKA. Overexpression of nuclear localization signal-tagged HSP27 also rescues mutant TBP-mediated down-regulation of TrkA in a PC12 cell model of SCA17. These results indicate that nuclear HSP27 can modulate SP1-dependent transcriptional activity to promote neuronal protection.

    Funded by: NIA NIH HHS: R01 AG031153; NINDS NIH HHS: NS041669, NS0465016, R01 NS041669, R01 NS045016

    The Journal of biological chemistry 2009;284;41;27944-51

  • Hsp-27 expression at diagnosis predicts poor clinical outcome in prostate cancer independent of ETS-gene rearrangement.

    Foster CS, Dodson AR, Ambroisine L, Fisher G, Møller H, Clark J, Attard G, De-Bono J, Scardino P, Reuter VE, Cooper CS, Berney DM and Cuzick J

    Division of Cellular Pathology and Molecular Genetics, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, UK. csfoster@liv.ac.uk

    Background: This study was performed to test the hypothesis that expression of small heat shock protein Hsp-27 is, at diagnosis, a reliable predictive biomarker of clinically aggressive prostate cancer.

    Methods: A panel of tissue microarrays constructed from a well-characterised cohort of 553 men with conservatively managed prostate cancer was stained immunohistochemically to detect Hsp-27 protein. Hsp-27 expression was compared with a series of pathological and clinical parameters, including outcome.

    Results: Hsp-27 staining was indicative of higher Gleason score (P<0.001). In tissue cores having a Gleason score >7, the presence of Hsp-27 retained its power to independently predict poor clinical outcome (P<0.002). Higher levels of Hsp-27 staining were almost entirely restricted to cancers lacking ERG rearrangements (chi2 trend=31.4, P<0.001), although this distribution did not have prognostic significance.

    Interpretation: This study has confirmed that, in prostate cancers managed conservatively over a period of more than 15 years, expression of Hsp-27 is an accurate and independent predictive biomarker of aggressive disease with poor clinical outcome (P<0.001). These findings suggest that apoptotic and cell-migration pathways modulated by Hsp-27 may contain targets susceptible to the development of biologically appropriate chemotherapeutic agents that are likely to prove effective in treating aggressive prostate cancers.

    Funded by: Cancer Research UK: A13239; Medical Research Council: G0501019

    British journal of cancer 2009;101;7;1137-44

  • Heat shock protein 27 is involved in SUMO-2/3 modification of heat shock factor 1 and thereby modulates the transcription factor activity.

    Brunet Simioni M, De Thonel A, Hammann A, Joly AL, Bossis G, Fourmaux E, Bouchot A, Landry J, Piechaczyk M and Garrido C

    INSERM U866, Dijon, France.

    Heat shock protein 27 (HSP27) accumulates in stressed cells and helps them to survive adverse conditions. We have already shown that HSP27 has a function in the ubiquitination process that is modulated by its oligomerization/phosphorylation status. Here, we show that HSP27 is also involved in protein sumoylation, a ubiquitination-related process. HSP27 increases the number of cell proteins modified by small ubiquitin-like modifier (SUMO)-2/3 but this effect shows some selectivity as it neither affects all proteins nor concerns SUMO-1. Moreover, no such alteration in SUMO-2/3 conjugation is achievable by another HSP, such as HSP70. Heat shock factor 1 (HSF1), a transcription factor responsible for HSP expression, is one of the targets of HSP27. In stressed cells, HSP27 enters the nucleus and, in the form of large oligomers, binds to HSF1 and induces its modification by SUMO-2/3 on lysine 298. HSP27-induced HSF1 modification by SUMO-2/3 takes place downstream of the transcription factor phosphorylation on S303 and S307 and does not affect its DNA-binding ability. In contrast, this modification blocks HSF1 transactivation capacity. These data show that HSP27 exerts a feedback inhibition of HSF1 transactivation and enlighten the strictly regulated interplay between HSPs and HSF1. As we also show that HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, our study raises the possibility that HSP27 may act as a SUMO-E3 ligase specific for SUMO-2/3.

    Oncogene 2009;28;37;3332-44

  • Modulation of HSP27 alters hypoxia-induced endothelial permeability and related signaling pathways.

    Liu T, Guevara OE, Warburton RR, Hill NS, Gaestel M and Kayyali US

    Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

    This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played 1f40 by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia-induced contractility which was ROCK-dependent rather than HSP27-dependent. Results described here show that increased HSP27 phosphorylation not only does not underlie hypoxia-induced permeability, but may actually augment the endothelial barrier. Hypoxia causes gap formation between RPMEC and increases MLC2 phosphorylation. The phosphorylation of MYPT1, which inhibits MLC2 phosphatase, is also increased in hypoxia. In addition, FAK phosphorylation, which alters focal adhesion signaling, is increased in hypoxia. Overexpressing phosphomimicking HSP27 (pmHSP27), which induces significant actin stress fiber formation, surprisingly renders RPMEC resistant to hypoxia- or TGFbeta-induced permeability. siRNA against pmHSP27 reverses the increased actin stress fiber formation in pmHSP27-overexpressing cells, and disrupting actin stress fibers in pmHSP27-overexpressing RPMEC renders them more susceptible to hypoxia. Finally, hypoxia-induced gap formation, as well as phosphorylation of MLC2, MYPT1 and FAK are almost abolished by overexpressing pmHSP27 in RPMEC. These effects of pmHSP27 overexpression might represent decreased cytoskeletal plasticity and increased tethering which counteracts permeability-inducing contractility. Thus hypoxia activates two pathways one leading to contractility and increased permeability, the other leading to actin stress fibers, stronger adhesion, and reduced permeability. Altering HSP27 phosphorylation, which tips the balance towards decreased permeability, might be targeted in managing endothelial barrier dysfunction.

    Funded by: NHLBI NIH HHS: HL-79320, R01 HL079320, R01 HL079320-05S2

    Journal of cellular physiology 2009;220;3;600-10

  • Heat shock protein 27 modification is increased in the human diabetic failing heart.

    Gawlowski T, Stratmann B, Stork I, Engelbrecht B, Brodehl A, Niehaus K, Körfer R, Tschoepe D and Milting H

    Heart and Diabetes Center NRW, Ruhr-University Bochum for the Meta-Card Research Group, Diabetes Center, 32545 Bad Oeynhausen, Germany.

    Chronic conditions like diabetes mellitus (DM) leading to altered metabolism might cause cardiac dysfunction. Hyperglycemia plays an important role in the pathogenesis of diabetic complications including accumulation of methylglyoxal (MG), a highly reactive alpha-dicarbonyl metabolite of glucose degradation pathways and increased generation of advanced glycation endproducts (AGEs). The aim of this investigation was to study the extent of the MG-modification argpyrimidine in human diabetic heart and in rat cardiomyoblasts grown under hyperglycemic conditions. Left ventricular myocardial samples from explanted hearts of patients with cardiomyopathy with (n=8) or without DM (n=8) as well as nonfailing donor organs (n=6), and rat cardiac myoblasts H9c2 treated with glucose were screened for the MG-modification argpyrimidine. The small heat shock protein 27 (Hsp27) revealed to be the major argpyrimidine containing protein in cardiac tissue. Additionally, the modification of arginine leading to argpyrimidine and the phosphorylation of Hsp27 are increased in the myocardium of patients with DM. In H9c2 cells hyperglycemia leads to a decrease of the Hsp27-expression and an increase in argpyrimidine content and phosphorylation of Hsp27, which was accompanied by the induction of oxidative stress and apoptosis. This study shows an association between diabetes and increased argpyrimid 1f40 ine-modification of myocardial Hsp27, a protein which is involved in apoptosis, oxidative stress, and cytoskeleton stabilization.

    Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 2009;41;8;594-9

  • Arachidonic acid stimulates cell adhesion through a novel p38 MAPK-RhoA signaling pathway that involves heat shock protein 27.

    Garcia MC, Ray DM, Lackford B, Rubino M, Olden K and Roberts JD

    Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, Nor a83 th Carolina 27709, USA.

    Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.

    The Journal of biological chemistry 2009;284;31;20936-45

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Human heat shock protein 27 overexpressing mice are protected against hepatic ischemia and reperfusion injury.

    Chen SW, Park SW, Kim M, Brown KM, D'Agati VD and Lee HT

    Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, NY 10032-3784, USA.

    Background: Hepatic ischemia reperfusion injury (IRI) is a major clinical problem during the perioperative period and occurs frequently after major hepatic resection or liver transplantation. Our laboratory previously demonstrated that exogenous A1 adenosine receptor activation protects against renal IRI by upregulation and phosphorylation of heat shock protein 27 (HSP27).

    Methods: This study used mice overexpressing human HSP27 (huHSP27 OE) to determine whether these mice are protected against liver IRI.

    Results: After hepatic IR, the huHSP27 OE mice had significant protection against liver injury (reduced alanine transferase) and necrosis (hematoxylin-eosin staining) compared with the HSP27 WT mice. The huHSP27 OE mice also showed less induction of proinflammatory messenger RNA MIP-2, reduced neutrophil infiltration, and decreased apoptosis (caspase 3 fragmentation and DNA laddering) compared with the HSP27 WT mice. Finally, the huHSP27 OE mice showed significantly less disruption of filamentous actin in hepatocytes and bile canaliculi of the ischemic lobes compared with the HSP27 WT mice. Depletion of Kupffer cells with gadolinium chloride provided significant protection against liver IRI in HSP27 WT mice but not in huHSP27 OE mice suggesting that the overexpression of huHSP27 in the Kupffer cells may be responsible for the hepatic protection observed in huHSP27 OE mice.

    Conclusions: Our results show that the overexpression of huHSP27 in Kupffer cells of the liver may be responsible for the protection against hepatic IRI in vivo by reducing necrosis and apoptosis and by stabilizing F-actin with subsequent reductions in inflammation and proinflammatory neutrophil infiltration. Harnessing the mechanisms of cytoprotection with HSP27 may lead to new therapies for the management of perioperative hepatic IRI.

    Funded by: NIDDK NIH HHS: R01 DK-58547, R01 DK058547, R01 DK058547-06A1, R01 DK058547-07, R01 DK058547-08

    Transplantation 2009;87;10;1478-87

  • The level of Hsp27 in lymphocytes is negatively associated with a higher risk of lung cancer.

    Wang F, Feng M, Xu P, Xiao H, Niu P, Yang X, Bai Y, Peng Y, Yao P, Tan H, Tanguay RM and Wu T

    Department of Occupational and Environmental Health, Huazhong University of Science and Technology, Hubei, 430030, People's Republic of China.

    Heat shock proteins (Hsps) can protect cells, organs, and whole organisms against damage caused by abnormal environmental hazards. Some studies have reported that lymphocyte Hsps may serve as biomarkers for evaluating disease status and exposure to environmental stresses; however, few epidemiologic studies have examined the associations between lymphocyte Hsps levels and lung cancer risk. We examined lymphocyte levels of Hsp27 and Hsp70 in 263 lung cancer cases and age- and gender-matched cancer-free controls by flow cytometry. Multivariate logistic regression models were used to estimate the association between lymphocyte Hsps levels and lung cancer risk. Our results showed that Hsp27 levels were significantly lower in lung cancer cases than in controls (16.5 vs 17.8 mean fluorescence intensity, P < 0.001). This was not observed for Hsp70 levels. Further stratification analysis revealed that lymphocyte Hsp27 levels were negatively associated with lung cancer risk especially in males and heavy smokers. There was a statistical trend of low odd ratios (95% confidence intervals) and upper tertile levels of Hsp27 [1.000, 0.904 (0.566-1.444) and 0.382 (0.221-0.658, P (trend) = 0.001) in males and 1.000, 0.9207 (0.465-1.822) and 0.419 (0.195-0.897, P (trend) = 0.036) in heavy smokers] after adjustment for confounding factors. These results suggest that lower lymphocyte Hsp27 levels might be associated with an increased risk of lung cancer. Our findings need to be validated in a large prospective study.

    Cell stress & chaperones 2009;14;3;245-51

  • Serum heat shock protein 27 is increased in chronic pancreatitis and pancreatic carcinoma.

    Liao WC, Wu MS, Wang HP, Tien YW and Lin JT

    Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.

    Objectives: A prior study suggested serum heat shock protein 27 (HSP27) as a potential marker for pancreatic carcinoma, but its accuracy in differentiating cancer from chronic pancreatitis was not evaluated. We aimed to analyze HSP27 levels in pancreatic carcinoma, chronic pancreatitis, and healthy subjects and assess its diagnostic efficacy.

    Methods: Pretreatment serums from 58 pancreatic carcinoma, 44 chronic pancreatitis, and 102 control subjects were collected. Serum HSP27 and carbohydrate antigen 19-9 (CA19-9) levels were analyzed using an enzyme-linked immunosorbent assay and radioimmunoassay, respectively.

    Results: Heat shock protein 27 levels were significantly higher in cancer and pancreatitis compared with control (P < 0.001 for both), but no significant difference was noted between cancer and pancreatitis (P = 0.978). By logistic regression, HSP27 was a significant predictor of differentiation between cancer and control (P < 0.0001) but not between cancer and pancreatitis (P = 0.885). At a cutoff of 1650 ng/L, the sensitivity and specificity for differentiating cancer from healthy control were 62.1% and 95.1%, respectively. Receiver operating characteristic analyses showed a greater area under curve for CA19-9 compared with HSP27 in differentiating between cancer and control (0.92 and 0.84, respectively, P = 0.014).

    Conclusions: Serum HSP27 is increased in both chronic pancreatitis and pancreatic carcinoma. It should not be recommended as a diagnostic marker for pancreatic carcinoma.

    Pancreas 2009;38;4;422-6

  • The 27-kDa heat shock protein confers cytoprotective effects through a beta 2-adrenergic receptor agonist-initiated complex with beta-arrestin.

    Rojanathammanee, Harmon EB, Grisanti LA, Govitrapong P, Ebadi M, Grove BD, Miyagi M and Porter JE

    Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, ND 58202-9037, USA.

    Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical beta adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with beta-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this beta-arrestin/Hsp27 complex in response to the selective beta adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the beta2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both beta-arrestin isoforms to confer this cytoprotective consequence of beta adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and beta-arrestin to form a previously unknown antiapoptotic "signalosome."

    Funded by: NCRR NIH HHS: RR016471, RR017699; NIDDK NIH HHS: DK062865; NIGMS NIH HHS: GM66726

    Molecular pharmacology 2009;75;4;855-65

  • Transduced HSP27 protein protects neuronal cell death by enhancing FALS-associated SOD1 mutant activity.

    An JJ, Lee YP, Kim DW, Sohn EJ, Jeong HJ, Kang HW, Shin MJ, Kim MJ, Ahn EH, Jang SH, Kang JH, Kang TC, Won MH, Kwon OS, Cho SW, Lee KS, Park J, Eum WS and Choi SY

    Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.

    Familial Amyotrophic lateral sclerosis (FALS) is a progressive neurodegenetative disorder induced by mutations of the SOD1 gene. Heat shock protein 27 (HSP27) is well-defined as a stress-inducible protein, however the its role in ALS protection has not yet been established. To investigate the role HSP27 may have in SOD1 mutant-mediated apoptosis, human SOD1 or HSP27 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame fusion protein, which was then transduced into cells. We found the purified PEP-1-HSP27 fusion proteins can be transduced efficiently into neuronal cells and protect against cell death by enhancing mutant SOD1 activity. Moreover, transduced PEP-1-HSP27 efficiently prevents protein aggregation produced by oxidative stress. These results suggest that transduced HSP27 fusion protein may be explored as a potential therapeutic agent for FALS patients.

    BMB reports 2009;42;3;136-41

  • Mice that overexpress human heat shock protein 27 have increased renal injury following ischemia reperfusion.

    Chen SW, Kim M, Kim M, Song JH, Park SW, Wells D, Brown K, Belleroche Jd, D'Agati VD and Lee HT

    Department of Anesthesiology, Anesthesiology Research Laboratories, College of Physicians and Surgeons, Columbia University, New York, New York 10032-3784, USA.

    We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

    Funded by: NIDDK NIH HHS: R01 DK058547, R01 DK058547-08

    Kidney international 2009;75;5;499-510

  • A clinical phenotype of distal hereditary motor neuronopathy type II with a novel HSPB1 mutation.

    Ikeda Y, Abe A, Ishida C, Takahashi K, K and Yamada M

    Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan. yoshi-i@med.kanazawa-u.ac.jp

    We report a Japanese family with distal hereditary motor neuronopathy type II (distal HMN II) due to a novel K141Q mutation in heat-shock 27-kDa protein 1 gene (HSPB1/HSP27). A 47-year-old man (proband) with diabetes mellitus (DM) developed distal wasting and weakness of the legs and severe autonomic dysfunctions in his early forties, while his father and grandfather, without DM, demonstrated slowly progressive muscular wasting and weakness in all limbs still later in life. This mutation appears linked with the late-onset clinical phenotype as distal HMN II. Severe autonomic disturbances in the proband were probably due to uncontrolled DM, but may have been related to HSPB1 mutation.

    Journal of the neurological sciences 2009;277;1-2;9-12

  • HSP27 regulates IL-1 stimulated IKK activation through interacting with TRAF6 and affecting its ubiquitination.

    Wu Y, Liu J, Zhang Z, Huang H, Shen J, Zhang S, Jiang Y, Luo L and Yin Z

    Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.

    Heat shock protein 27 (HSP27) is an ubiquitiously expressed protein, which has been mediated in various biological functions. Here, we present a novel mechanism utilized by HSP27 in regulating IL-1beta induced NF-small ka, CyrillicB activation. Both over-expression and RNAi experiments indicate that HSP27 physically interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) and promotes TRAF6 ubiquitination. Over-expressed HSP27 augments IL-1beta induced TRAF6 ubiquitination and Ismall ka, CyrillicB kinase (IKK) activation. On the other hand, IL-1beta stimulation reduces endogenous HSP27/TRAF6 association, but inhibiting HSP27 phosphorylation by using SB202190, an inhibitor of p38, and MAPKAPK2 RNAi increases HSP27/TRAF6 association and thereby enhances TRAF6 ubiquitination, IKK phosphorylation as well as NF-small ka, CyrillicB activation. Furthermore, co-transfection study shows that HSP27 S78/82A, two phosphorylated serine site deficient mutants, but not wild-type HSP27 (HSP27 WT) and HSP27 S15A mutant increases TRAF6 ubiquitination and thereby mediates IL-1beta triggered IKK phosphorylation. Taken together, our data indicate that HSP27 regulates IL-1beta triggered NF-small ka, CyrillicB activation via a feedback loop which includes the interaction between HSP27 phosphorylation and ability of HSP27 to bind with TRAF6. The findings of this study reveal a novel mechanism by which HSP27 controls cytokine stimulation.

    Cellular signalling 2009;21;1;143-50

  • Elevated HSP27, HSP70 and HSP90 alpha in chronic obstructive pulmonary disease: markers for immune activation and tissue destruction.

    Hacker S, Lambers C, Hoetzenecker K, Pollreisz A, Aigner C, Lichtenauer M, Mangold A, Niederpold T, Zimmermann M, Taghavi S, Klepetko W and Ankersmit HJ

    Department of Surgery, Medical University of Vienna, Austria.

    Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death. Although the underlying pathomechanism remains poorly understood, COPD is accompanied by increased cellular stress and inflammation. We investigated serum contents of heat shock proteins (HSP 27, 60, 70, 90 alpha), 20S proteasomes, C-reactive protein (CRP), and interleukin-6 (IL-6) in patients with mild or severe COPD, healthy smokers and nonsmokers. HSP27, HSP70 and HSP90 alpha were significantly altered in patients suffering from COPD as compared to controls. HSP27 and HSP70 are potential novel serum markers for the diagnosis of COPD in the smoking population. This is the first study to demonstrate elevated serum levels of the described heat shock proteins in patients with COPD. We showed sensitivity and specificity of serum HSP27 and HSP70 as diagnostic markers for COPD.

    Clinical laboratory 2009;55;1-2;31-40

  • Protein expression profiling in esophageal adenocarcinoma patients indicates association of heat-shock protein 27 expression and chemotherapy response.

    Langer R, Ott K, Specht K, Becker K, Lordick F, Burian M, Herrmann K, Schrattenholz A, Cahill MA, Schwaiger M, Hofler H and Wester HJ

    Institute of Pathology, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany. rupert.langer@lrz.tu-muenchen.de

    Purpose: To identify pretherap 1efd eutic predictive biomarkers in tumor biopsies of patients with locally advanced esophageal adenocarcinomas treated with neoadjuvant chemotherapy, we used an explorative proteomic approach to correlate pretherapeutic protein expression profiles with tumor response to neoadjuvant chemotherapy.

    Thirty-four patients with locally advanced esophageal adenocarcinomas who received neoadjuvant platin/5-fluorouracil-based chemotherapy before surgical resection were enrolled in this study. Response to chemotherapy was determined (a) by the amount of decline of [18F]fluorodeoxyglucose tumor uptake 2 weeks after the start of chemotherapy measured by positron emission tomography and (b) by histopathologic evaluation of tumor regression after surgical resection. Explorative quantitative and qualitative protein expression analysis was done through a quantitative differential protein expression analysis that used dual-isotope radioactive labeling of protein extracts. Selected identified biomarkers were validated by immunohistochemistry and quantitative real time reverse transcription-PCR.

