G2Cdb::Gene report

Gene id
G00001517
Gene symbol
HSPA9 (HGNC)
Species
Homo sapiens
Description
heat shock 70kDa protein 9 (mortalin)
Orthologue
G00000268 (Mus musculus)

Databases (6)

Gene
ENSG00000113013