    Results: Proteomic analysis revealed four cellular stress response-associated proteins [heat-shock protein (HSP) 27, HSP60, glucose-regulated protein (GRP) 94, GRP78] and a number of cytoskeletal proteins whose pretherapeutic abundance was significantly different (P < 0.001) between responders and nonresponders. Immunohistochemistry and gene expression analysis confirmed these data, showing a significant association between low HSP27 expression and nonresponse to neoadjuvant chemotherapy (P = 0.049 and P = 0.032, respectively).

    Conclusions: Albeit preliminary, our encouraging data suggest that protein expression profiling may distinguish cancers with a different response to chemotherapy. Our results suggest that response to chemotherapy may be related to a different activation of stress response and inflammatory biology in general. Moreover, the potential of HSPs and GRPs as biomarkers of chemotherapy response warrants further validation.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;24;8279-87

  • Heat shock proteins HSP70 and HSP27 in the cerebral spinal fluid of patients undergoing thoracic aneurysm repair correlate with the probability of postoperative paralysis.

    Hecker JG, Sundram H, Zou S, Praestgaard A, Bavaria JE, Ramchandren S and McGarvey M

    Department of Anesthesia, University of Pennsylvania, Philadelphia, PA 19104-6112, USA. heckerj@uphs.upenn.edu

    An understanding of the time course and correlation with injury of heat shock proteins (HSPs) released during brain and/or spinal cord cellular stress (ischemia) is critical in understanding the role of the HSPs in cellular survival, and may provide a clinically useful biomarker of severe cellular stress. We have analyzed the levels of HSPs in the cerebrospinal fluid (CSF) from patients who are undergoing thoracic aneurysm repair. Blood and CSF samples were collected at regular intervals, and CSF was analyzed by enzyme-linked immunosorbent assay for HSP70 and HSP27. These results were correlated with intraoperative somatosensory-evoked potentials measurements and postoperative paralysis. We find that the levels of these proteins in many patients are elevated and that the degree of elevation correlates with the 1f40 risk of permanent paralysis. We hypothesize that sequential measurement intraoperatively of the levels of the heat shock proteins HSP70 and HSP27 in the CSF can predict those patients who are at greatest risk for paralysis during thoracic aneurysm surgery and will allow us to develop means of preventing or attenuating this severe and often fatal complication.

    Cell stress & chaperones 2008;13;4;435-46

  • Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2.

    Houlden H, Laura M, Wavrant-De Vrièze F, Blake J, Wood N and Reilly MM

    Department of Molecular Neurosciences, Institute of Neurology and The National Hospital for Neurology and Neurosurgery, Queen Square, London, UK. h.houlden@ion.ucl.ac.uk

    Background: Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disorder and is characterized by significant clinical and genetic heterogeneity. Recently, mutations in both the small heat shock protein 27 (HSP27 or HSPB1) and 22 (HSP22 or HSPB8) genes have been reported to cause autosomal dominant CMT with minimal sensory involvement (CMT 2F/CMT2L) and autosomal dominant distal hereditary motor neuropathy type II (dHMN II).

    Methods: We analyzed the HSPB1 and HSPB8 genes in a large clinically well-characterized series of dHMN and CMT type 2 (CMT2) cases and families using linkage analysis and direct sequencing of these genes.

    Results: We identified a novel homozygous mutation in the alpha-crystallin domain of HSPB1 segregating in an autosomal recessive fashion in a family with distal HMN/CMT2. A further four heterozygous HSPB1 mutations were identified in four autosomal dominant families dHMN/CMT2, and two sporadic cases were identified with probable de novo mutations. In the autosomal dominant and autosomal recessive families, there were no clinical sensory findings, but reduced sural nerve action potential amplitudes were found in some affected individuals, indicating that long sensory axons are mildly affected in this predominantly motor disorder.

    Conclusions: This extends the clinical and electrophysiologic spectrum of HSPB1 mutations and identifies four unreported dominant HSPB1 mutations and the first family where the HSPB1 mutation acts in a recessive way to cause distal HMN.

    Funded by: Medical Research Council: G0601943, G0802760, G108/638

    Neurology 2008;71;21;1660-8

  • Heat shock protein 27, a novel regulator of 5-fluorouracil resistance in colon cancer.

    Tsuruta M, Nishibori H, Hasegawa H, Ishii Y, Endo T, Kubota T, Kitajima M and Kitagawa Y

    Department of Surgery, Keio University, Shinjuku-ku, Tokyo, Japan. championtsuru@yahoo.co.jp

    The resistance of colon cancer to 5-fluorouracil (5-FU) is a critical issue, and the cause of this resistance cannot always be explained based on the known molecules. Heat shock protein 27 (HSP27) mRNA expression has recently been shown to be correlated with 5-FU resistance in 5-FU-resistant cell lines. In this study, we attempted to elucidate the functional mechanism of HSP27 in 5-FU resistance in colon cancer. HSP27 protein levels in several human colon cancer cell lines (LoVo, HCT15, WiDr, HCT116, HT-29 and SW480) were determined by immunoblot and densitometry analysis. The in vitro growth inhibition rates (IR) of the cell lines at various concentrations of 5-FU were assessed by MTT assay. The degree of 5-FU resistance was estimated as the drug concentration inducing 50% IR (IC50). The HSP27 protein level and IC50 were significantly correlated in these cell lines (p=0.010). The effect of HSP27 overexpression on IC50 was evaluated in LoVo cells. HSP27 transfectants significantly increased IC50 and reduced HSP27 resistance. The effect of HSP27 down-regulation by HSP27 siRNA on IC50 was confirmed in HCT15 cells. HSP27 siRNA suppressed HSP27 protein levels and reduced IC50 in a dose-dependent manner. These data indicated that HSP27 is closely connected with 5-FU resistance in colon cancer and suggested that HSP27 levels predicted 5-FU resistance. HSP27 down-regulation overcame 5-FU resistance and HSP27 may be a clinical target in patients with 5-FU-resistant colon cancer.

    Oncology reports 2008;20;5;1165-72

  • Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin.

    Dai S, Jia Y, Wu SL, Isenberg JS, Ridnour LA, Bandle RW, Wink DA, Roberts DD and Karger BL

    Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.

    Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.

    Funded by: Intramural NIH HHS: Z01 SC009174-04; NIGMS NIH HHS: F32 GM015847, GM 15847, R01 GM015847, R01 GM015847-38, R37 GM015847

    Journal of proteome research 2008;7;10;4384-95

  • Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis.

    Adly MA, Assaf HA and Hussein MR

    Department of Zoology, Sohag Faculty of Science, South Valley University, Egypt.

    Cell biology international 2008;32;10;1247-55

  • Chaperone Hsp27, a novel subunit of AUF1 protein complexes, functions in AU-rich element-mediated mRNA decay.

    Sinsimer KS, Gratacós FM, Knapinska AM, Lu J, Krause CD, Wierzbowski AV, Maher LR, Scrudato S, Rivera YM, Gupta S, Turrin DK, De La Cruz MP, Pestka S and Brewer G

    Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.

    Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3' untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1- and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (ARE-mRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.

    Funded by: NIAID NIH HHS: P01 AI057596, R01 AI059465, T32 AI00743; NIGMS NIH HHS: R25 GM058389

    Molecular and cellular biology 2008;28;17;5223-37

  • Cyclic strain-induced HSP27 phosphorylation modulates actin filaments in airway smooth muscle cells.

    Chaudhuri S and Smith PG

    Division of Pharmacology and Critical Care, Department of Pediatrics, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106, USA. sxc30@case.edu

    Mechanical stress (cyclic deformational strain) increases proteins of cytoskeletal and contractile domains in airway smooth muscle (ASM) cells in a manner that increases cell contractility. Here we studied the role of HSP27 in strain-induced microfilament formation and stability. Cultured ASM cells showed rapid phosphorylation of HSP27 upon cyclic strain within a few minutes that continued for 30 to 40 minutes. Such increases in HSP27 phosphorylation were abolished with SB 202190, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by PD 98059 (an inhibitor of extracellular regulated kinase), GF109203X (an inhibitor of protein kinase C), or Y27632 (an inhibitor of Rho kinase). Direct activation of RhoA by GTPgammaS did not alter the level of HSP27 phosphorylation. Confocal microscopy revealed that cells pre-incubated with SB 202190, and/or Y27632 resulted in disorganization of stress fibers upon strain, unlike PD 98059 and GF 1092030X, suggesting that both p38 MAPK and Rho kinase were necessary for strain-induced microfilament formation. To determine the relationship between HSP27 and RhoA in strain-induced microfilament formation, cells were transfected with various isoforms of HSP27 and RhoA before strain. Co-expression of inactive HSP27 (3A-HSP27) with constitutively active EGF-RhoA (RhoV14) caused diminution of microfilaments compared with constitutive active EGFP-RhoA (RhoV14) alone, suggesting that HSP27 is necessary for microfilament stability. Similarly, expression of phosphomimicking HSP27 (3D-HSP27) was sufficient for retaining microfilament formation even when co-expressed with the dominant-negative RhoA (EGFP-RhoN17). Thus, HSP27 activation is necessary for microfilament stability independently of RhoA activation.

    American journal of respiratory cell and molecular biology 2008;39;3;270-8

  • HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasion.

    Golembieski WA, Thomas SL, Schultz CR, Yunker CK, McClung HM, Lemke N, Cazacu S, Barker T, Sage EH, Brodie C and Rempel SA

    Barbara Jane Levy Laboratory of Molecular Neuro-Oncology, Department of Neurosurgery, Henry Ford Hospital, Detroit, Michigan 48202, USA.

    Secreted protein acidic and rich in cysteine (SPARC) regulates cell-extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC-green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC-induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC-regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC-induced glioma invasion.

    Funded by: NCI NIH HHS: CA095809, CA109196, CA86997, R24 CA095809

    Glia 2008;56;10;1061-75

  • Phosphorylated heat shock protein 27 represses growth of hepatocellular carcinoma via inhibition of extracellular signal-regulated kinase.

    Matsushima-Nishiwaki R, Takai S, Adachi S, Minamitani C, Yasuda E, Noda T, Kato K, Toyoda H, Kaneoka Y, Yamaguchi A, Kumada T and Kozawa O

    Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.

    Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-alpha, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.

    The Journal of biological chemistry 2008;283;27;18852-60

  • Distal hereditary motor neuropathy in Korean patients with a small heat shock protein 27 mutation.

    Chung KW, Kim SB, Cho SY, Hwang SJ, Park SW, Kang SH, Kim J, Yoo JH and Choi BO

    Department of Biological Science, Kongju National University, Gongju, Korea.

    Distal hereditary motor neuropathy (dHMN) is a heterogeneous disorder characterized by degeneration of motor nerves in the absence of sensory abnormalities. Recently, mutations in the small heat shock protein 27 (HSP27) gene were found to cause dHMN type II or Charcot-Marie-Tooth disease type 2F (CMT2F). The authors studied 151 Korean axonal CMT or dHMN families, and found a large Korean dHMN type II family with the Ser135Phe mutation in HSP27. This mutation was inherited in an autosomal dominant manner, and was well associated with familial members with the dHMN phenotype. This mutation site is located in the alpha-crystallin domain and is highly conserved between different species. The frequency of this HSP27 mutation in Koreans was 0.6%. Magnetic resonance imaging analysis revealed that fatty infiltrations tended to progressively extend distal to proximal muscles in lower extremities. In addition, fatty infiltrations in thigh muscles progressed to affect posterior and anterior compartments but to lesser extents in medial compartment, which differs from CMT1A patients presenting with severe involvements of posterior and medial compartments but less involvement of anterior compartment. The authors describe the clinical and neuroimaging findings of the first Korean dHMN patients with the HSP27 Ser135Phe mutation. To our knowledge, this is the first report of the neuroimaging findings of dHMN type II.

    Experimental & molecular medicine 2008;40;3;304-12

  • HSP27 regulates cell adhesion and invasion via modulation of focal adhesion kinase and MMP-2 expression.

    Lee JW, Kwak HJ, Lee JJ, Kim YN, Lee JW, Park MJ, Jung SE, Hong SI, Lee JH and Lee JS

    Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.

    Heat-shock protein 27 (HSP27), a member of the small heat-shock protein family, is a molecule involved in cellular protection in response to a variety of stresses such as heat shock, toxicants, and oxidative stress. HSP27 is also known to modulate cell functions via intera 1f40 ction with the actin cytoskeleton. To elucidate the functions of HSP27 in adhesion and invasion in more detail, we examined NIH3T3 cells overexpressing HSP27. HSP27 overexpression affected FAK phosphorylation and focal adhesion formation, depending on integrin-mediated actin cytoskeleton polymerization. In addition, the HSP27-overexpressing cells showed a retarded cell migration and invasion in wound-healing assays. Such HSP27-mediated retarded wound healing was correlated with reduced matrix metalloproteinase-2 (MMP-2) expression. The transcription factor for MMP-2 expression, signal transducer and activator or transcription 3 (STAT3), was correspondingly less phosphorylated. When a phosphomimetic form of HSP27 was transiently transfected, migration and invasion were similarly decreased via the regulation of the FAK/STAT3/MMP-2 signaling pathway, whereas a non-phosphorylatable form of HSP27 blocked HSP27-mediated phenotypes probably due to a dominant-negative effect on phosphorylation of endogenous HSP27. Altogether, our results suggest that HSP27 can enhance cell adhesion and modulate cell migration and invasion via the coordination of FAK-dependent actin organization and STAT3-dependent MMP-2 expression, and that phosphorylation of HSP27 is indispensable to regulate this signal pathway.

    European journal of cell biology 2008;87;6;377-87

  • Kinase activity, heat shock protein 27 phosphorylation, and lung epithelial cell glutathione.

    Jackson RM and Garcia-Rojas R

    Miami VA Hospital System, Miami, Florida, USA.

    The 27-kDa heat shock protein (Hps27) is phosphorylated in a way that appears to regulate antioxidant defenses by mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a component of the p38(MAPK) pathway. To investigate the role of Hsp27 in cellular resistance to oxidant stress, lung cells (A549) were incubated with MAPK inhibitors to investigate the pathway's role in antioxidant defense. Cells were harvested for measurement of reduced gluthathione and glutathione disulfide (GSH and GSSH); or, exposed to 2,3-dimethoxy-1,4-napthoquinone (DMNQ). Inhibition of MAPK with SB203580 decreased total cellular glutathione (mean +/- SE): Vehicle, 150 +/- 20 mu M; SB203580, 57 +/- 10* (*P < .01). Inhibition of MAPK tripled [GSSG]/[GSH]: Vehicle, 0.29 +/- 0.09; SB203580, 1.06 +/- 0.43* (*P > .05; n = 6 per group). Hsp27 protein content did not change significantly after MAPK inhibition: Vehicle 2.20 +/- 0.24 ng/mg protein; SB203580, 2.03 +/- 0.34 (P > .05). Transfection of epithelial cells with wild-type (pcDNA-HA-Hsp27) or phosphomimic (pcDNA-HA-Hsp27-S3D) vector increased Hsp27 protein, which significantly protected cells from oxidant stress. Inhibition of the MAPK system, including p38(MAPK), results in cellular oxidant stress. Hsp27, which is phosphorylated by MK2 in the MAPK pathway, protects epithelial cells from oxidant stress.

    Experimental lung research 2008;34;5;245-62

  • Hsp27 regulates pro-inflammatory mediator release in keratinocytes by modulating NF-kappaB signaling.

    Sur R, Lyte PA and Southall MD

    Preclinical Pharmacology, Johnson and Johnson Skin Research Center, CPPW, a Unit of Johnson & Johnson Consumer Companies Inc., Skillman, New Jersey 08558, USA.

    Heat-shock protein 27 (Hsp27) is a member of the small Hsp family that functions as molecular chaperones and protects cells against environmental stress. Hsp27 is expressed in the upper epidermal layers of normal human skin and has been reported to play a role in keratinocyte differentiation and apoptosis. In this investigation, we show an additional role of Hsp27 in the regulation of inflammatory pathways in keratinocytes. Downregulation of Hsp27 using Hsp27-specific small interfering RNA increased prostaglandin E(2) (PGE(2)) production in both unstimulated and tumor necrosis factor-alpha (TNF-alpha)-stimulated keratinocytes. Moreover, downregulation of Hsp27 increased the release of the pro-inflammatory cytokine IL-8 from TNF-alpha-stimulated and UV-irradiated keratinocytes, and this increase was inhibited by pretreatment with the NF-kappaB inhibitor BAY11-7082. Further studies showed that downregulation of Hsp27 resulted in induction of NF-kappaB reporter activity in keratinocytes. This correlated with enhanced degradation of IkappaB-alpha protein and accumulation of phosphorylated IkappaB-alpha in Hsp27 knockdown cells. Moreover, Hsp27 associated with the IkappaB kinase (IKK) complex. As synthesis of the pro-inflammatory cytokine IL-8 and the prostanoid PGE(2) are regulated by NF-kappaB, this could be a probable mechanism by which Hsp27 modulates the production of these inflammatory cytokines. Thus, Hsp27 plays a protective role in regulating inflammatory responses in skin.

    The Journal of investigative dermatology 2008;128;5;1116-22

  • Asymmetrical late onset motor neuropathy associated with a novel mutation in the small heat shock protein HSPB1 (HSP27).

    James PA, Rankin J and Talbot K

    MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

    Distal hereditary motor neuropathy, also known as distal spinal muscular atrophy, is characterised by slowly progressive weakness and wasting of the hands and feet and has a heterogeneous genetic basis. One form of distal hereditary motor neuropathy is associated with mutations in the gene for the small heat shock protein HSPB1 (hsp27). Families have been described in which slowly progressive, symmetrical, lower limb predominant motor weakness is usually evident by middle age. Here we report a novel mutation, G84R, in an elderly patient presenting with strikingly asymmetrical weakness. Expression of this and other known mutations in cell culture demonstrated enhanced aggregation of mutant HSPB1 protein compared with wild-type.

    Funded by: Medical Research Council

    Journal of neurology, neurosurgery, and psychiatry 2008;79;4;461-3

  • Control of estradiol-directed gene transactivation by an intracellular estrogen-binding protein and an estrogen response element-binding protein.

    Chen H, Hewison M and Adams JS

    Division of Endocrinology, Metabolism, and Lipids, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    New World primates exhibit a form of resistance to estrogens that is associated with overexpression of an estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol (E(2))-binding protein (IEBP). Both proteins suppress E(2)-mediated transcription when overexpressed in estrogen receptor-alpha (ERalpha)-positive cells. Although ERE-BP acts as a competitor for ERE occupancy by liganded ERalpha, the function of IEBP and its human homolog, heat-shock protein 27 (hsp27), is less clear. In data presented here, we have used E(2)-responsive human MCF-7 breast cancer cells to show that IEBP/hsp27 can regulate estrogen signaling as a cytosolic decoy for E(2) and as a protein chaperone for ERalpha. Furthermore, co-immunoprecipitation, colocalization, y 1f40 east two-hybrid, and glutathione S-transferase pull-down analyses indicate that IEBP/hsp27 also interacts with ERE-BP to form a dynamic complex that appears to cycle between the cytoplasm and nucleus during normal estrogen signaling. Overexpression of either IEBP/hsp27 or ERE-BP in MCF-7 cells resulted in abnormal subcellular distribution of the IEBP/hsp27 and ERE-BP, with concomitant dysregulation of ERE occupancy as determined by chromatin immunoprecipitation. We hypothesize that IEBP/hsp27 and ERE-BP not only cause hormone resistance in New World primates but are also crucial to normal estrogen signaling in human cells. This appears to involve a physical association between the two proteins to form a complex that is able to interact with both E(2) and ERalpha in cytosolic and nuclear compartments.

    Funded by: NIAMS NIH HHS: R01 AR037399, R01 AR037399-20; NIDDK NIH HHS: R01 DK055843, R01 DK055843-04, R01DK055843

    Molecular endocrinology (Baltimore, Md.) 2008;22;3;559-69

  • Induction of p38MAPK and HSP27 phosphorylation in pemphigus patient skin.

    Berkowitz P, Diaz LA, Hall RP and Rubenstein DS

    Funded by: NIAID NIH HHS: R01 AI49427; NIAMS NIH HHS: AR30281, AR32599

    The Journal of investigative dermatology 2008;128;3;738-40

  • Involvement of protein kinase C beta-extracellular signal-regulating kinase 1/2/p38 mitogen-activated protein kinase-heat shock protein 27 activation in hepatocellular carcinoma cell motility and invasion.

    Guo K, Liu Y, Zhou H, Dai Z, Zhang J, Sun R, Chen J, Sun Q, Lu W, Kang X and Chen P

    Liver Cancer Institute, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai 200032, China.

    To understand the molecular mechanism that underlies the role of various prominent signal pathways in hepatocellular carcinoma (HCC) metastasis, a human signal transduction oligonucleotide microarray analysis was carried out in cultured HCC cell models with increasing spontaneous metastatic potential (MHCC97L, MHCC97H, and HCCLM6). The results revealed that the mitogen-activated protein kinase (MAPK) pathway is the prominently upregulated pathway in HCC metastasis. Further study showed that basal phosphorylated levels of extracellular signal-regulating kinase (ERK)(1/2) and p38 MAPK consecutively increased from MHCC97L to MHCC97H to HCCLM6 cells, but not c-Jun N-terminal kinase. The phosphorylation of ERK(1/2) and p38 MAPK was regulated by upregulated protein kinase C beta (PKC beta) in HCC cells through the integrated use of PKC beta RNA interference, the PKC beta specific inhibitor enzastaurin and a PKC activator phorbol-12-myristate-13-acetate. Heat shock protein 27 (HSP27) was also verified as a downstream common activated protein of PKC beta-ERK(1/2) and PKC beta-p38 MAPK. In vitro migration and invasion assay further showed that the depletion of PKC beta or inhibition of PKC beta activation effectively decreased HCC cell motility and invasion. Moreover, the motility and invasion of phorbol-12-myristate-13-acetate-stimulated PKC beta-mediated HCC cells was significantly negated by an ERK inhibitor, 1.4-diamino-2.3-dicyano-1.4-bis[2-aminophenylthio] butadiene, or a p38 MAPK inhibitor, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole. It also showed that HSP27 is critical in PKC beta-mediated HCC cell motility and invasion. Taken together, this study reveals the important role of this PKC beta-ERK(1/2)/p38MAPK-HSP27 pathway, which was verified for the first time, in modulating HCC cell motility and invasion.

    Cancer science 2008;99;3;486-96

  • The Role of heat shock protein 27 in extravillous trophoblast differentiation.

    Matalon ST, Drucker L, Fishman A, Ornoy A and Lishner M

    Oncogenetic Laboratory, Meir Medical Center, Kfar-Saba, Israel. matalon.shelly@clalit.org.il

    Trophoblast cells from placental explants differentiate in culture to extravillous trophoblast cells (EVT cells). During trophoblast differentiation heat-shock-protein-27 (HSP27) mRNA and multidrug-resistance-protein-5 (MRP5, transporter of cyclic nucleotides) expression are increased. HSP27 is a regulator of actin filaments structure and dynamic, has a role in cell differentiation and may affect NF-kB activity. In this study we aimed to assess HSP27 level in trophoblast cells and its correlation with motility and differentiation related processes [MMPs activity, nitric oxide (NO), inducible nitric oxide synthase (iNOS), proliferation and MRP5 levels]. We evaluated HSP27 expression in a first trimester human trophoblast explants model designed to assess EVT cells differentiation/migration with/without 6-mercaptopurine (6MP, an EVT inhibitor of migration). We found that HSP27 level is expressed in the nucleous and cytoplasm of non-proliferting villous-trophoblast cells (negative for Ki67) and in the cell periphery and cytoplasm of motile EVT c 17cd ells. Moreover, 6MP decreased HSP27 nucleous expression that was associated with inhibited MMP2 activity and NO production. Also decreased iNOS expression and increased MRP5 mRNA levels were observed. In conclusion, HSP27 expression is modulated in concordance with migration dependent parameters in trophoblast cells.

    Journal of cellular biochemistry 2008;103;3;719-29

  • PKD, PKD2, and p38 MAPK mediate Hsp27 serine-82 phosphorylation induced by neurotensin in pancreatic cancer PANC-1 cells.

    Yuan J and Rozengurt E

    Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine; CURE: Digestive Diseases Research Center and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.

    It is widely recognized that Hsp27 is a downstream substrate of the p38 MAPK cascade whereas the role of PKD family members in mediating receptor-stimulated Hsp27 Ser-82 phosphorylation has not been evaluated. Here, we show that neurotensin induced a rapid and striking increase in Hsp27 Ser-82 phosphorylation in PANC-1 cells, which was closely correlated with stimulation of activation loop phosphorylation of PKDs and p38 MAPK Thr180/Tyr182 phosphorylation. Treatment of PANC-1 cells with either the selective PKC inhibitor GF-I or the p38 MAPK inhibitor SB202190 partially reduced neurotensin-induced Hsp27 Ser-82 phosphorylation. However, treatment of the cells with a combination of 17ed GF-I and SB202190 virtually abolished neurotensin-induced Hsp27 Ser-82 phosphorylation. Overexpression of PKD in stably transfected PANC-1 cells increased the magnitude and prolonged the duration of Hsp27 Ser-82 phosphorylation in response to neurotensin. Either PKD or PKD2 gene silencing utilizing siRNAs targeting distinct PKD or PKD2 sequences reduced neurotensin-stimulated Hsp27 Ser-82 phosphorylation, but cotransfection of siRNAs targeting both, PKD and PKD2, markedly decreased neurotensin-induced Hsp27 Ser-82 phosphorylation. Knockdown of PKD and PKD2 abolished Hsp27 phosphorylation in cells treated with SB202190. Thus, neurotensin induces Hsp27 Ser-82 phosphorylation through p38 MAPK- and PKC/PKD-dependent pathways in PANC-1 cells. Our results demonstrate, for the first time, that neurotensin induces a striking increase in Hsp27 phosphorylation on Ser-82 in PANC-1 cells through convergent p38 MAPK, PKD, and PKD2 signaling.

    Funded by: NIDDK NIH HHS: DK 41301, DK 55003, DK 56930, P30 DK041301

    Journal of cellular biochemistry 2008;103;2;648-62

  • Proteomic identification of heat shock protein 27 as a differentiation and prognostic marker in neuroblastoma but not in Ewing's sarcoma.

    Zanini C, Pulerà F, Carta F, Giribaldi G, Mandili G, Maule MM, Forni M and Turrini F

    Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 5 bis, 10126, Turin, Italy. cristina.zanini@unito.it

    Virchows Archiv : an international journal of pathology 2008;452;2;157-67

  • Responses of the 27-kDa heat shock protein to UVB irradiation in human epidermal melanocytes.

    Shi B, Grahn JC, Reilly DA, Dizon TC and Isseroff RR

    Department of Dermatology, University of California Davis School of Medicine, Davis, CA 95616, USA.

    Solar ultraviolet radiation (UVR) is a major environmental hazard for the skin, and UVB (280-320 nm) has been proposed to be a main factor for melanoma development. In response to sunlight exposure, the skin has adapted a number of innate resistance mechanisms. Among them is the small heat shock protein of 27 kDa (HSP27) known to play a role in the protection of cells from variety of environmental insults including UV irradiation. In this study, we demonstrated that UVB irradiation of cultured normal epidermal melanocytes initiates changes in HSP27 phosphorylation and localization. In unstressed melanocytes, HSP27 was present as the non-phosphorylated isoform. UVB irradiation with a physiological dose (7-25 mJ/cm(2)) resulted in the formation of a mono-phosphorylated isoform and sometimes a bi-phosphorylated isoform. The UVB-induced HSP27 phosphorylation was inhibited when melanocytes were treated with the antioxidant N-acetyl cysteine or inhibitor of p38 MAP kinase prior to UVB exposure, suggesting that UVB induced HSP27 phosphorylation through reactive oxygen species/p38 MAP kinase pathway. In response to UBV irradiation, HSP27 in melanocytes translocated from the cytoplasm to the nucleus. The HSP27 responses may provide some protective role against UVB-induced cell damage in the skin.

    Experimental dermatology 2008;17;2;108-14

  • Contraction in human myometrium is associated with changes in small heat shock proteins.

    MacIntyre DA, Tyson EK, Read M, Smith R, Yeo G, Kwek K and Chan EC

    Mothers and Babies Research Centre, The University of Newcastle, John Hunter Hospital, 1 Lookout Road, New Lambton Heights, Newcastle 2305, Australia.

    The myometrium undergoes substantial remodeling at the time of labor including rearrangement of the cellular contractile machinery. The regulation of this process in human myometrium at the time of labor is poorly defined, but evidence in other muscle types suggests modulation by small heat shock proteins (sHSP). The aim of this study was to investigate whether similar changes in sHSP occur in the myometrium at labor. Using a quantitative proteomic approach (two-dimensional difference gel electrophoresis), we found a 69% decrease in the sHSP alphaB-crystallin in the myometrium at labor plus multiple isoforms of HSP27. Immunoblotting using phosphospecific HSP27 antibodies (HSP27-serine15, -78, and -82) detected marked changes in HSP27 phosphorylation at labor. Although total HSP27 levels were unchanged, HSP27-Ser15 was 3-fold higher at labor. Coimmunoprecipitation studies showed that HSP27 coprecipitates with alphaB-crystallin and also smooth muscle alpha-actin. Coimmunofluorescence studies demonstrated a relocation of HSP27 from the perinuclear region to the actin cytoskeleton at labor. The functional significance of these changes was demonstrated in vitro where myometrial strips stimulated to contract with oxytocin exhibited increased HSP27-Ser15 phosphorylation. Our findings provide data consistent with a novel pathway regulating human myometrial contraction at labor and identify HSP27 and alphaB-crystallin as potential targets for future tocolytic design.

    Endocrinology 2008;149;1;245-52

  • P-cadherin and beta-catenin are useful prognostic markers in breast cancer patients; beta-catenin interacts with heat shock protein Hsp27.

    Fanelli MA, Montt-Guevara M, Diblasi AM, Gago FE, Tello O, Cuello-Carrión FD, Callegari E, Bausero MA and Ciocca DR

    Oncology Laboratory, Institute of Experimental Medicine and Biology of Cuyo, Regional Center for Scientific and Technological Research, National Research Council (CONICET), Mendoza, Argentina. mfanelli@lab.cricyt.edu.ar

    The cadherin-catenin proteins have in common with heat shock proteins (HSP) the capacity to bind/interact proteins of other classes. Moreover, there are common molecular pathways that connect the HSP response and the cadherin-catenin protein system. In the present study, we have explored whether in breast cancer the HSP might interact functionally with the cadherin-catenin cell adhesion system. Beta-catenin was immunoprecipitated from breast cancer biopsy samples, and the protein complexes isolated in this way were probed with antibodies against HSP family members. We are thus the first to demonstrate a specific interaction between beta-catenin and Hsp27. However, beta-catenin did not bind Hsp60, Hsp70, Hsp90, gp96, or the endoplasmic reticulum stress response protein CHOP. To confirm the finding of Hsp27-beta-catenin interaction, the 27-kDa immunoprecipitated band was excised from one-dimensional polyacrylamide gel electrophoresis gels and submitted to liquid chromatography-tandem mass spectrometry with electrospray ionization, confirming a role for Hsp27. In addition, beta-catenin interacted with other proteins including heat shock transcription factor 1, P-cadherin, and caveolin-1. In human breast cancer biopsy samples, beta-catenin was coexpressed in the same tumor areas and in the same tumor cells that expressed Hsp27. However, this coexpression was strong when beta-catenin was present in the cytoplasm of the tumor cells and not when beta-catenin was expressed at the cell surface only. Furthermore, murine breast cancer cells transfected with hsp25 showed a redistribution of beta-catenin from the cell membrane to the cytoplasm. When the prognostic significance of cadherin-catenin expression was examined by immunohistochemistry in breast cancer patients (n = 215, follow-up = >10 years), we found that the disease-free survival and overall survival were significantly shorter for patients expressing P-cadherin and for patients showing expression of beta-catenin in the cytoplasm only (not at the cell surface). The interactions of beta-catenin with Hsp27 and with HSF1 may explain some of the molecular pathways that influence tumor cell survival and the clinical significance in the prognosis of the breast cancer patients.

    Cell stress & chaperones 2008;13;2;207-20

  • Heat shock protein 27 regulates oxidative stress-induced apoptosis in cardiomyocytes: mechanisms via reactive oxygen species generation and Akt activation.

    Liu L, Zhang XJ, Jiang SR, Ding ZN, Ding GX, Huang J and Cheng YL

    Department of Geriatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.

    Background: Increased reactive oxygen species (ROS) formation, which in turn promotes cardiomyocytes apoptosis, is associated with the pathogenesis and progression of various cardiac diseases such as ischemia and heart failure. Recent studies have shown that over expression of heat shock protein 27 (Hsp27) confers resistance to cardiac ischemia/reperfusion injury. However, not much is known about the regulation of myocyte survival by Hsp27.

    Methods: The rat cardiac cell line H9c2, with a stable overexpression of Hsp27, was established, with empty vector transfected H9c2 cells as controls. Following the cells challenged by Hydrogen Peroxide (H2O2), lactate dehydrogenase (LDH) release, apoptosis, intracellular ROS, cell morphology, mitochondrial transmembrane potential and the activation of serine/threonine kinase Akt were determined.

    Results: Along with marked suppression of H2O2-induced injury by Hsp27 overexpression in H9c2 cells, ROS generation and the loss of mitochondrial membrane potential were also significantly depressed. Furthermore, augmented Akt activation was observed in Hsp27 overexpressed H9c2 cells following H2O2 exposure.

    Conclusions: Hsp27 inhibits oxidative stress-induced H9c2 damage and inhibition of ROS generation and the augmentation of Akt activation may be involved in the protective signaling.

    Chinese medical journal 2007;120;24;2271-7

  • Hsp27 modulates p53 signaling and suppresses cellular senescence.

    O'Callaghan-Sunol C, Gabai VL and Sherman MY

    Department of Biochemistry, Boston University Medical School, Boston, Massachusetts 02118, USA.

    The small heat shock protein Hsp27 is expressed at high levels in many tumors and provides protection against anticancer drugs. Here, we show that expression of recombinant Hsp27 at elevated levels leads to protection of MCF10A human mammary epithelial cells from doxorubicin. The protection was associated with suppression of the doxorubicin-induced senescence, where Hsp27 inhibited p53-mediated induction of p21, the major regulator of the senescence program. Similarly, Hsp27 inhibited accumulation of p21 and suppressed senescence in response to the p53 activator nutlin-3, indicating that Hsp27 has a general effect on the p53 pathway. In line with these findings, down-regulation of Hsp27 in HCT116 human colon carcinoma cells that express this heat shock protein at high levels caused senescence in a population of cells and sensitized the rest of the cells to doxorubicin-induced senescence (at low doses) or apoptosis (at high doses of doxorubicin). Induction of senescence by Hsp27 down-regulation associated with activation of the p53 pathway and induction of p21. Interestingly, depletion of Hsp27 caused neither significant proteotoxic nor genotoxic stress, and therefore this heat shock protein seems to have a specific effect on the p53 signaling. Indeed, Hsp27 down-regulation was associated with destabilization of HDM2 and stabilization of p53. These data suggest that Hsp27 may play a general role in regulation of cellular senescence by modulating the p53 pathway.

    Cancer research 2007;67;24;11779-88

  • Proteomic alterations in heat shock protein 27 and identification of phosphoproteins in ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve.

    Matt P, Fu Z, Carrel T, Huso DL, Dirnhofer S, Lefkovits I, Zerkowski HR and Van Eyk JE

    Johns Hopkins Bayview Proteomics Center, Department of Medicine, Johns Hopkins University, 5200 Eastern Avenue, Baltimore, MD 21224, USA. pmatt1@jmhi.edu

    Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients 1f40 (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.

    Funded by: NHLBI NIH HHS: HV 28120, RC1 HL100021, RC1 HL100021-01

    Journal of molecular and cellular cardiology 2007;43;6;792-801

  • Cooperative interactions between androgen receptor (AR) and heat-shock protein 27 facilitate AR transcriptional activity.

    Zoubeidi A, Zardan A, Beraldi E, Fazli L, Sowery R, Rennie P, Nelson C and Gleave M

    The Prostate Centre, Vancouver General Hospital, and Department of Urological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

    Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser(78) and Ser(82) residues in an AR- and p38 kinase-dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer.

    Cancer research 2007;67;21;10455-65

  • Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells.

    Bryantsev AL, Kurchashova SY, Golyshev SA, Polyakov VY, Wunderink HF, Kanon B, Budagova KR, Kabakov AE and Kampinga HH

    Department of Electron Microscopy, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia.

    In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

    The Biochemical journal 2007;407;3;407-17

  • Exogenous heat shock protein 27 uniquely blocks differentiation of monocytes to dendritic cells.

    Laudanski K, De A and Miller-Graziano C

    Department of Surgery, University of Rochester Medical Center, Rochester, NY 14642, USA.

    Circulating heat shock protein (HSP)-27 is associated with tumor progression and increased post-injury infection. Extracellular HSP-27 might alter monocyte (MO)-derived DC and/or MPhi function to mediate immunosuppression. HSP-27 treatment inhibited expression of CD1a and CD1b/c, antigen uptake, and allogeneic T cell induction (MLR) by IL-4 + GM-CSF-differentiated human DC while increasing some MPhi characteristics ( upward arrowCD14, upward arrowCD16, upward arrowCD163). MO cytokine receptor profiles elicited by 24-h exogenous HSP-27 treatment remained supportive of immature DC (iDC) emergence ( upward arrowIL-4R, downward arrowIL-6R, downward arrowM-CSFR). IL-10, IL-6, and M-CSF (which promote MPhi differentiation) were significantly increased in IL-4 + GM-CSF + HSP-27 MO-->iDC differentiation cultures. However, HSP-27 treatment during MO differentiation to DC increased programmed cell death ligand 1 coinhibitor and depressed CD86 costimulator expression in parallel to decreased iDC MLR activity. This suggested that increased MPhi differentiation was not solely responsible for HSP-27 reduction of differentiating DC activity. HSP-27 treatment actually depressed the phagocytic capacity of MO differentiated to MPhi by IL-10 or M-CSF culture. CD163 (hemoglobin receptor) expression was depressed on M-CSF + HSP-27 MO-derived MPhi. HSP-27-mediated inhibition of MO-->iDC differentiation was reversed by p38alpha & beta inhibitor (SB202190) addition or TLR4 receptor modulation. HSP-27 impaired appropriate MO-->iDC and MO-->MPhi differentiation modulating expression of receptors necessary for their proper functions. This suggests that endogenous HSP-27 has immunoregulatory activities which could contribute to immunopathology.

    Funded by: NIGMS NIH HHS: GM36214, GM65237

    European journal of immunology 2007;37;10;2812-24

  • Binding of caspase-3 prodomain to heat shock protein 27 regulates monocyte apoptosis by inhibiting caspase-3 proteolytic activation.

    Voss OH, Batra S, Kolattukudy SJ, Gonzalez-Mejia ME, Smith JB and Doseff AI

    Heart and Lung Research Institute, Division of Pulmonary and Critical Care and Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA.

    Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.

    Funded by: NHLBI NIH HHS: R01HL075040

    The Journal of biological chemistry 2007;282;34;25088-99

  • Genetic variant in the HSPB1 promoter region impairs the HSP27 stress response.

    Dierick I, Irobi J, Janssens S, Theuns J, Lemmens R, Jacobs A, Corsmit E, Hersmus N, Van Den Bosch L, Robberecht W, De Jonghe P, Van Broeckhoven C and Timmerman V

    Peripheral Neuropathy Group, University of Antwerp, Universiteitsplein 1, Antwerpen, Belgium.

    Human mutation 2007;28;8;830

  • Maximal eccentric exercise induces a rapid accumulation of small heat shock proteins on myofibrils and a delayed HSP70 response in humans.

    Paulsen G, Vissing K, Kalhovde JM, Ugelstad I, Bayer ML, Kadi F, Schjerling P, Hallén J and Raastad T

    Norwegian School of Sport Sciences, P.O. Box 4014 U.S., N-0806 Oslo, Norway. goran.paulsen@nih.no

    In this study the stress protein response to unaccustomed maximal eccentric exercise in humans was investigated. Eleven healthy males performed 300 maximal eccentric actions with the quadriceps muscle. Biopsies from vastus lateralis were collected at 30 min and 4, 8, 24, 96, and 168 h after exercise. Cellular regulation and localization of heat shock protein (HSP) 27, alpha B-crystallin, and HSP70 were analyzed by immunohistochemistry, ELISA technique, and Western blotting. Additionally, mRNA levels of HSP27, alpha B-crystallin, and HSP70 were quantified by Northern blotting. After exercise (30 min), 81 +/- 8% of the myofibers showed strong HSP27 staining (P < 0.01) that gradually decreased during the following week. alpha B-Crystallin mimicked the changes observed in HSP27. After exercise (30 min), the ELISA analysis showed a 49 +/- 13% reduction of the HSP27 level in the cytosolic fraction (P < 0.01), whereas Western blotting revealed a 15-fold increase of the HSP27 level in the myofibrillar fraction (P < 0.01). The cytosolic HSP70 level increased to 203 +/- 37% of the control level 24 h after exerci 882 se (P < 0.05). After 4 days, myofibrillar-bound HSP70 had increased approximately 10-fold (P < 0.01) and was accompanied by strong staining on cross sections. mRNA levels of HSP27, alpha B-crystallin, and HSP70 were all elevated the first day after exercise (P < 0.01); HSP70 mRNA showed the largest increase (20-fold at 8 h). HSP27 and alpha B-crystallin seemed to respond immediately to maximal eccentric exercise by binding to cytoskeletal/myofibrillar proteins, probably to function as stabilizers of disrupted myofibrillar structures. Later, mRNA and total HSP protein levels, especially HSP70, increased, indicating that HSPs play a role in skeletal muscle recovery and remodeling/adaptation processes to high-force exercise.

    American journal of physiology. Regulatory, integrative and comparative physiology 2007;293;2;R844-53

  • Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma.

    Takai S, Matsushima-Nishiwaki R, Tokuda H, Yasuda E, Toyoda H, Kaneoka Y, Yamaguchi A, Kumada T and Kozawa O

    Department of Pharmacology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu, Japan.

    We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.

    Life sciences 2007;81;7;585-91

  • Inhibition of heat shock protein 27-mediated resistance to DNA damaging agents by a novel PKC delta-V5 heptapeptide.

    Kim EH, Lee HJ, Lee DH, Bae S, Soh JW, Jeoung D, Kim J, Cho CK, Lee YJ and Lee YS

    Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.

    Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase C delta (PKC delta)-V5 region with ensuing inhibition of PKC delta activity and PKC delta-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKC delta activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKC delta-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27.

    Cancer research 2007;67;13;6333-41

  • 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

  • The interaction and cellular localization of HSP27 and ERbeta are modulated by 17beta-estradiol and HSP27 phosphorylation.

    Al-Madhoun AS, Chen YX, Haidari L, Rayner K, Gerthoffer W, McBride H and O'Brien ER

    University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada.

    Recently, we identified heat shock protein 27 (HSP27) as an estrogen receptor-beta (ERbeta) associated protein that acts as a co-repressor of estrogen signaling and serves as a biomarker of atherosclerosis. In this study, we sought to further characterize the subcellular interaction of HSP27 and ERbeta, as well as explore the factors that may modulate this interaction. In vitro we determined that phosphorylated HSP27 is retained in the cytoplasm after treatment with 17beta-estradiol and to a lesser extent with heat shock. Under all experimental conditions ERbeta was found to be slightly more abundant in the cytoplasm compared to the nucleus. HSP27 and ERbeta associate in both the cytoplasm and nucleus, however, co-localization studies reveal that in the presence of 17beta-estradiol, a significant portion of this interaction occurs outside of the nucleus. These data highlight an extranuclear interaction between ERbeta and HSP27 that may be of potential importance in modulating estrogen signaling.

    Funded by: NHLBI NIH HHS: HL077726

    Molecular and cellular endocrinology 2007;270;1-2;33-42

  • HSP27 and HSP70 interact with CD10 in C4-2 prostate cancer cells.

    Dall'Era MA, Oudes A, Martin DB and Liu AY

    Department of Urology, University of Washington, Seattle, Washington, USA. mdallera@urology.ucsf.edu

    Background: CD10 is an approximately 100 kDa transmembrane metallo-endopeptidase. CD10 is strongly expressed by normal prostate epithelium. While only 30% of primary prostate tumors express CD10, it is strongly expressed by most lymph node metastases. The function of CD10 and the interaction between CD10 and other cellular proteins in prostate cancer (CaP) is not well defined. Cellular context may ultimately determine its biologic function in CaP. In this study, we compared CD10 mRNA and protein expression between benign and malignant prostate cells and employed proteomic analysis to identify proteins that interact with CD10 in C4-2 prostate cancer cells.

    Methods: CD10 mRNA and protein expression was compared using RT-PCR and Western blotting. CD10-protein complexes were isolated by immunoprecipitation using anti-CD10 monoclonal antibodies. Eluted fractions were combined, trypsinized, and the resulting peptides analyzed by microLC-ESI-MS/MS. The parent proteins were identified by searching MS/MS spectra against a human protein database using SEQUEST.

    Results: There were no differences in CD10 mRNA length or CD10 protein molecular weight between normal tissue and CaP. We identified 75 proteins unique to or heavily enriched in the CD10 immunoprecipitates by proteomic analysis. The 27 kDa heat shock protein (HSP27) and HSP70 were identified in three separate precipitations. Protein identification by proteomics was confirmed by Western blotting. Protein complexes immunopurified from C4-2 protein extracts using anti-HSP27 and anti-HSP70 antibodies were found to contain CD10.

    Conclusions: The function of CD10 in prostate cancer is largely unknown. In the C4-2 CaP cell line, CD10 was found to interact with both HSP27 and HSP70.

    Funded by: NCI NIH HHS: CA111244, CA85859, CA98699; NIDDK NIH HHS: DK63630

    The Prostate 2007;67;7;714-21

  • Anthrax lethal toxin paralyzes actin-based motility by blocking Hsp27 phosphorylation.

    During RL, Gibson BG, Li W, Bishai EA, Sidhu GS, Landry J and Southwick FS

    Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.

    Inhalation of anthrax causes fatal bacteremia, indicating a meager host immune response. We previously showed that anthrax lethal toxin (LT) paralyzes neutrophils, a major component of innate immunity. Here, we have found that LT also inhibits actin-based motility of the intracellular pathogen Listeria monocytogenes. LT inhibition of actin assembly is mediated by blockade of Hsp27 phosphorylation, and can be reproduced by treating cells with the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580. Nonphosphorylated Hsp27 inhibits Listeria actin-based motility in cell extracts, and binds to and sequesters purified actin monomers. Phosphorylation of Hsp27 reverses these effects. RNA interference knockdown of Hsp27 blocks LT inhibition of Listeria actin-based motility. Rescue with wild-type Hsp27 accelerates Listeria speed in knockdown cells, whereas introduction of Hsp27 mutants incapable of phosphorylation or dephosphorylation causes slowing down. We propose that Hsp27 facilitates actin-based motility through a phosphorylation cycle that shuttles actin monomers to regions of new actin filament assembly. Our findings provide a previously unappreciated mechanism for LT virulence, and emphasize a central role for p38 MAP kinase-mediated phosphorylation of Hsp27 in actin-based motility and innate immunity.

    Funded by: NIAID NIH HHS: R01 AI023262, R01 AI034276, R01 AI064891, R01AI-23262, R01AI-34276

    The EMBO journal 2007;26;9;2240-50

  • Anti-vpr activities of heat shock protein 27.

    Liang D, Benko Z, Agbottah E, Bukrinsky M and Zhao RY

    Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    HIV-1 Vpr plays a pivotal role in viral pathogenesis and is preferentially targeted by the host immune system. In this report, we demonstrate that a small heat shock protein, HSP27, exhibits Vpr-specific antiviral activity, as its expression is specifically responsive to vpr gene expression and increased levels of HSP27 inhibit Vpr-induced cell cycle G2 arrest and cell killing. We further show that overexpression of HSP27 reduces viral replication in T-lymphocytes in a Vpr-dependent manner. Mechanistically, Vpr triggers HSP27 expression through heat shock factor (HSF) 1, but inhibits prolonged expression of HSP27 under heat-shock conditions. Together, these data suggest a potential dynamic and antagonistic interaction between HIV-1 Vpr and a host cell HSP27, suggesting that HSP27 may contribute to cellular intrinsic immunity against HIV infection.

    Funded by: NIAID NIH HHS: AI33776, AI40891, R01 AI033776, R01 AI040891, R21 AI033776, R21 AI040891, R29 AI033776, R29 AI040891; NIGMS NIH HHS: GM63080, R01 GM063080

    Molecular medicine (Cambridge, Mass.) 2007;13;5-6;229-39

  • Photodynamic therapy resistant human colon carcinoma HT29 cells show cross-resistance to UVA but not UVC light.

    Zacal N and Rainbow AJ

    Department of Biology, McMaster University, Hamilton, ON, Canada.

    The isolation of photodynamic therapy (PDT)-resistant HT29 human colon adenocarcinoma cells has been reported previously. These PDT-resistant variants show increased expression of the Hsp27 and BNip3 proteins and a decreased expression of mutant p53 protein compared with parental HT29 cells. Because mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. In the present report, we examined the colony survival of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells to ultraviolet light (UV) treatment. The HT29 PDT-resistant variants showed cross-resistance to long-wavelength UVA (320-400 nm) but not to short-wavelength UVC (200-280 nm) light. Cell sensitivity to UVA or UVC was then correlated with Hsp27, BNip3 and mutant p53 protein levels in the PDT-resistant variants as well as in several clonal variants of HT29 cells that express different levels of Hsp27, BNip3 and mutant p53. We show that increased expression of Hsp27 and BNip3 and decreased expression of mutant p53 correlated with increased resistance to UVA. In contrast, increased expression of Hsp27 and BNip3 correlated with increased sensitivity to UVC, whereas increased expression of mutant p53 showed no significant correlation with sensitivity to UVC. These results suggest that the PDT-resistant HT29 cell variants are differentially sensitized to UVA compared with UVC due, in part at least, through the altered expression levels of BNip3, Hsp27 and mutant p53.

    Photochemistry and photobiology 2007;83;3;730-7

  • Markers of oxidative stress in the skeletal muscle of patients on haemodialysis.

    Crowe AV, McArdle A, McArdle F, Pattwell DM, Bell GM, Kemp GJ, Bone JM, Griffiths RD and Jackson MJ

    Division of Metabolic and Cellular Medicine, University of Liverpool, and Renal Unit, Royal Liverpool University and Broadgreen Hospitals NHS Trust, Liverpool L69 3GA, UK.

    Background: Increased oxidative stress may play a role in morbidity and mortality of patients with renal failure. Most studies have examined serum markers of oxidation, but it is unclear whether oxidative stress is involved in skeletal muscle atrophy.

    Methods: This study examined markers of oxidative stress in the skeletal muscle of 10 haemodialysed patients and 10 control subjects. Biopsies from the quadriceps femoris were analysed for reduced and oxidized glutathione, protein thiols, malonaldehyde and heat shock proteins (HSP27, HSP60 and HSP70), superoxide dismutase and catalase activities. A novel microdialysis procedure was used to examine hydroxyl radical activity in the interstitial fluid of the tibialis anterior.

    Results: Patients had muscle atrophy with a reduced diameter of both type I and II fibres (by 15 and 20%, respectively). Muscle microdialysates contained 2,3- and 2,5-dihydroxybenzoates formed from salicylate indicating hydroxyl radical activity, with no differences between patients and control subjects. Muscle protein thiol and oxidized glutathione contents were unchanged in patients, but malonaldehyde content was reduced. In contrast, total muscle glutathione and heat shock protein contents were increased. Muscle superoxide dismutase activity was unchanged, but catalase activity was reduced in patients.

    Conclusions: The muscle of patients undergoing haemodialysis undergoes some adaptive responses in total glutathione content, heat shock protein content and catalase activity that are potentially related to chronic oxidative stress. However, there is no evidence of gross oxidation, nor any clear relationship between oxidative stress and muscle fibre atrophy, arguing against a direct role of oxidants in the degenerative processes.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2007;22;4;1177-83

  • Heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (TAK1)-mediated signaling.

    Alford KA, Glennie S, Turrell BR, Rawlinson L, Saklatvala J and Dean JL

    Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London W6 8LH, United Kingdom.

    Heat shock protein (HSP) 27 has long been known to be a component of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. p38 MAPK has important functions in the inflammatory response, but the role of HSP27 in inflammation has remained unknown. We have used small interfering RNAs to suppress HSP27 expression in HeLa cells and fibroblasts and found that it is required for pro-inflammatory cell signaling and the expression of pro-inflammatory genes. HSP27 is needed for the activation by interleukin (IL)-1 of TAK1 and downstream signaling by p38 MAPK, JNK, and their activators (MKK-3, -4, -6, -7) and IKKbeta. IL-1-induced ERK activation appears to be independent of HSP27. HSP27 is required for both IL-1 and TNF-induced signaling pathways for which the most upstream common signaling protein is TAK1. HSP27 is also required for IL-1-induced expression of the pro-inflammatory mediators, cyclooxygenase-2, IL-6, and IL-8. HSP27 functions to drive cyclooxygenase-2 and IL-6 expression by augmenting the activation of the kinase downstream of p38 MAPK, MK2, resulting in stabilization of cyclooxygenase-2 and IL-6 mRNAs. The mechanism may not occur in cells of myeloid lineage because HSP27 protein was undetectable in human monocytes and murine macrophages.

    Funded by: Medical Research Council: G8623776

    The Journal of biological chemistry 2007;282;9;6232-41

  • Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms.

    Jog NR, Jala VR, Ward RA, Rane MJ, Haribabu B and McLeish KR

    Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY 40202, USA.

    The targets of the p38 MAPK pathway responsible for regulation of neutrophil chemotaxis and exocytosis are unknown. One target of this pathway is the actin-binding protein, heat shock protein 27 (Hsp27). Therefore, we tested the hypothesis that Hsp27 mediates p38 MAPK-dependent chemotaxis and exocytosis in human neutrophils through regulation of actin reorganization. Sequestration of Hsp27 by introduction of anti-Hsp27 Ab, but not an isotype Ab, inhibited fMLP-stimulated chemotaxis, increased cortical F-actin in the absence of fMLP stimulation, and inhibited fMLP-stimulated exocytosis. Pretreatment with latrunculin A prevented actin reorganization and the changes in fMLP-stimulated exocytosis induced by Hsp27 sequestration. To determine the role of Hsp27 phosphorylation, wild-type, phosphorylation-resistant, or phosphorylation-mimicking recombinant Hsp27 was introduced into neutrophils by electroporation. The phosphorylation-resistant mutant significantly reduced migration toward fMLP, whereas none of the Hsp27 proteins affected fMLP-stimulated or TNF-alpha-stimulated exocytosis or actin polymerization. Endogenous Hsp27 colocalized with F-actin in unstimulated and fMLP-stimulated neutrophils, whereas phosphorylated Hsp27 showed cytosolic localization in addition to colocalization with F-actin. Our results suggest that Hsp27 regulates neutrophil chemotaxis and exocytosis in an actin-dependent, phosphorylation-independent manner. Phosphorylation of Hsp27 regulates chemotaxis, but not exocytosis, independent of regulation of actin reorganization.

    Funded by: NIDDK NIH HHS: DK62389

    Journal of immunology (Baltimore, Md. : 1950) 2007;178;4;2421-8

  • Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

    Hirose H, Sakuma N, Kaji N, Nakayama K, Inoue K, Sekijima M, Nojima T and Miyakoshi J

    Research Division for Advanced Technology, Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., Kamisu, Japan. h-hirose@ankaken.co.jp

    An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the Internatio 1f40 nal Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.

    Bioelectromagnetics 2007;28;2;99-108

  • Overexpression of human 27 kDa heat shock protein in laryngeal cancer cells confers chemoresistance associated with cell growth delay.

    Lee JH, Sun D, Cho KJ, Kim MS, Hong MH, Kim IK, Lee JS and Lee JH

    Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, 137-701, South Korea. leejh@catholic.ac.kr

    Purpose: Among the family of heat shock proteins (HSPs), HSP70 and HSP27 have been implicated in tumorigenesis and chemoresistance, probably via the prevention of apoptosis. HSP27 levels are frequently increased in large populations of tumors of the head and neck, but the mechanism of its chemoresistance is not yet fully understood. In the present study, the role of HSP27 in the resistance to cytotoxic stress was studied in Hep-2 human laryngeal cancer cells.

    Method: We established a Hep-2 cell line overexpressing HSP27 and examined whether the expression of HSP27 provides resistance to heat shock and several cytotoxic agents using a MTT colorimetic assay. Cell cycle progression was assessed by flow cytometry and fluorescence staining was performed for F-actin filaments.

    Results: HSP27 overexpression induced cellular resistance to heat shock at 45 degrees C for 1 h as well as against several cytotoxic agents, including cisplatin, staurosporin and H(2)O(2). However, no difference in sensitivity to irradiation or serum starvation was found. Moreover, HSP27 overexpressing Hep-2 cells showed a delayed cell growth, compared to control cells. To determine if the decreased cell proliferation in HSP27 overexpressing cells contributed to chemoresistance, control Hep-2 cells were synchronized at the late G1 phase by treatment with mimosine. The synchronized Hep-2 cells were resistant to cisplatin and H(2)O(2), but not to irradiation or serum starvation, correlating the protection effect shown in HSP27 overexpressing cells. These results suggest that the overexpression of HSP27 in Hep-2 cells confers chemoresistance which is associated with the delay in cell growth. We also propose that the stabilization of F-actin observed in Hep-2/hsp27 cells is partly related to the delay in cell cycle progression, by showing that the induction of actin polymerization in Hep-2/neo cells results in the retardation of cell growth as well as a cytoprotective effect as observed in Hep-2/hsp27.

    Journal of cancer research and clinical oncology 2007;133;1;37-46

  • HSF2 binds to the Hsp90, Hsp27, and c-Fos promoters constitutively and modulates their expression.

    DC, Skaggs HS and Sarge KD

    Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.

    Although the vast majority of genomic DNA is tightly compacted during mitosis, the promoter regions of a number of genes remain in a less compacted state throughout this stage of the cell cycle. The decreased compaction of these promoter regions, which is referred to as gene bookmarking, is thought to be important for the ability of cells to express these genes during the following interphase. Previously, we reported a role for the DNA-binding protein heat shock factor (HSF2) in bookmarking the stress-inducible 70,000-Da heat shock protein (hsp70) gene. In this report, we have extended those studies and found that during mitosis, HSF2 is bound to the HSE promoter elements of other heat shock genes, including hsp90 and hsp27, as well as the proto-oncogene c-fos. The presence of HSF2 is important for expression of these genes because blocking HSF2 levels by RNA interference techniques leads to decreased levels of these proteins. These results suggest that HSF2 is important for constitutive as well as stress-inducible expression of HSE-containing genes.

    Funded by: NICHD NIH HHS: F32 HD050043, F32HD050043; NIEHS NIH HHS: ES007266, T32 ES007266; NIGMS NIH HHS: R01 GM061053, R01 GM064606

    Cell stress & chaperones 2007;12;3;283-90

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27.

    Rafiee P, Theriot ME, Nelson VM, Heidemann J, Kanaa Y, Horowitz SA, Rogaczewski A, Johnson CP, Ali I, Shaker R and Binion DG

    Dept. of Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA. prafiee@mcw.edu

    The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration).

    American journal of physiology. Cell physiology 2006;291;5;C931-45

  • 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

  • Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants.

    Fontaine JM, Sun X, Hoppe AD, Simon S, Vicart P, Welsh MJ and Benndorf R

    Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Pl., Ann Arbor, MI 48109-2200, USA.

    Two mutations (K141E, K141N) in the small heat shock protein (sHSP) HSP22 (HSPB8) are associated with the inherited peripheral motor neuron disorders distal hereditary motor neuropathy type II and axonal Charcot-Marie-Tooth disease type 2L. HSP22 is known to form homodimers, heterodimers with other sHSPs, and larger oligomers. In an effort to elucidate the cellular basis for these diseases, we have determined the ability of mutant HSP22 to interact with itself, with wild-type HSP22, and with other sHSPs that are abundant in neurons. Using the yeast two-hybrid method, quantitative fluorescence resonance energy transfer in live cells, and cross-linking, we found aberrantly increased interactions of mutant HSP22 forms with themselves, with wild-type HSP22, and with the other sHSPs, alphaB-crystallin, and HSP27. Interaction with HSP20 was not affected by the mutations. The data suggest that each mutant form of HSP22 has a characteristic pattern of abnormal interaction properties. A mutation (S135F) in HSP27 that is also associated with these disorders showed increased interaction with wild-type HSP22 also, suggesting linkage of these two etiologic factors, HSP22 and HSP27, into one common pathway. Increased interactions involving mutant sHSPs may be the molecular basis for their increased tendency to form cytoplasmic protein aggregates, and for the occurrence of the associated neuropathies.

    Funded by: NIEHS NIH HHS: P01ES11188

    FASEB journal : official publication of the Federation of American Societies for 199d Experimental Biology 2006;20;12;2168-70

  • Suppression of NF-kappaB activation by Entamoeba histolytica in intestinal epithelial cells is mediated by heat shock protein 27.

    Kammanadiminti SJ and Chadee K

    Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

    Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) 27 and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp27 and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-kappaB activation induced by interleukin-1beta; specifically, the NF-kappaB-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of IkappaB-alpha were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp27 in suppressing NF-kappaB activation at IkappaB kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp27 interacts with IKK-alpha and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp27 amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp27 abrogated SAP-induced NF-kappaB inhibition. In conditioned epithelial cells, Hsp27 was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.

    The Journal of biological chemistry 2006;281;36;26112-20

  • Expression of heat shock protein 27 in human atherosclerotic plaques and increased plasma level of heat shock protein 27 in patients with acute coronary syndrome.

    Park HK, Park EC, Bae SW, Park MY, Kim SW, Yoo HS, Tudev M, Ko YH, Choi YH, Kim S, Kim DI, Kim YW, Lee BB, Yo cd6 on JB and Park JE

    Division of Cardiology, Samsung Medical Center and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul 135-710, South Korea.

    Background: We intended to identify proteins that are differentially expressed in human atherosclerotic plaques.

    Comparative 2-dimensional electrophoretic analysis on carotid atherosclerotic endarterectomy specimens (n = 10) revealed that heat shock protein 27 (Hsp27) expression was significantly increased in the nearby normal-appearing area compared with the plaque core area from the same vessel specimen, which was further confirmed by Western blot analysis. The Hsp27 expression in the adjacent normal-appearing vessel areas was much higher than that in nonatherosclerotic reference arteries. The phosphorylation of Hsp27 showed a gradation in the degree of phosphorylation: greatest in the reference arteries, intermediate in the adjacent normal-appearing area, and lowest in plaque core area. Immunohistochemical analysis showed that the phosphorylation of Hsp27 of smooth muscle cells in the carotid endarterectomy specimens was decreased compared with that in the reference artery specimen. The mean plasma level of Hsp27 was significantly higher in patients with acute coronary syndrome (ACS) (n = 27; 106.1 +/- 74.1 ng/mL) than in the normal reference subjects (n = 29; 45.8 +/- 29.5 ng/mL; P < 0.005). The plasma levels of Hsp27 were significantly correlated with those of heat shock protein 70 (Hsp70) (r = 0.422, P < 0.0005), with adjustment for ACS/reference status.

    Conclusions: In the atherosclerotic lesion, Hsp27 expression is increased in the normal-appearing vessel adjacent to atherosclerotic plaque, whereas levels in the plaque itself are significantly decreased. Both plaque and adjacent artery show decreased Hsp27 phosphorylation compared with reference vessel. In ACS, plasma Hsp27 and Hsp70 are increased, and levels of Hsp27 correlate with Hsp70, C-reactive protein, and CD40L levels.

    Circulation 2006;114;9;886-93

  • Elevated serum kininogen in patients with Paget's disease of bone: a role in marrow stromal/preosteoblast cell proliferation.

    Tsuruga E, Rao DS, Baatz JE and Reddy SV

    Children's Research Institute, Medical University of South Carolina, Charleston, South Carolina, USA.

    Paget's disease (PD) of bone is a chronic focal skeletal disorder characterized by excessive bone resorption followed by abundant new bone formation. Enhanced levels of IL-6, RANKL, M-CSF, and endothelin-1 have been associated with PD. In the present study, we identified increased serum levels (2 to 5-fold) of inflammatory cytokine, kininogen (KNG) in patients with PD compared to normal subjects. Treatment of pagetic bone marrow derived stromal/preosteoblast cells with recombinant KNG (25 ng/ml) for 24 h period resulted in a 5-fold increase in the levels of phospho-HSP27 and a 3-fold increase in ERK1/2 phosphorylation in these cells. However, pagetic stromal cells stimulated with KNG in the presence of ERK activation inhibitor peptide did not significantly affect the levels of phospho-HSP27. KNG increased normal and pagetic marrow stromal cell proliferation at 1.4-fold and 2.5-fold, respectively. KNG in the presence of an ERK inhibitor peptide did not stimulate pagetic marrow stromal cell proliferation. Furthermore, siRNA suppression of HSP27 expression significantly decreased KNG inhibition of etoposide-induced caspase-3 activation and apoptosis in these cells. In summary, KNG modulate bone marrow derived stromal/preosteoblast cell proliferation and suppress etoposide-induced apoptosis through ERK and HSP27 activation, respectively. These results implicate a pathophysiologic role for KNG in patients with PD.

    Funded by: NIAMS NIH HHS: AR 049363

    Journal of cellular biochemistry 2006;98;6;1681-8

  • Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.

    Kim SC, Sprung R, Chen Y, Xu Y, Ball H, Pei J, Cheng T, Kho Y, Xiao H, Xiao L, Grishin NV, White M, Yang XJ and Zhao Y

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.

    Funded by: NCI NIH HHS: CA107943

    Molecular cell 2006;23;4;607-18

  • Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation.

    Seo HR, Chung DY, Lee YJ, Lee DH, Kim JI, Bae S, Chung HY, Lee SJ, Jeoung D and Lee YS

    Laboratory of Radiation Effect and Laboratory of Radiation Experimental Therapeutics, Korea Institute of Radiological and Medical Sciences, Seoul 139-706.

    The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.

    The Journal of biological chemistry 2006;281;25;17220-7

  • Biological significance of decreased HSP27 in human atherosclerosis.

    Martin-Ventura JL, Nicolas V, Houard X, Blanco-Colio LM, Leclercq A, Egido J, Vranckx R, Michel JB and Meilhac O

    Inserm, U698, University of Paris 7 and CHU X-Bichat, Paris, France.

    Objective: Because culprit atherosclerotic plaques contain proteases, we hypothesized that the diminished heat shock protein 27 (HSP27) released by atherosclerotic plaques could be due to proteolysis. We assessed the role of HSP27 in human vascular smooth muscle cells (VSMCs) under proteolytic injury.

    Active plasmin is present in culprit atherosclerotic plaques. Recombinant HSP27 was cleaved by plasmin and this effect was prevented by different inhibitors. Fragments and aggregated forms of HSP27 appeared after incubation of mammary control endarteries with plasmin. Coincubation of atherosclerotic plaques with recombinant HSP27 or mammary endarteries led to HSP27 proteolysis. After incubation of VSMCs with plasmin, HSP27 was overexpressed, phosphorylated, aggregated, and redistributed from the cytoskeleton to the cytosol, nucleus, and cell membrane. Plasmin-induced VSMC apoptosis was significantly higher in VSMCs treated by HSP27 siRNA. Immunohistochemical analysis of atherosclerotic plaques showed that plasmin(ogen) and apoptotic cells are localized in the core/shoulder whereas HSP27 and VSMCs are mainly expressed in the cap/media.

    Conclusions: Extracellular HSP27 can be degraded by enzymes released from atherosclerotic plaques and may reflect a proteolytic imbalance. Intracellular HSP27 downregulation decreases VSMCs resistance to proteolytically-induced apoptosis. HSP27 might play a pivotal role in the prevention of plaque instability and rupture.

    Arteriosclerosis, thrombosis, and vascular biology 2006;26;6;1337-43

  • MAPKAPK2 and HSP27 are downstream effectors of p38 MAP kinase-mediated matrix metalloproteinase type 2 activation and cell invasion in human prostate cancer.

    Xu L, Chen S and Bergan RC

    Department of Medicine, Division of Hematology/Oncology, Northwestern University Medical School, Chicago, IL, USA.

    Although cell invasion is a necessary early step in cancer metastasis, its regulation is not well understood. We have previously shown, in human prostate cancer, that transforming growth factor beta (TGFbeta)-mediated increases in cell invasion are dependent upon activation of the serine/threonine kinase, p38 MAP kinase. In the current study, downstream effectors of p38 MAP kinase were sought by first screening for proteins phosphorylated after TGFbeta treatment, only in the absence of chemical inhibitors of p38 MAP kinase. This led us to investigate mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2), a known substrate of p38 MAP kinase, as well as heat-shock protein 27 (HSP27), a known substrate of MAPKAPK2, in both PC3 and PC3-M human prostate cells. After transient transfection, wild-type MAPKAPK2 and HSP27 both increased TGFbeta-mediated matrix metalloproteinase type 2 (MMP-2) activity, as well as cell invasion, which in turn was inhibited by SB203580, an inhibitor of p38 MAP kinase. Conversely, dominant-negative MAPKAPK2 blocked phosphorylation of HSP27, whereas dominant-negative MAPKAPK2 or mutant, non-phosphorylateable, HSP27 each blocked TGFbeta-mediated increases in MMP-2, as well as cell invasion. Similarly, knock down of MAPKAPK2, HSP27 or both together, by siRNA, also blocked TGFbeta-mediated cell invasion. This study demonstrates that both MAPKAPK2 and HSP27 are necessary for TGFbeta-mediated increases in MMP-2 and cell invasion in human prostate cancer.

    Funded by: NCI NIH HHS: CA90386

    Oncogene 2006;25;21;2987-98

  • Phosphoproteome analysis of the human mitotic spindle.

    Nousiainen M, Silljé HH, Sauer G, Nigg EA and Körner R

    Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

    During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;14;5391-6

  • Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection.

    Leong WF and Chow VT

    Human Genome Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 117597.

    Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.

    Cellular microbiology 2006;8;4;565-80

  • Self-association and chaperone activity of Hsp27 are thermally activated.

    Lelj-Garolla B and Mauk AG

    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

    The small heat shock protein 27 (Hsp27) is an oligomeric, molecular chaperone in vitro. This chaperone activity and other physiological roles attributed to Hsp27 have been reported to depend on the state of self-association. In the present work, we have used sedimentation velocity experiments to demonstrate that the self-association of Hsp27 is independent of pH and ionic strength but increases significantly as the temperature is increased from 10 to 40 degrees C. The largest oligomers formed at 10 degrees C are approximately 8-12 mer, whereas at 40 degrees C oligomers as large as 22-30 mer are observed. Similarly, the chaperone activity of Hsp27 as indicated by its ability to inhibit dithiothreitol-induced insulin aggregation also increases with increased temperature, with a particularly sharp increase in activity as temperature is increased from 34 to 43 degrees C. Similar studies of an Hsp27 triple variant that mimics the behavior of the phosphorylated protein establish that this protein has greatly diminished chaperone activity that responds minimally to increased temperature. We conclude that Hsp27 can exploit a large number of oligomerization states and that the range of oligomer size and the magnitude of chaperone activity increase significantly as temperature is increased over the range that is relevant to the physiological heat shock response.

    The Journal of biological chemistry 2006;281;12;8169-74

  • Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells.

    Schalkwijk CG, v 1f40 an Bezu J, van der Schors RC, Uchida K, Stehouwer CD and van Hinsbergh VW

    Department of Clinical Chemistry, Vrije Universiteit Medical Center, Amsterdam, The Netherlands. c.schalkwijk@intmed.unimaas.nl

    In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.

    FEBS letters 2006;580;6;1565-70

  • Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27).

    Shi B and Isseroff RR

    Department of Dermatology, University of California Davis School of Medicine, Davis, California 95616, USA.

    Exposure to ultraviolet (UV) light poses a health risk for eye disease, and solar ultraviolet in the B range (UVB, 280-320 nm) is known to be related to various corneal disorders. In this study, we investigated whether pre-conditioning of cells with arsenite (AsO2(-1)) can reduce UVB-induced apoptosis in human corneal epithelial cells, and whether the anti-apoptotic activity of 27 kDa heat shock protein (HSP27), a small heat shock protein, plays a role in this protection. UVB at levels comparable to physiologic solar exposure induces apoptosis of corneal epithelial cells in culture, demonstrated by activation of caspase 9 and caspase 3, and DNA fragmentation. When cells were pre-conditioned with arsenite prior to UVB exposure, the UVB-induced cell death was reduced, and UVB-induced activation of caspases and DNA fragmentation was inhibited. When cells were pre-treated with SB 203580, which inhibits HSP27 phosphorylation through inhibition of p38 MAP kinase activation, the arsenite-induced reduction of UVB-induced apoptosis was partially reversed. Arsenite pre-conditioning inhibited UVB-induced apoptosis in a two-phase pattern, which was temporally correlated with arsenite-induced HSP27 expression and phosphorylation. Neutralization of intracellular HSP27 with its antibody reduced arsenite's inhibition of UVB-induced caspase3 activation. Our results suggest that forms of stress that upregulate HSP27 and its phosphorylation may be useful as novel approaches to prevent adverse ocular effects arising from UV exposure in humans.

    Funded by: NIAMS NIH HHS: AR 44518; NIEHS NIH HHS: ES05707

    Journal of cellular physiology 2006;206;2;301-8

  • Hsp27 enhances recovery of splicing as well as rephosphorylation of SRp38 after heat shock.

    Marin-Vinader L, Shin C, Onnekink C, Manley JL and Lubsen NH

    Department of Biochemistry, Radboud University Nijmegen, 6500 HB Nijmegen, The Netherlands.

    A heat stress causes a rapid inhibition of splicing. Exogenous expression of Hsp27 did not prevent that inhibition but enhanced the recovery of splicing afterward. Another small heat shock protein, alphaB-crystallin, had no effect. Hsp27, but not alphaB-crystallin, also hastened rephosphorylation of SRp38-dephosphorylated a potent inhibitor of splicing-after a heat shock, although it did not prevent dephosphorylation by a heat shock. The effect of Hsp27 on rephosphorylation of SRp38 required phosphorylatable Hsp27. A Hsp90 client protein was required for the effect of Hsp27 on recovery of spicing and on rephosphorylation of SRp38. Raising the Hsp70 level by either a pre-heat shock or by exogenous expression had no effect on either dephosphorylation of SRp38 during heat shock or rephosphorylation after heat shock. The phosphatase inhibitor calyculin A prevented dephosphorylation of SRp38 during a heat shock and caused complete rephosphorylation of SRp38 after a heat shock, indicating that cells recovering from a heat shock are not deficient in kinase activity. Together our data show that the activity of Hsp27 in restoring splicing is not due to a general thermoprotective effect of Hsp27, but that Hsp27 is an active participant in the c84 (de)phosphorylation cascade controlling the activity of the splicing regulator SRp38.

    Funded by: NIGMS NIH HHS: R37 GM048259, R37 GM48259

    Molecular biology of the cell 2006;17;2;886-94

  • Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values.

    Luk JM, Lam CT, Siu AF, Lam BY, Ng IO, Hu MY, Che CM and Fan ST

    Centre for the Study of Liver Disease and Department of Surgery, The University of Hong Kong, Hong Kong SAR, P. R. China. jmluk@hkucc.hku.hk

    To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.

    Proteomics 2006;6;3;1049-57

  • A mutation in the small heat-shock protein HSPB1 leading to distal hereditary motor neuronopathy disrupts neurofilament assembly and the axonal transport of specific cellular cargoes.

    Ackerley S, James PA, Kalli A, French S, Davies KE and Talbot K

    Department of Human Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, UK.

    Distal hereditary motor neuronopathies (dHMNs) are a clinically and genetically heterogeneous group of disorders in which motor neurons selectively undergo age-dependant degeneration. Mutations in the small heat-shock protein HSPB1 (HSP27) are responsible for one form of dHMN. In this study, we have analysed the effect of expressing a form of mutant HSPB1 in primary neuronal cells in culture. Mutant (P182L) but not wild-type HSPB1 led to the formation of insoluble intracellular aggregates and to the sequestration in the cytoplasm of selective cellular components, including neurofilament middle chain subunit (NF-M) and p150 dynactin. These findings suggest a possible pathogenic mechanism for HSPB1 whereby the mutation may lead to preferential motor neuron loss by disrupting selective components essential for axonal structure and transport.

    Funded by: Medical Research Council: MC_U137761449

    Human molecular genetics 2006;15;2;347-54

  • Silencing the hsp25 gene eliminates migration capability of the highly metastatic murine 4T1 breast adenocarcinoma cell.

    Bausero MA, Bharti A, Page DT, Perez KD, Eng JW, Ordonez SL, Asea EE, Jantschitsch C, Kindas-Muegge I, Ciocca D and Asea A

    Center for Molecular Stress Response, Boston University Medical Center and Boston University School of Medicine, Mass., USA.

    The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration.

    Funded by: NCI NIH HHS: R01 CA091889, R01 CA091889-05

    Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2006;27;1;17-26

  • Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis.

    Feng JT, Liu YK, Song HY, Dai Z, Qin LX, Almofti MR, Fang CY, Lu HJ, Yang PY and Tang ZY

    Liver Cancer Institute & Zhong Shan Hospital, Fudan University, Shanghai, China.

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.

    Proteomics 2005;5;17;4581-8

  • Dynamic expression of Hsp27 in the presence of mutant ataxin-3.

    Chang WH, Cemal CK, Hsu YH, Kuo CL, Nukina N, Chang MH, Hu HT, Li C and Hsieh M

    Department of Life Science, Tunghai University, Taichung, Taiwan.

    Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. The molecular mechanisms underlying the selective neuronal death typical of MJD/SCA3 are unknown. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD with 78 CAG repeats were assayed for the dynamic expression of Hsp27, known as a suppressor of poly-Q mediated cell death, in the presence of mutant ataxin-3 in different passages of cultured cells. A dramatic decrease of Hsp27 expression was observed in the earlier passage of cultured SK-N-SH-MJD78 cells, however, the later passage of cells showed a significant increase of Hsp27 to almost the same level of the parental cells. Furthermore, immunohistochemical analysis of MJD transgenic mice and post-mortem human brain tissues showed increased expression of Hsp27 compared to normal control brain, suggesting an up-regulation of Hsp27 in the end stage of MJD. However, mutant cells of earlier passages were more susceptible to serum deprivation than mutant cells of later passages, indicating weak tolerance toward stress in cells with reduced Hsp27. While heat shock was used to assess the stress response, cells expressing mutant ataxin-3 displayed normal response upon heat shock stimuli when compared to the parental cells. Taken together, we proposed that during the early disease stage, the reduction of Hsp27 synthesis mitigated the ability of neuron cells to cope with cytotoxicity induced by mutant ataxin-3, triggering the cell death process during the disease progress. In the late stage of disease, after prolonged stressful conditions of polyglutamine cytotoxicity, the increased level of Hsp27 may reflect a dynamic process of the survived cells to unfold and remove mutant ataxin-3. However, this increased Hsp27 still cannot reverse the global dysfunction of cellular proteins due to accumulation of cytotoxic effects.

    Biochemical and biophysical research communications 2005;336;1;258-67

  • [Mutation analysis of small heat shock protein 27 gene in Chinese patients with Charcot-Marie-Tooth disease].

    Liu XM, Tang BS, Zhao GH, Xia K, Zhang FF, Pan Q, Cai F, Hu ZM, Zhang C, Chen B, Shen L, Zhang RX and Jiang H

    Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, PR China.

    Objective: To investigate 1f40 the features of small heat shock protein 27 (HSP27) gene mutation in Chinese patients with Charcot-Marie-Tooth disease (CMT).

    Methods: DNA samples from 114 CMT probands were screened for mutations in HSP27 gene by polymerase chain reaction and direct sequencing, and haplotype analysis was further carried out on the mutation detected families.

    Results: One missense mutation C379T was detected in 4 autosomal dominant CMT2 families. Haplotype analysis indicated that the 4 families probably had a common ancestor.

    Conclusion: To the authors' knowledge, this is the first report of HSP27 gene mutation in Chinese patients with CMT, but it may be not common(0.90%). The C379T mutation in HSP27 gene also causes CMT2 except for distal hereditary motor neuropathy, thus providing further evidence that even the same mutation in the same gene may lead to distinct phenotypes.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2005;22;5;510-3

  • 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

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Decreased expression of Hsp27 and Hsp70 in transformed lymphoblastoid cells from patients with spinocerebellar ataxia type 7.

    Tsai HF, Lin SJ, Li C and Hsieh M

    School of Medical Technology, Chung Shan Medical University, Taichung, Taiwan, ROC.

    Spinocerebellar ataxia type 7 (SCA7) is caused by an expansion of unstable CAG repeats within the coding region of the novel gene, ataxin-7, on chromosome 3. This disease is also associated with an accumulation of abnormal proteins, including expanded polyglutamine-containing proteins, molecular chaperones, and the ubiquitin-proteasome system. In this study, two SCA7 lymphoblastoid cell lines (LCLs) with 100 and 41 polyglutamine repeats were utilized to examine the effects of polyglutamine expansion on heat shock proteins. Interestingly, under basal conditions, Western blot and immunocytochemical analysis showed a significant decrease of Hsp27 and Hsp70 protein expression in cells containing expanded ataxin-7, as compared with that of the normal LCL. On the other hand, the protein levels of Hsp60 and Hsp90 were not significantly altered in the mutant LCLs. Results from semi-quantitative RT-PCR indicated that the differences in Hsp70 protein levels were due to transcriptional defects while the reduction of Hsp27 in the mutant cells was not caused by transcriptional defects. Our results further demonstrated that despite of defective protein expression of Hsp27 and Hsp70, a normal heat shock response was observed in lymphoblastoid cells expressing mutant ataxin-7. Taken together, our results indicated that expanded ataxin-7 that leads to neurodegeneration significantly impaired the expression of Hsp27 and Hsp70 protein, which may be, at least in part, responsible for the toxicity of mutant ataxin-7 proteins and ultimately resulted in an increase of stress-induced cell death.

    Biochemical and biophysical research communications 2005;334;4;1279-86

  • Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC.

    Gevaert K, Staes A, Van Damme J, De Groot S, Hugelier K, Demol H, Martens L, Goethals M and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, Ghent, Belgium.

    We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+-immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O-18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190 phosphorylated peptides from 152 different proteins. This dataset includes 38 novel protein phosphorylation sites.

    Proteomics 2005;5;14;3589-99

  • Mutation analysis of the small heat shock protein 27 gene in chinese patients with Charcot-Marie-Tooth disease.

    Tang B, Liu X, Zhao G, Luo W, Xia K, Pan Q, Cai F, Hu Z, Zhang C, Chen B, Zhang F, Shen L, Zhang R and Jiang H

    Department of Neurology, Xiangya Hospital, Central South University, Changsha, People's Republic of China. bstang7398@yahoo.com.cn

    Background: Charcot-Marie-Tooth (CMT) disease, the most common hereditary peripheral neuropathy, is highly clinically and genetically heterogeneous, and mutations in at least 18 genes have been identified. Recently, mutations in small heat shock protein 27 (Hsp27) were reported to cause CMT disease type 2F and distal hereditary motor neuropathy.

    Objective: To investigate the frequency and phenotypic features of an Hsp27 mutation in Chinese patients with CMT disease.

    Design: DNA samples from 114 unrelated patients with CMT disease were screened for mutations in Hsp27 by polymerase chain reaction and direct sequencing. A cosegregated study was performed using the MbiI restriction endonuclease, and 50 healthy control subjects were analyzed. Haplotype analysis was performed using 5 short tandem repeat markers to analyze whether the families with the same mutation probably had a common ancestor.

    Results: One missense mutation, C379T, was detected in 4 autosomal dominant families with CMT disease type 2, and haplotype analysis indicated that the 4 families probably had a common founder. The frequency of the Hsp27 mutation is 0.9% (1/111) in Chinese patients with CMT disease in our study, and the phenotypes were characterized by later onset (age, 35-60 years) and mild sensory impairments. Electrophysiological findings showed moderately to severely slowed nerve conduction velocities in lower limb nerves but normal or mildly reduced velocities in upper limb nerves.

    Conclusions: To our knowledge, this is the first report of an Hsp27 mutation in the People's Republic of China. The C379T mutation in Hsp27 also causes CMT disease type 2, except for distal hereditary motor neuropathy, and the phenotypes are distinct from the family with CMT disease type 2F described previously. A mutation of Hsp27 may be uncommon in Chinese patients with CMT disease.

    Archives of neurology 2005;62;8;1201-7

  • Heat shock protein 27 is associated with freedom from graft vasculopathy after human cardiac transplantation.

    De Souza AI, Wait R, Mitchell AG, Banner NR, Dunn MJ and Rose ML

    Transplant Immunology, National Heart and Lung Institute, Imperial College London, Harefield Hospital, Harefield, Middlesex, UB9 6JH, United Kingdom.

    Experimental studies have suggested that protective genes protect allografts from cardiac allograft vasculopathy (CAV), the major complication after cardiac transplantation. Here we have sought to confirm this hypothesis using long-term heart transplant recipients. Twenty-two patients that were 9 years or older after transplant were investigated; 11 of these were without angiographic evidence of CAV; 11 had developed early CAV at 1 to 3 years after transplant. To identify proteins that may act as protectors from CAV, a global proteomic approach was used comparing cardiac biopsies from 12 patients taken within the first 2 weeks after transplant and those taken after 9 years from the same patient. Proteins were separated by 2-D gel-electrophoresis, detected by silver staining, and analyzed using Progenesis software. A particular protein spot was found in 4/6 biopsies from patients without CAV, but absent from 5/6 biopsies from those with CAV (P=0.24); however, quantitati 1f40 ve analysis of spot intensity showed a significant difference (0.061+/-0.05 versus 0.003+/-0.01, P=0.04). This spot was identified by mass spectrometry and a combination of techniques as a diphosphorylated form of HSP27. Immunohistochemistry of further biopsies not only validated that HSP27 was more abundantly expressed on biopsies without CAV but also showed it to be localized to blood vessels. In contrast, vessels from patients with CAV did not express HSP27 (P=0.028x10(-4)). Immunohistochemistry of 12 further early biopsies and nontransplanted heart showed HSP27 to be present in normal blood vessels. These findings suggest that expression of a specific diphosphorylated form of HSP27 is associated with healthy blood vessels; it appears to be lost from vessels of patients with graft vasculopathy.

    Circulation research 2005;97;2;192-8

  • Activation of p38 has opposing effects on the proliferation and migration of endothelial cells.

    McMullen ME, Bryant PW, Glembotski CC, Vincent PA and Pumiglia KM

    Center for Cell Biology, Albany Medical College, Albany, New York 12208, USA.

    Pathological conditions such as hypertension and hyperglycemia as well as abrasions following balloon angioplasty all lead to endothelial dysfunction that impacts disease morbidity. These conditions are associated with the elaboration of a variety of cytokines and increases in p38 activity in endothelial cells. However, the relationship between enhanced p38 activity and endothelial cell function remains poorly understood. To investigate the effect of enhanced p38 MAPK activity on endothelial cell function, we expressed an activated mutant of MEK6 (MEK6E), an upstream regulator of p38. Expression of MEK6E activated p38 and resulted in phosphorylation of its downstream substrate, heat shock protein 27 (Hsp27). Activation of p38 was not sufficient to induce apoptosis; however, it did induce p38-dependent cell cycle arrest. MEK6E expression was sufficient to inhibit ERK phosphorylation triggered by growth factors and integrin engagement. MAPK phosphatase-1 (MKP-1) expression was increased upon p38 activation, and expression of a "substrate-trapping" MKP-1 was sufficient to restore ERK activity. Activation of p38 was sufficient to induce cell migration, which was accompanied by alterations in actin architecture characterized by enhanced lamellipodia. Co-expression of a mutant form of Hsp27, lacking all three phosphorylation sites, reversed MEK6E-induced cell migration and altered the cytoskeletal changes induced by p38 activation. Collectively, these results suggest that cellular decisions regarding migration and proliferation are influenced by p38 activity and that prolonged activation of p38 may result in an anti-angiogenic phenotype that contributes to endothelial dysfunction.

    Funded by: NCI NIH HHS: CA-90653, R01 CA081419, R01 CA081419-06, R01-CA-81419; NHLBI NIH HHS: HL075573, T32-HL-07194; NINDS NIH HHS: NS/HL20537

    The Journal of biological chemistry 2005;280;22;20995-1003

  • HSP25 inhibits radiation-induced apoptosis through reduction of PKCdelta-mediated ROS production.

    Lee YJ, Lee DH, Cho CK, Chung HY, Bae S, Jhon GJ, Soh JW, Jeoung DI, Lee SJ and Lee YS

    Laboratory of Radiation Effect, Division of Radiation Biology, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, Korea.

    Since radiation-induced caspase-dependent apoptosis and ROS generation were partially prevented by HSP25 overexpression, similar to the treatment of control cells with antioxidant agents such as DPI and tiron, questions arise whether radiation-mediated ROS generation contributes to the apoptotic cell death, and also whether HSP25 overexpression can reduce ROS mediated apoptotic cell death. In the present study, radiation-induced cytochrome c release from mitochondria and activation of caspases accompanied by a decrease of mitochondrial membrane potential in Jurkat T cells were shown to be inhibited by mitochondrial complex I inhibitor rotenone, suggesting that mitochondrial ROS might be important in radiation-induced caspase-dependent apoptosis. When HSP25 was overexpressed, effects similar to the treatment of cells with the antioxidants were obtained, indicating that HSP25 suppressed radiation-induced mitochondrial alteration that resulted in apoptosis. Furthermore, activation of p38 MAP kinase by radiation was associated with radiation-induced cell death and ROS production and PKCdelta was an upstream molecule for p38 MAP kinase activation, ROS generation and subsequent caspase-dependent apoptotic events. However, in the HSP25 overexpressed cells, the above-described effects were blocked. In fact, radiation-induced membrane translocation of PKCdelta and tyrosine phosphorylation were inhibited by HSP25. Based on the above data, we suggest that HSP25 downregulates PKCdelta, which is a key molecule for radiation-induced ROS generation and mitochondrial-mediated caspase-dependent apoptotic events.

    Oncogene 2005;24;23;3715-25

  • 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

  • A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D.

    Döppler H, Storz P, Li J, Comb MJ and Toker A

    Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

    The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.

    Funded by: NCI NIH HHS: CA75134

    The Journal of biological chemistry 2005;280;15;15013-9

  • Modulation of estrogen signaling by the novel interaction of heat shock protein 27, a biomarker for atherosclerosis, and estrogen receptor beta: mechanistic insight into the vascular effects of estrogens.

    Miller H, Poon S, Hibbert B, Rayner K, Chen YX and O'Brien ER

    University of Ottawa Heart Institute, Ontario, Canada.

    Objective: We sought to discover proteins that associate with estrogen receptor beta (ERbeta) and modulate estrogen signaling.

    Using a yeast 2-hybrid screen, we identified heat shock protein 27 (HSP27) as an ERbeta-associated protein. HSP27 is a recently identified biomarker of atherosclerosis that is secreted at reduced levels from atherosclerotic compared with normal arteries. In vitro protein-binding assays confirmed the specific interaction of HSP27 with ERbeta and not ERalpha. HSP27 expression was absent in coronary arteries with complex atherosclerotic lesions. Interestingly, HSP27 expression was also absent in 60% of coronary arteries from young males and females (27+/-6.5 years) with normal histology or nonobstructive fatty streaks/atheromas. Moreover, the absence of HSP27 in these normal or minimally diseased arteries coincided with the loss of ERbeta expression. Only 35% of arteries showed coexpression of HSP27 and ERbeta. Relative to controls, estradiol-mediated transcription was reduced 20% with overexpression of HSP27 and increased 44% when HSP27 protein levels were reduced with HSP27 siRNA.

    Conclusions: HSP27, an ERbeta-associated protein, shows attenuated expression with coronary atherosclerosis and modulates estrogen signaling.

    Arteriosclerosis, thrombosis, and vascular biology 2005;25;3;e10-4

  • Mechanism of chaperone function in small heat shock proteins: dissociation of the HSP27 oligomer is required for recognition and binding of destabilized T4 lysozyme.

    Shashidharamurthy R, Koteiche HA, Dong J and McHaourab HS

    Department of Molecular Physiology and Biophysics, Vanderbilt University, 741 Light Hall, Nashville, TN 37232, USA.

    Mammalian small heat shock proteins (sHSP) form polydisperse and dynamic oligomers that undergo equilibrium subunit exchange. Current models of their chaperone activity hypothesize that recognition and binding of protein non-native states involve changes in the oligomeric state. The equivalent thermodynamic representation is a set of three coupled equilibria that includes the sHSP oligomeric equilibrium, the substrate folding equilibrium, and the equilibrium binding between the sHSP and the substrate non-native states. To test this hypothesis and define the binding-competent oligomeric state of human Hsp27, we have perturbed the two former equilibria and quantitatively determined the consequences on binding. The substrate is a set of T4 lysozyme (T4L) mutants that bind under conditions that favor the folded state over the unfolded state by 10(2)-10(4)-fold. The concentration-dependent oligomer equilibrium of Hsp27 was perturbed by mutations that alter the relative stability of two major oligomeric states including phosphorylation-mimicking mutations that result in the dissociation to a small multimer over a wide range of concentrations. Correlation of binding isotherms with size exclusion chromatography analysis of the Hsp27 oligomer equilibrium demonstrates that the multimer is the binding-competent state. Binding occurs through two modes, each characterized by different affinity and number of binding sites, and results in T4L.Hsp27 complexes of different hydrodynamic properties. Mutants of the Hsp27 phosphorylation mimic that reverse the reduction in oligomer size also reduce the extent of T4L binding. Taken together, these results suggest a central role for the oligomeric equilibrium in regulating the chaperone activity of sHSP. The mutants identify sequence features important for modulating this equilibrium.

    Funded by: NEI NIH HHS: EY-R0112018

    The Journal of biological chemistry 2005;280;7;5281-9

  • The kaposin B protein of KSHV activates the p38/MK2 pathway and stabilizes cytokine mRNAs.

    McCormick C and Ganem D

    Howard Hughes Medical Institute, Department of Microbiology and Immunology, and Department of Medicine, University of California, San Francisco, CA 94143, USA.

    Cytokine production plays a critical role in diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Here we show that a latent KSHV gene product, kaposin B, increases the expression of cytokines by blocking the degradation of their messenger RNAs (mRNAs). Cytokine transcripts are normally unstable because they contain AU-rich elements (AREs) in their 3' noncoding regions that target them for degradation. Kaposin B reverses this instability by binding to and activating the kinase MK2, a target of the p38 mitogen-activated protein kinase signaling pathway and a known inhibitor of ARE-mRNA decay. These findings define an important mechanism linking latent KSHV infection to cytokine production, and also illustrate a distinctive mode by which viruses can selectively modulate mRNA turnover.

    Science (New York, N.Y.) 2005;307;5710;739-41

  • Cytotoxic effects induced by oxidative stress in cultured mammalian cells and protection provided by Hsp27 expression.

    Arrigo AP, Firdaus WJ, Mellier G, Moulin M, Paul C, Diaz-latoud C and Kretz-remy C

    Laboratoire Stress Oxydant, Chaperons et Apoptose, Centre de Génétique Moléculaire et Cellulaire, CNRS UMR 5534, Université Claude Bernard Lyon-1, Villeurbanne 69622, France. arrigo@univ-lyon1.fr

    There is currently great interest in the development of methods to analyze intracellular redox state and the cellular damages generated by oxidative stress. General methods for analyzing reactive oxygen species and glutathione level are presented together with more recently developed protocols to analyze the consequences of oxidative stress on the oxidation of macromolecules. Finally, techniques to study modalities of constitutive expression of Hsp27 in mammalian cells are considered as well as methods used to determine the protective activity of this small heat shock protein against the deleterious effects induced by oxidative stress.

    Methods (San Diego, Calif.) 2005;35;2;126-38

  • Self-association of a small heat shock protein.

    Lelj-Garolla B and Mauk AG

    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

    Human Hsp27 oligomerizes in vivo in a phosphorylation-dependent manner that regulates the functional activity of the protein. We have studied the self-association of wild-type Hsp27 by both sedimentation velocity and sedimentation equilibrium analysis and established that the protein forms an equilibrium mixture of monomers/dimers, tetramers, 12-mers and 16-mers (20 mM Tris-HCl (pH 8.4), 100 mM NaCl, 20 degrees C). Corresponding analysis of the S15D/S78D/S82D triple variant, which is believed to mimic the behavior of phosphorylated Hsp27, establishes that this form of the protein forms primarily monomers and dimers but also forms a small fraction of very large oligomers. Variants in which critical N-terminal sequences have been deleted exhibit oligomerization behavior that is intermediate between that of the triple variant and the wild-type protein. On the other hand a C-terminal sequence deletion variant forms larger oligomers than does the wild-type protein, but also exhibits a greater fraction of smaller oligomers. Notably, the presence of an N-terminal His6-tag induces formation of much larger oligomers than observed for any other form of the protein. The results of this work establish that the wild-type protein forms smaller oligomers than previously believed, define the roles played by various structural domains in Hsp27 oligomerization, and provide improved molecular probes with better-defined properties for the design of future experiments.

    Journal of molecular biology 2005;345;3

  • Heat shock protein 27 expression in patients with chronic liver damage.

    Federico A, Tuccillo C, Terracciano F, D'Alessio C, Galdiero M, Finamore E, D'Isanto M, Peluso L, Del Vecchio Blanco C and Loguercio C

    Centro Interuniversitario per Ricerche su Alimenti, Nutrizione e Apparato Digerente (CIRANAD), Cattedra di Gastroenterologia, II Università di Napoli, via Pansini 5, Napoli, Italy. alessandro.federico@unina2.it

    The aim of this study was to evaluate a possible relationship between lymphomonocyte expression of heat shock proteins (HSP) 60/27 and plasma levels of pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and markers of antioxidant/oxidative status [glutathione (GSH), alpha glutathione-S-transferase activity (alpha GST), malonyldialdeyde (MDA), 4-hydroxinonenal (4-HNE), and S-nitrosothiols (S-NO)] in 666 patients with chronic liver diseases. Entered into the study were 47 subjects: 10 healthy controls, 16 patients with HCV-related chronic hepatitis (CH), and 16 patients with HCV-related and 5 with alcohol-related liver cirrhosis (10 Child A and 11 Child B+C). HSP60 was clearly expressed only in 5% of patients and lowly in the control group. HSP27 was clearly expressed in 46.7% of CH and 71.4% of cirrhotic patients but was lowly present in healthy subjects. A significant difference was found between patients with a low expression of HSP27 (negative patients) and those with a high HSP27 expression (positive patients) of plasma levels both of antioxidants (GSH, p < 0.05), and of markers of enhanced production of free radicals and cytokines (alpha GST, TNF-alpha and IL-6, p < 0.05; MDA, 4-HNE and S-NO, p < 0.01) as well as for alcohol use and degree of liver impairment. The present data are the first showing that, particularly in conditions of enhanced oxidative stress, lymphomonocytes from liver disease patients present an increased expression of HSP27.

    Immunobiology 2005;209;10;729-35

  • Regulation of Hsp27 expression and cell survival by the POU transcription factor Brn3a.

    Farooqui-Kabir SR, Budhram-Mahadeo V, Lewis H, Latchman DS, MS and Heads RJ

    Cell death and differentiation 2004;11;11;1242-4

  • 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

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Protective role of HSP27 against UVC-induced cell death in human cells.

    Wano C, Kita K, Takahashi S, Sugaya S, Hino M, Hosoya H and Suzuki N

    Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

    It is an intriguing problem whether heat shock proteins (HSPs) play a protective role in UVC-induced cell death in human cells, and the problem has not been solved. To search for the HSPs involved in UVC resistance, gene expression profiles using cDNA array were compared between UVC-sensitive human RSa cells and their UVC-resistant variant AP(r)-1 cells. The expression levels of heat shock protein 27 (HSP27) were lower in RSa cells than in AP(r)-1 cells. RSa cells transfected with sense HSP27 cDNA showed slightly lower sensitivity to UVC-induced cell death than the control cells transfected with a vector alone and much lower sensitivity than RSa cells transfected with the antisense HSP27 cDNA. Furthermore, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4)photoproducts) in the cells with the up-regulation of HSP27 were moderately elevated compared with those in the control cells, while those in the cells with down-regulation were remarkably suppressed. These results suggest that HSP27 is involved in the UVC-resistance of human cells, at least those tested, possibly via functioning in nucleotide excision repair.

    Experimental cell research 2004;298;2;584-92

  • Expression of heat shock protein 27 in human renal cell carcinoma.

    Sarto C, Valsecchi C, Magni F, Tremolada L, Arizzi C, Cordani N, Casellato S, Doro G, Favini P, Perego RA, Raimondo F, Ferrero S, Mocarelli P and Galli-Kienle M

    University Department of Laboratory Medicine, University Milano-Bicocca, Hospital of Desio, Desio, Italy. sarto@uds.unimb.it

    Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 pI range and 18-29 kDa M(r) range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.

    Proteomics 2004;4;8;2252-60

  • Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy.

    Irobi J, Van Impe K, Seeman P, Jordanova A, Dierick I, Verpoorten N, Michalik A, De Vriendt E, Jacobs A, Van Gerwen V, Vennekens K, Mazanec R, Tournev I, Hilton-Jones D, Talbot K, Kremensky I, Van Den Bosch L, Robberecht W, Van Vandekerckhove J, Van Broeckhoven C, Gettemans J, De Jonghe P and Timmerman V

    Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen, Belgium.

    Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein alphaA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.

    Nature genetics 2004;36;6;597-601

  • Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy.

    Evgrafov OV, Mersiyanova I, Irobi J, Van Den Bosch L, Dierick I, Leung CL, Schagina O, Verpoorten N, Van Impe K, Fedotov V, Dadali E, Auer-Grumbach M, Windpassinger C, Wagner K, Mitrovic Z, Hilton-Jones D, Talbot K, Martin JJ, Vasserman N, Tverskaya S, Polyakov A, Liem RK, Gettemans J, Robberecht W, De Jonghe P and Timmerman V

    Department of Psychiatry, New York State Psychiatric Institute/Research Foundation for Mental Hygiene, Unit 28, 1051 Riverside Drive, New York, New York 10032, USA. Evgrafo@pi.cpmc.columbia.edu

    Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11-q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20-alpha-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.

    Nature genetics 2004;36;6;602-6

  • Differential expression of the MAD2, BUB1 and HSP27 genes in Barrett's oesophagus-their association with aneuploidy and neoplastic progression.

    Doak SH, Jenkins GJ, Parry EM, Griffiths AP, Baxter JN and Parry JM

    Human Molecular Pathology Group, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK. s.h.doak@swansea.ac.uk

    Chromosomal instability (CIN) leading to aneuploidy is a ubiquitous and early event in the progression of Barrett's oesophagus, but its origins are unknown. Hence, the transcriptional levels of components of the mitotic spindle checkpoint (important in ensuring precise chromosome segregation) were examined in Barrett's lesions and correlated with the degree of aneuploidy present in the tissues. Gene expression levels of the MAD2 and BUB1 mitotic spindle checkpoint genes were assessed in 37 Barrett's patients (with histology ranging from metaplasia to adenocarcinoma) by real-time RT-PCR. In addition, the transcriptional levels of HSP27 were also examined as firstly, its expression is known to be down regulated in Barrett's metaplasia (BM) and thus was included as a positive control for the real-time RT-PCR assay. While, secondly, the expression pattern of this gene during Barrett's neoplastic progression was investigated, as this has not been previously assessed. Both over and under expression of the MAD2 and BUB1 mitotic spindle checkpoint genes were detected at all Barrett's histological stages with no apparent selective trend with neoplastic progression. In addition, no correlation with aneuploidy was established, indicating an alternative mechanism must underlie Barrett's associated chromosomal instability. HSP27 expression was reduced in metaplasia and then significantly increased with progression. Gender-related differences were observed and HSP27 expression was higher in poorly-differentiated ad 1f40 enocarcinomas than in well-differentiated forms. HSP27 transcriptional patterns therefore present potential as a prognostic tool to predict the aggressiveness of oesophageal adenocarcinomas (OA).

    Mutation research 2004;547;1-2;133-44

  • Stat3 modulates heat shock 27kDa protein expression in breast epithelial cells.

    Song H, Ethier SP, Dziubinski ML and Lin J

    Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Michigan Comprehensive Cancer Center, Cellular and Molecular Biology Graduate Program, Ann Arbor, MI 48109-0936, USA.

    The constitutive activation of signal transducer and activator of transcription 3 (Stat3) is frequently detected in breast carcinoma cell lines but not in normal breast epithelial cells. Stat3 has been classified as an oncogene because activated Stat3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. In this study, we investigated potential Stat3 regulated genes in breast cells. Upon expression of Stat3-C, a constitutively active Stat3 form, in nonmalignant telomerase immortalized breast cells (TERT), cell lysate was subjected to 2-dimensional (2-D) protein gel analysis. Our results showed that heat shock 27kDa protein (HSP27) was markedly induced by Stat3-C expression. Further analysis demonstrated that phosphorylation of HSP27 at serine residue 78 was induced by Stat3-C in TERT breast cells as well as in MCF-10A and MDA-MB-453 breast cells. RT-PCR result confirmed that HSP27 mRNA was induced by Stat3-C in TERT cells. As the result of Stat3 knock-down by Stat3 short interfering RNA oligonucleotides in MDA-MB-468 human breast carcinoma cells, HSP27 was markedly reduced consistent with Stat3 reduction. Furthermore, we observed that Stat3 was physically associated with HSP27 and HSP90 in MDA-MB-468 breast carcinoma cells. Taken together, our findings demonstrate that constitutively activated Stat3 up-regulates HSP27 and may facilitate phosphorylation of HSP27 at serine residue 78. The up-regulation of HSP27 may be one of the underlying mechanisms with which aberrant Stat3 signaling induces cell malignancies.

    Funded by: NCI NIH HHS: CA096714

    Biochemic d7e al and biophysical research communications 2004;314;1;143-50

  • Interaction of human HSP22 (HSPB8) with other small heat shock proteins.

    Sun X, Fontaine JM, Rest JS, Shelden EA and Benndorf R

    Departments of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.

    Funded by: NIEHS NIH HHS: 5R01ES011196-02, P01ES11188

    The Journal of biological chemistry 2004;279;4;2394-402

  • Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance.

    Chauhan D, Li G, Hideshima T, Podar K, Mitsiades C, Mitsiades N, Catley L, Tai YT, Hayashi T, Shringarpure R, Burger R, Munshi N, Ohtake Y, Saxena S and Anderson KC

    Jerome Lipper Multiple Myeloma Center, Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

    Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Heat shock protein 27 (Hsp27) negatively regulates another mitochondrial protein, cytochrome c, during apoptosis; however, the role of Hsp27 in modulating Smac rele 1996 ase is unknown. Here we show that Hsp27 is overexpressed in both dexamethasone (Dex)-resistant multiple myeloma (MM) cell lines (MM.1R, U266, RPMI-8226) and primary patient cells. Blocking Hsp27 by an antisense (AS) strategy restores the apoptotic response to Dex in Dex-resistant MM cells by triggering the release of mitochondrial protein Smac, followed by activation of caspase-9 and caspase-3. Moreover, AS-Hsp27 overcomes interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis. These data demonstrate that Hsp27 inhibits the release of Smac, and thereby confers Dex resistance in MM cells.

    Blood 2003;102;9;3379-86

  • Suppression of thermotolerance in mumps virus-infected cells is caused by lack of HSP27 induction contributed by STAT-1.

    Yokota S, Yokosawa N, Kubota T, Okabayashi T, Arata S and Fujii N

    Department of Microbiology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan.

    Viral infection modulates the regulation of apoptosis in host cells. Here, we report a novel mechanism by which human cells infected with mumps virus become susceptible to apoptosis caused by extracellular stresses. Mumps virus stimulates proteasome-dependent degradation of STAT-1 by action of viral accessory protein V, resulting in a severe decrease in STAT-1 protein in infected cells. We exposed mumps virus-infected and uninfected cells to heat and chemical stress. The infected cells failed to acquire resistance to apoptotic stimuli (thermotolerance) after exposure to these mild stresses. The induction of HSP27 by stress exposure was dramatically suppressed in the infected cells, but HSP70 induction was not affected. STAT-1 was required for transcriptional activation of the HSP27 gene, but not for the HSP70 gene, and cDNA transfection of STAT-1 in mumps virus-infected cells restored thermotolerance. Phosphorylated heat shock factor-1 (HSF-1) and STAT-1 phosphorylated on neither tyrosine nor serine residues were co-transported to the nucleus in response to stress. Furthermore, overexpression of unphosphorylatable mutants of STAT-1 also restored thermotolerance in mumps virus-infected cells. These lines of evidence indicate that the induction of HSP27 by stress requires STAT-1 in addition to the activated HSF-1. Furthermore, STAT-1 required for the induction of HSP27 worked independent to its phosphorylation. Thus, HSP27-dependent thermotolerance is suppressed by mumps virus infection through the destruction of STAT-1. The lack of thermotolerance should allow the infected cells to be eliminated by apoptosis and might be a host defense against viral infection.

    The Journal of biological chemistry 2003;278;43;41654-60

  • HSP27 is a ubiquitin-binding protein involved in I-kappaBalpha proteasomal degradation.

    Parcellier A, Schmitt E, Gurbuxani S, Seigneurin-Berny D, Pance A, Chantôme A, Plenchette S, Khochbin S, Solary E and Garrido C

    INSERM U517, UFR Médecine et Pharmacie, 21033 Dijon, France.

    HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-alpha). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappaB by degrading its main inhibitor, I-kappaBalpha. HSP27 overexpression increases NF-kappaB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin 1beta. HSP27 does not affect I-kappaBalpha phosphorylation but enhances the degradation of phosphorylated I-kappaBalpha by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-kappaBalpha. A protein complex that includes HSP27, phosphorylated I-kappaBalpha, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-kappaBalpha. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-kappaB activity.

    Molecular and cellular biology 2003;23;16;5790-802

  • Heat shock protein 27 gene: chromosomal and molecular location and relationship to Williams syndrome.

    Stock AD, Spallone PA, Dennis TR, Netski D, Morris CA, Mervis CB and Hobart HH

    Department of Pediatrics, Division of Genetics, Laboratory of Molecular Cytogenetics, University of Nevada School of Medicine, Las Vegas 89102, USA.

    Heat shock protein 27 (HSP27) is one of a number of actin-binding proteins that regulate actin polymerization. Three related HSP27 sequences had previously been mapped to chromosomes 3, 9, and X. We have used fluorescent in-situ hybridization (FISH) to correct and refine the map position of the transcribed HSP27 gene (locus HSPB1) to chromosome 7q11.23. This band also contains the site of the deletion associated with Williams syndrome (WS). To define the relationship between HSP27 and the WS deletion, we used two-color FISH on previously G-banded and photographed metaphase chromosomes from WS cell-lines and peripheral blood. Six WS patients with longer deletions that extend telomeric to the classical WS deletion region were analyzed for deletion length using HSP27, cosmids generated from P193O22 (cos11) and B350L10 (cos64 and 82), B350L10, B161A02, and B363M4. The BAC 363M4 was selected from the Washington University database and contains HSP27. Our results indicated that HSP27 was deleted in three patients and that HSP27 is telomeric to cos11, cos64, cos82, and B350L10. B363M4 was demonstrated to overlap the telomeric end of B161A02 and HSP27 may be contained partially within the telomeric end of B161A02. The possible role of HSP27 in the cognitive features of WS is discussed.

    Funded by: NINDS NIH HHS: R01 NS035102, R01-NS35102

    American journal of medical genetics. Part A 2003;120A;3;320-5

  • Heat shock protein 27 controls apoptosis by regulating Akt activation.

    Rane MJ, Pan Y, Singh S, Powell DW, Wu R, Cummins T, Chen Q, McLeish KR and Klein JB

    Department of Medicine, University of Louisville, Louisville, Kentucky 40202-1764, USA.

    Activation of the serine-threonine kinase Akt by cytokines, chemokines, and bacterial products delays constitutive neutrophil apoptosis, resulting in a prolonged inflammatory response. We showed previously that Akt exists in a signaling complex with p38 MAPK, MAPK-activated protein kinase-2 (MAPKAPK-2), and hea 1f40 t shock protein-27 (Hsp27); and Hsp27 dissociates from the complex upon neutrophil activation. To better understand the regulation of this signaling module, the hypothesis that Akt phosphorylation of Hsp27 regulates its interaction with Akt was tested. The present study shows that Akt phosphorylated Hsp27 on Ser-82 in vitro and in intact cells, and phosphorylation of Hsp27 resulted in its dissociation from Akt. Additionally, the interaction between Hsp27 and Akt was necessary for activation of Akt in intact neutrophils. Constitutive neutrophil apoptosis was accelerated by sequestration of Hsp27 from Akt, and this enhanced rate of apoptosis was reversed by introduction of constitutively active recombinant Akt. Our results define a new mechanism by which Hsp27 regulates apoptosis, through control of Akt activity.

    Funded by: NHLBI NIH HHS: HL66358-02

    The Journal of biological chemistry 2003;278;30;27828-35

  • The DNA sequence of human chromosome 7.

    Hillier LW, Fulton RS, Fulton LA, Graves TA, Pepin KH, Wagner-McPherson C, Layman D, Maas J, Jaeger S, Walker R, Wylie K, Sekhon M, Becker MC, O'Laughlin MD, Schaller ME, Fewell GA, Delehaunty KD, Miner TL, Nash WE, Cordes M, Du H, Sun H, Edwards J, Bradshaw-Cordum H, Ali J, Andrews S, Isak A, Vanbrunt A, Nguyen C, Du F, Lamar B, Courtney L, Kalicki J, Ozersky P, Bielicki L, Scott K, Holmes A, Harkins R, Harris A, Strong CM, Hou S, Tomlinson C, Dauphin-Kohlberg S, Kozlowicz-Reilly A, Leonard S, Rohlfing T, Rock SM, Tin-Wollam AM, Abbott A, Minx P, Maupin R, Strowmatt C, Latreille P, Miller N, Johnson D, Murray J, Woessner JP, Wendl MC, Yang SP, Schultz BR, Wallis JW, Spieth J, Bieri TA, Nelson JO, Berkowicz N, Wohldmann PE, Cook LL, Hickenbotham MT, Eldred J, Williams D, Bedell JA, Mardis ER, Clifton SW, Chissoe SL, Marra MA, Raymond C, Haugen E, Gillett W, Zhou Y, James R, Phelps K, Iadanoto S, Bubb K, Simms E, Levy R, Clendenning J, Kaul R, Kent WJ, Furey TS, Baertsch RA, Brent MR, Keibler E, Flicek P, Bork P, Suyama M, Bailey JA, Portnoy ME, Torrents D, Chinwalla AT, Gish WR, Eddy SR, McPherson JD, Olson MV, Eichler EE, Green ED, Waterston RH and Wilson RK

    Genome Sequencing Center, Washington University School of Medicine, Campus Box 8501, 4444 Forest Park Avenue, St Louis, Missouri 63108, USA.

    Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

    Nature 2003;424;6945;157-64

  • 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

  • Hsp27 associates with actin and limits injury in energy depleted renal epithelia.

    Van Why SK, Mann AS, Ardito T, Thulin G, Ferris S, Macleod MA, Kashgarian M and Siegel NJ

    Departments of Pediatrics and Pathology, Yale University School of Medicine, New Haven, Connecticut, USA. svanwhy@mcw.edu

    The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia. LLC-PK1 cells were stably transfected to overexpress human Hsp27 tagged with green fluorescence protein (GFP). Transfected expression of the labeled Hsp27 did not reduce endogenous Hsp25 levels in the cells compared with either nontransfected cells or cells transfected with GFP alone used as the transfectant control (G). By fluorescence energy transfer (FRET) between GFP-tagged Hsp27 and rhodamine ph 1caa alloidin-decorated actin, minimal interaction was found in uninjured control cells. In ATP-depleted cells, Hsp27 was associated closely with F-actin at lateral cell boundaries and with aggregated actin within the cell body. Less Hsp27 interaction with actin was found during recovery; but when adjusted for total phalloidin fluorescence, FRET between Hsp27 and F-actin did not change between 2-h ATP depletion and 4-h recovery. Where Hsp27 association with actin persisted during recovery, it was principally with the residual aggregates of actin in the cell body. Detachment of Na,K-ATPase from the cytoskeleton at 2-h ATP depletion was significantly less in Hsp27 cells compared with transfectant control G cells but not at 4-h ATP depletion. Detachment of ezrin from the cytoskeleton during ATP depletion was nearly complete and was not prevented in the Hsp27 cells. Protection of the Hsp27 cells was not attributable to preservation of cellular ATP levels. Hsp27 appears to have specific actions in renal epithelia subjected to energy depletion, including interacting with actin to preserve architecture in specific intracellular domains.

    Funded by: NICHD NIH HHS: HD 32573; NIDDK NIH HHS: DK 44336

    Journal of the American Society of Nephrology : JASN 2003;14;1;98-106

  • Critical involvement of p38 MAP kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability.

    Garcia JG, Wang P, Schaphorst KL, Becker PM, Borbiev T, Liu F, Birukova A, Jacobs K, Bogatcheva N and Verin AD

    Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. drgarcia@jhmi.edu

    Bordetella pertussis is an important cause of infection in humans worldwide, with full expression of the syndrome associated with characteristic increases in lung permeability and airway edema. The exact cellular mechanisms by which pertussis toxin (PTX) exerts pulmonary toxicity remain unknown, but may involve its ability to ADP-ribosylate-specific G-proteins. We determined that PTX directly and reproducibly reduced lung endothelial and epithelial cell barrier function in vitro and in vivo assessed by decreases in transmonolayer electrical resistance (TER) and isolated perfused lung preparations. Alterations in lung permeability began approximately 30 min after PTX and were dependent on intrinsic ADP-ribosyltransferase activity, as neither the cell binding beta-oligomer subunit or a genetically engineered PTX mutant (devoid of ADP-ribosyltransferase activity) altered TER. PTX-induced barrier dysfunction was associated with mild increases in F-actin stress fiber formation and causally linked to p38 MAP kinase activities. PTX-mediated p38 MAP kinase activation did not involve either p42/p44 ERK, p60src, Rho family of GTPases, or phosphatidylinositol-3' kinase pathways. PTX-mediated decreases in TER were temporally linked to phosphorylation of the actin binding proteins Hsp27 and caldesmon, known substrates for the Ser/Thr kinase MAPKAP2, whose activity is regulated by p38 MAP kinase. In addition to defining novel signaling pathways involved in PTX-induced respiratory pathophysiology, these data suggest that the direct cell-activating effects of PTX be carefully considered as a potential limitation to its use as a tool in signal transduction analysis.

    Funded by: NHLBI NIH HHS: HL 03666, HL 58064, HL 60628

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2002;16;9;1064-76

  • Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress-mediated apoptosis: possible implication in asthma.

    Merendino AM, Paul C, Vignola AM, Costa MA, Melis M, Chiappara G, Izzo V, Bousquet J and Arrigo AP

    Instituto di Medicina Generale e Pneumologia, Università di Palermo, Palermo, Italy.

    Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide-induced apoptosis in human bronchial epithelial 16-HBE cells when they were genetically modified to express reduced levels of Hsp27.

    Cell stress & chaperones 2002;7;3;269-80

  • Subcorneal colocalization of the small heat shock protein, hsp27, with keratins and proteins of the cornified cell envelope.

    Jonak C, Klosner G, Kokesch C, FOdinger D, HOnigsmann H and Trautinger F

    Division of Special and Environmental Dermatology, University of Vienna, Währinger Gürtel 18-20, Austria. constanze.jonak@akh-wien.ac.at

    Background: hsp27 is a member of the small heat shock protein family. Its expression in epidermal keratinocytes in situ and in tissue culture correlates with differentiation. Experimental evidence points to the fact that hsp27 is a molecular chaperone and is involved in the regulation of cell growth and differentiation.

    Objectives: To investigate whether epidermal hsp27 through its chaperone function plays a role in the assembly of keratin filaments and the cornified cell envelope.

    Methods: We performed double staining immunofluorescence and immunogold microscopy on normal human skin (n = 15). We analysed the colocalization of hsp27 with actin, keratins and proteins of the cornified cell envelope (loricrin, filaggrin, transglutaminase 1).

    Results: Actin staining did not reveal detectable colocalization with hsp27. For keratins, transglutaminase, loricrin and filaggrin colocalization was found in more than 60% of the samples. Colocalization was confined to a narrow subcorneal layer with varying patterns of expression. Electron microscopy revealed that loricrin and filaggrin colocalize with hsp27 indirectly through binding to intermediate filaments.

    Conclusions: These results provide morphological evidence that in normal human skin hsp27 might act as a chaperone of cornification. Investigations of the molecular hsp27 interactions with the proteins of the cornified cell envelope are necessary to gain further insight into terminal keratinocyte differentiation and disorders of keratinization.

    The British journal of dermatology 2002;147;1;13-9

  • Differential expression of heat shock protein 27 in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma.

    Leonardi, Pannone G, Magro G, Kudo Y, Takata T and Lo Muzio L

    Department of Medical and Surgical Sciences, II Dental Unit, University of Catania, Catania, Italy.

    The aim of this study was to evaluate HSP27 expression in fetal, normal and inflamed oral mucosal epithelium and in oral premalignant epithelial lesions and in their ensuing invasive cancers. In developing human oral epithelia, immunoreactions for HSP27 were moderately observed in suprabasal keratinocytes of palate and tongue. Normal oral epithelium had an intense suprabasal positivity. In inflamed oral mucosa, HSP27 staining was stronger in basal and suprabasal keratinocytes than in normal epithelium. Most oral premalignant lesions showed no (5 cases, 29%) or low (8 cases, 46.4%) staining. In OSCC both low and high HSP27 levels of expression were observed. HSP27 immunolabelling was down-regulated in poorly differentiated areas and up-regulated in highly differentiated ones. These findings indicated that HSP27 expression seems to protect cells from apoptosis during inflammation, while the down-regulation in dysplasia could impair the protective mechanism against mutagenesis induced by environmental factors and thus enhancing the transformation of oral epithelial dysplasia into OSCCs.

    Oncology reports 2002;9;2;261-6

  • Detection of protein-protein interactions among lens crystallins in a mammalian two-hybrid system assay.

    Fu L and Liang JJ

    Center for Ophthalmic Research, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    alpha-Crystallin consists of two subunits, alphaA and alphaB, and each can form an oligomer by itself or with the other. The aggregation arises from interdomain interactions. However, it is not known whether such interactions also exist among alpha-, beta-, and gamma-crystallins. This heterogeneous crystallin interaction is far weaker than the homogeneous crystallin interaction and is difficult to detect by conventional spectroscopic measurements. We used a mammalian two-hybrid system in this study. The major crystallin components, alphaA-, alphaB-, betaB2-, and gammaC-crystallin genes, were subcloned into the DNA binding domain and transcription activation domain vectors of the two-hybrid system, and they were cotransfected along with a chloramphenicol acetyltransferase (CAT) reporter vector into HeLa cells. Chloramphenicol acetyltransferase activity indicated that there were interactions between alphaA- (or alphaB-) and betaB2- or gammaC-crystallins but with an intensity of one-third that of alphaA-alphaB interactions. Hsp27, a member of the family of the small heat-shock proteins, showed a similar interaction property with alphaB-crystallin. Using the N- and C-terminal domain-truncated mutants, we demonstrated that both domains were important in the alphaA-crystallin self-interaction, but that only the C-terminal domain was important in the alphaB-crystallin self-interaction. These results show that the two-hybrid system can detect interactions among various crystallins and may be used in mapping interaction domains.

    Funded by: NEI NIH HHS: EY05803

    The Journal of biological chemistry 2002;277;6;4255-60

  • Hsp27 as a negative regulator of cytochrome C release.

    Paul C, Manero F, Gonin S, Kretz-Remy C, Virot S and Arrigo AP

    Centre de Génétique Moléculaire et Cellulaire, CNRS-UMR-5534, Université Claude Bernard Lyon I, F-69622 Villeurbanne, France.

    We previously showed that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a manner dependent on level of expression, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and procaspase 3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacts with Hsp27, hence excluding a role of this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular relocalization was altered by changes in Hsp27 level of expression, suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizing agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspase activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway, which induces Bid intracellular redistribution, is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine-treated cells, phalloidin only partially inhibited cytochrome c release and caspase activation. Moreover, in etoposide-treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria and Bid intracellular redistribution in conditions where F-actin was not altered.

    Molecular and cellular biology 2002;22;3;816-34

  • Characterization of proteins associated with heat shock protein hsp27 in the squamous cell carcinoma cell line A431.

    Kindas-Mügge I, Rieder C, Fröhlich I, Micksche M, Trautinger F and Riedler C

    Department of Applied and Exp 1f40 erimental Oncology, Institute of Tumorbiology/Cancer Research, Vienna, Austria. margaret.kindas-muegge@univie.ac.at

    Heat shock protein hsp27 is a molecular chaperone and identification of hsp27-binding proteins might help to elucidate its functional role in keratinocyte biology. In the present investigation we used a human epidermal cell carcinoma cell line (A431) transfected with hsp27 (A431/16) to study interference between hsp27 protein and other proteins. Immunoprecipitation experiments with anti-hsp27 antibody revealed a multicomponent complex when analysed by silver staining. By immunoblotting analysis we could demonstrate that hsp27 associates with actin, the mutant form of p53, hsp70 and hsp90. Immunofluorescence analysis showed a co-localization between hsp27 and p53, hsp70 and hsp90. To control for the specificity of the observed interactions, immuno-precipitations with antibodies to actin, p53, hsp70 and hsp90 respectively, were performed. All of the tested proteins demonstrated a coimmunoprecipitation with hsp27. We conclude that hsp27, like the other heat shock proteins, is part of a complex system of molecular chaperones in epidermal keratinocytes.

    Cell biology international 2002;26;1;109-16

  • Expression of heat-shock protein 25 immunoreactivity in the dental pulp and enamel organ during odontogenesis in the rat molar.

    Ohshima H, Nakakura-Ohshima K and Maeda T

    Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan. histoman@dent.niigata-u.ac.jp

    The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.

    Connective tissue research 2002;43;2-3;220-3

  • Identification and characterization of hic-5/ARA55 as an hsp27 binding protein.

    Jia Y, Ransom RF, Shibanuma M, Liu C, Welsh MJ and Smoyer WE

    Cellular and Molecular Biology Program, Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.

    hsp27 has been reported to participate in a wide variety of activities, including resistance to thermal and metabolic stress, regulation of growth and differentiation, and acting as a molecular chaperone or a regulator of actin polymerization. We hypothesized that these diverse functions are regulated in a cell- or tissue-specific manner via interaction with various binding proteins. To investigate this hypothesis, we used hsp27 as a "bait" 1f40 to screen a yeast two-hybrid cDNA library from rat kidney glomeruli and identified a novel hsp27 binding protein, hic-5 (also known as ARA55), a focal adhesion protein and steroid receptor co-activator. Biochemical interaction between hsp27 and hic-5 was confirmed by co-immunoprecipitation, and critical protein.protein interaction regions were mapped to the hic-5 LIM domains and the hsp27 C-terminal domain. Initial analysis of the functional role of hsp27.hic-5 interaction revealed that hic-5 significantly inhibited the protection against heat-induced cell death conferred by hsp27 overexpression in co-transfected 293T cells. In contrast, when a non-hsp27-interacting hic-5 truncation mutant (hic-5/DeltaLIM4) was co-expressed with hsp27, the hic-5 inhibition of hsp27 protection was absent. We conclude that hic-5 is a true hsp27 binding protein and inhibits the ability of hsp27 to provide protection against heat shock in an interaction-dependent manner.

    Funded by: NIDDK NIH HHS: K08 DK02455-01; NIEHS NIH HHS: ES06265

    The Journal of biological chemistry 2001;276;43;39911-8

  • Pho 1f40 sphatase inhibitors prevent HSP27 dephosphorylation, destruction of stress fibrils, and morphological changes in endothelial cells during ATP depletion.

    Loktionova SA and Kabakov AE

    Laboratory of Cell Adhesion, Institute of Experimental Cardiology, Russian Research-and-Production Cardiology Complex, Russian Ministry of Health, Moscow. calab@cardio.ru

    Pretreatment with phosphatase inhibitors did not affect the decrease in ATP content in endothelial cells, but inhibited HSP27 dephosphorylation and redistribution, damages to actin cytoskeleton, and morphological changes in cells. Our results suggest that inhibition of stress-induced HSP27 dephosphorylation protects cells from ischemia-induced damages.

    Bulletin of experimental biology and medicine 2001;132;3;914-7

  • A new locus for autosomal dominant Charcot-Marie-Tooth disease type 2 (CMT2F) maps to chromosome 7q11-q21.

    Ismailov SM, Fedotov VP, Dadali EL, Polyakov AV, Van Broeckhoven C, Ivanov VI, De Jongh 1f40 e P, Timmerman V and Evgrafov OV

    Russian State Medical University, Moscow, Russia.

    Charcot-Marie-Tooth disease (CMT) constitutes a genetically heterogeneous group of inherited motor and sensory peripheral neuropathies. The axonal type of CMT is designated CMT type 2 (CMT2). Four loci for autosomal dominant CMT2 have been reported so far. Only in CMT2E, linked to chromosome 8p21, disease-causing mutations in the gene for neurofilament light chain (NEFL) were identified. In this study we report a multigenerational Russian family with autosomal dominant CMT2 and assign the locus to chromosome 7q11-q21. The CMT2 neuropathy in this family represents a novel genetic entity designated CMT2F.

    European journal of human genetics : EJHG 2001;9;8;646-50

  • HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27).

    Benndorf R, Sun X, Gilmont RR, Biederman KJ, Molloy MP, Goodmurphy CW, Cheng H, Andrews PC and Welsh MJ

    Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. rbenndo@umich.edu

    Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser(15), Ser(78), and Ser(82)) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as "bait" construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the alpha-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser(14) and Thr(63)) and by p44 mitogen-activated protein kinase (at residues Ser(27) and Thr(87)) but not by MAPKAPK-2.

    Funded by: NCI NIH HHS: R01CA77078-01; NHGRI NIH HHS: R01HG01709-01; NIEHS NIH HHS: ES06265

    The Journal of biological chemistry 2001;276;29;26753-61

  • p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils.

    Rane MJ, Coxon PY, Powell DW, Webster R, Klein JB, Pierce W, Ping P and McLeish KR

    Department of Medicine, University of Louisville Health Sciences Center and the Veterans Affairs Medical Center, Louisville, Kentucky 40202, USA. mrane@louisville.edu

    Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.

    Funded by: NCRR NIH HHS: 1S10RR11368-01A1; NHLBI NIH HHS: HL63901

    The Journal of biological chemistry 2001;276;5;3517-23

  • Protein-protein interactions with subunits of human nuclear RNase P.

    Jiang T and Altman S

    Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA.

    A yeast two-hybrid system was used to analyze interactions among the protein subunits of human nuclear RNase P themselves and with other interacting partners encoded in a HeLa cell cDNA library. Subunits hpop1, Rpp21, Rpp29, Rpp30, Rpp38, and Rpp40 are involved in extensive, but weak, protein-protein interactions in the holoenzyme complex. Rpp14, Rpp20, and Rpp30 were found to have strong interactions with proteins encoded in the cDNA library. The small heat shock protein 27, which interacts with Rpp20 in the two-hybrid assay, binds to Rpp20 during affinity chromatography and can be found to be associated with, and enhances the activity of, highly purified RNase P. RNase P activity in HeLa cell nuclei also increases under the stress of heat shock.

    Funded by: NIGMS NIH HHS: GM19422, R01 GM019422, R37 GM019422

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;3;920-5

  • Inhibition of Daxx-mediated apoptosis by heat shock protein 27.

    Charette SJ, Lavoie JN, Lambert H and Landry J

    Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec, Canada G1R 2J6.

    Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.

    Funded by: Canadian Institutes of Health Research: 7088-4, 7088-5, 7088-6

    Molecular and cellular biology 2000;20;20;7602-12

  • Hsp27 negatively regulates cell death by interacting with cytochrome c.

    Bruey JM, Ducasse C, Bonniaud P, Susin SA, Diaz-Latoud C, Gurbuxani S, Arrigo AP, Kroemer G, Solary E and Garrido C

    INSERM U-517, Faculty of Medicine and Pharmacy, 7 Boulevard Jeanne d'Arc, 21033 Dijon, France.

    Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

    Nature cell biology 2000;2;9;645-52

  • Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27.

    Liu C, Gilmont RR, Benndorf R and Welsh MJ

    Departments of Cell and Developmental Biology and Plastic and Reconstructive Surgery, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function. We hypothesize that hsp27 function depends on specific association with other proteins. To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system. We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1). BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence. In vitro, bacterially expressed glutathione S-transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co-immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines. The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1. Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells. The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1. 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ. Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell.

    Funded by: NCRR NIH HHS: MO1RR00042; NIEHS NIH HHS: ES06265

    The Journal of biological chemistry 2000;275;25;18724-31

  • Chaperone hsp27 inhibits translation during heat shock by binding eIF4G and facilitating dissociation of cap-initiation complexes.

    Cuesta R, Laroia G and Schneider RJ

    Department of Microbiology, New York University School of Medicine and Kaplan Cancer Center, New York, New York 10016 USA.

    Inhibition of protein synthesis during heat shock limits accumulation of unfolded proteins that might damage eukaryotic cells. We demonstrate that chaperone Hsp27 is a heat shock-induced inhibitor of cellular protein synthesis. Translation of most mRNAs requires formation of a cap-binding initiation complex known as eIF4F, consisting of factors eIF4E, eIF4A, eIF4E kinase Mnk1, poly(A)-binding protein, and adaptor protein eIF4G. Hsp27 specifically bound eIF4G during heat shock, preventing assembly of the cap-initiation/eIF4F complex and trapping eIF4G in insoluble heat shock granules. eIF4G is a specific target of Hsp27, as eIF4E, eIF4A, Mnk1, poly(A)-binding protein, eIF4B, and eIF3 were not bound by Hsp27 and were not recruited into insoluble complexes. Dissociation of eIF4F was enhanced during heat shock by ectopic overexpression of Hsp25, the murine homolog of human Hsp27. Overexpression of Hsc70, a constitutive homolog of Hsp70, prevented loss of cap-initiation complexes and maintained eIF4G solubility. Purified Hsp27 specifically bound purified eIF4G in vitro, prevented in vitro translation, eliminated eIF4G interaction with protein binding factors, and promoted eIF4G insolubilization. These results therefore demonstrate that Hsp27 is a heat-induced inhibitor of eIF4F-dependent mRNA translation.

    Funded by: NCI NIH HHS: CA42357, R01 CA042357

    Genes & development 2000;14;12;1460-70

  • Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells.

    Hino M, Kurogi K, Okubo MA, Murata-Hori M and Hosoya H

    Department of Biological Science, Faculty of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan.

    One of the monoclonal antibodies raised against mitotic HeLa cells (termed as mH3) recognized a 27-kDa protein and stained microtubules in the mitotic spindles of HeLa cells. Immunoscreening of a HeLa cDNA library revealed that mH3 antigen is a small heat shock protein, HSP27. Immunoprecipitation analysis using mH3 suggested that both alpha- and beta-tubulin are associated with HSP27. Further, sucrose-cushioned ultra centrifugation revealed that HSP27 is co-sedimented with taxol-stabilized microtubules. These results indicate that HSP27 assoc 1f40 iates with tubulin/microtubules in HeLa cells.

    Biochemical and biophysical research communications 2000;271;1;164-9

  • The interaction of HSP27 with Daxx identifies a potential regulatory role of HSP27 in Fas-induced apoptosis.

    Charette SJ and Landry J

    Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec, Canada G1R 2J6.

    The heat shock protein HSP27 protects cells against a wide variety of toxic treatments and blocks apoptosis induced by exposures to anticancer drugs and activation of the death receptor Fas. The molecular mechanisms of protection are unknown but appear to be regulated by phosphorylation of HSP27. Two apoptotic pathways can be activated downstream of Fas. The Fas-adaptor FADD mediates a caspase-dependent pathway. Fas also activates a caspase-independent pathway which correlates with Fas-induced translocation of Daxx from the nucleus to the cytoplasm and involves the interaction of Daxx with Fas and Ask1. We found that phosphorylated dimers of HSP27 interact with Daxx, preventing its interaction with Ask1 and Fas and blocking Daxx-mediated apoptosis. Expression of HSP27 also prevents the translocation of Daxx from the nucleus to the cytoplasm which is induced upon expression of Ask1 or stimulation of Fas. The observations reveal a new level of regulation of the Fas pathway. Whereas the FADD axis can be modulated by expression of FLIP, a natural inhibitor of FADD, our results show that HSP27 can accomplish a similar function for the Daxx axis.

    Annals of the New York Academy of Sciences 2000;926;126-31

  • Two-dimensional gel analysis of human endometrial proteins: characterization of proteins with increased expression in hyperplasia and adenocarcinoma.

    Byrjalsen I, Mose Larsen P, Fey SJ, Nilas L, Larsen MR and Christiansen C

    Center for Clinical and Basic Research, Ballerup Byvej 222, DK-2750 Ballerup, Denmark.

    In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [(35)S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMP-dependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, alpha enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.

    Molecular human reproduction 1999;5;8;748-56

  • PRAK, a novel protein kinase regulated by the p38 MAP kinase.

    New L, Jiang Y, Zhao M, Liu K, Zhu W, Flood LJ, Kato Y, Parry GC and Han J

    Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

    We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo.

    Funded by: NIAID NIH HHS: AI41637; NIGMS NIH HHS: GM51417

    The EMBO journal 1998;17;12;3372-84

  • Assignment of the mouse Hsp25 and Hsp105 genes to the distal region of chromosome 5 by linkage analysis.

    Hunt CR, Goswami PC and Kozak CA

    Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri 63108, USA. hunt@radonc.wustl.edu

    Funded by: NCI NIH HHS: CA 60757

    Genomics 1997;45;2;462-3

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress.

    Clifton AD, Young PR and Cohen P

    MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Scotland, UK.

    MAPKAP kinase-2 and MAPKAP kinase-3 were both activated in response to cellular stress, interleukin-1 and tumour necrosis factor in KB and HeLa cells, and with identical kinetics. Activation of MAPKAP kinase-3, like MAPKAP kinase-2, was prevented by SB 203580, a specific inhibitor of SAPK-2, the upstream activator of MAPKAP kinase-2. MAPKAP kinase-3 and MAPKAP kinase-2 phosphorylated peptide substrates with similar kinetic constants and phosphorylated the same serine residues in HSP27 at the same relative rates. These results establish that MAPKAP kinase-3 lies 'downstream' of SAPK-2 and that it is likely to have overlapping or identical substrates to MAPKAP kinase-2 in vivo.

    FEBS letters 1996;392;3;209-14

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

    Lavoie JN, Lambert H, Hickey E, Weber LA and Landry J

    Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Canada.

    Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.

    Funded by: NIGMS NIH HHS: GM 43167

    Molecular and cellular biology 1995;15;1;505-16

  • The 28-kDa protein whose phosphorylation is induced by protein kinase C activators in MCF-7 cells belongs to the family of low molecular mass heat shock proteins and is the estrogen-regulated 24-kDa protein.

    Faucher C, Capdevielle J, Canal I, Ferrara P, Mazarguil H, McGuire WL and Darbon JM

    Institut National de la Santé et de la Recherche Médicale, Unité 133, Faculté de Médecine, Toulouse, France.

    We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.

    The Journal of biological chemistry 1993;268;20;15168-73

  • Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

    Stokoe D, Engel K, Campbell DG, Cohen P and Gaestel M

    Department of Biochemistry, University of Dundee, Scotland, UK.

    MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.

    FEBS letters 1992;313;3;307-13

  • 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

  • Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

    Kato K, Shinohara H, Goto S, Inaguma Y, Morishita R and Asano T

    Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.

    Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a co 1f40 lumn of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.

    The Journal of biological chemistry 1992;267;11;7718-25

  • Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II.

    Landry J, Lambert H, Zhou M, Lavoie JN, Hickey E, Weber LA and Anderson CW

    Centre de Recherche en Cancérologie, Université Laval, Québec, Canada.

    The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled pho 1f40 sphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82-Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78-Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress.

    Funded by: NIGMS NIH HHS: GM43167

    The Journal of biological chemistry 1992;267;2;794-803

  • The 29-kDa proteins phosphorylated in thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein.

    Mendelsohn ME, Zhu Y and O'Neill S

    Department of Medicine, Brigham and Women's Hospital, Boston, MA.

    Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. We have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, we isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the "estrogen receptor-related protein" is HSP27, and the three major 29-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.

    Funded by: NHLBI NIH HHS: HL02154

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;24;11212-6

  • cDNA sequence of a human heat shock protein HSP27.

    Carper SW, Rocheleau TA and Storm FK

    Department of Surgery, University of Wisconsin, Madison 53792.

    Nucleic acids research 1990;18;21;6457

  • Identification of two related markers for common acute lymphoblastic leukemia as heat shock proteins.

    Strahler JR, Kuick R, Eckerskorn C, Lottspeich F, Richardson BC, Fo eed x DA, Stoolman LM, Hanson CA, Nichols D, Tueche HJ et al.

    Department of Pediatrics, University of Michigan Medical School, Ann Arbor 48109.

    By direct analysis of the polypeptide constituents of leukemic cells, we have previously detected several polypeptides that are restricted in their expression to acute lymphoblastic leukemia (ALL). In this study, we provide evidence that two polypeptides designated L2 and L4 are structurally related and represent novel markers for common ALL. Partial amino acid sequence analysis did not uncover differences between L2 and L4. The sequences obtained correspond to a previously cloned human gene designated hsp 27 that is expressed, following heat shock treatment, in a variety of cells. 32Pi incorporation studies indicate that L4 is an unphosphorylated form and L2 is a phosphorylated form of hsp27. The two forms were inducible by heat shock in leukemic and nonleukemic lymphoid cells. Thus, in acute leukemia, the common ALL subtype is uniquely characterized by the constitutive expression of a polypeptide that represents a major cellular phosphoprotein.

    Funded by: NCI NIH HHS: CA-26803, CA-32146; NHLBI NIH HHS: HL-16008

    The Journal of clinical investigation 1990;85;1;200-7

  • Induction of the estrogen-regulated "24K" protein by heat shock.

    Fuqua SA, Blum-Salingaros M and McGuire WL

    University of Texas Health Science Center, Department of Medicine/Oncology, San Antonio 78284-7884.

    We have previously demonstrated an estrogen-regulated protein, termed 24K, in human breast cancer cell lines and human tumor biopsies. The presence of the protein correlates well with the presence of steroid hormone receptors. We have cloned a partial complementary DNA to 24K and show that the nucleotide and deduced amino acid sequence contains a striking homology to the low molecular weight heat shock proteins of Drosophila and the mammalian alpha-crystallins. Our complementary DNA is identical to that reported for the 3' region of human heat shock protein 27/28 with the mRNAs for the gene significantly induced by both heat shock and estradiol treatment in MCF-7 breast cancer cells. A variant polymorphism of the gene was detected in two estrogen-unresponsive cell lines, but not in other human breast cancer cell lines or human placenta; the polymorphism did not affect heat shock induction of the gene. The heat shock protein 27/28 model system in MCF-7 cells will afford a valuable tool to analyze the molecular events underlying the hormonal regulation of gene expression; heat shock may offer an approach to manipulate the hormonal regulation of the heat shock protein 27/28 gene.

    Funded by: NCI NIH HHS: CA11378

    Cancer research 1989;49;15;4126-9

  • Sequence and organization of genes encoding the human 27 kDa heat shock protein.

    Hickey E, Brandon SE, Potter R, Stein G, Stein J and Weber LA

    The 27 kDa human heat shock protein (hsp27) is encoded by a gene family of 4 members. Two genomic fragments hybridizing to cDNA encoding hsp27 have been isolated, characterized, and sequenced. One clone is a member of a cluster of three genes linked within a 14-18 kb region of the genome and encodes a transcript interrupted by two intervening sequences. A single open reading frame encodes a polypeptide of 22,300 deduced molecular weight. The 5' flanking region contains two transcription start sites and sequences homologous to the Drosophila consensus heat inducible control element. Induction of both potential transcripts follows heat shock in vivo. Accurate heat inducible transcription occurs at both start sites after injection into Xenopus oocytes. The second genomic clone is a processed pseudogene lacking promoter elements and is unlinked with the other members of the hsp27 gene family. The amino acid sequence of human hsp27 shows striking homology with mammalian alpha crystallin, and contains a region towards the carboxy terminus which shares homology with the small hsp of Drosophila and other organisms.

    Funded by: NIGMS NIH HHS: GM32381

    Nucleic acids research 1986;14;10;4127-45

Gene lists (4)

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
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
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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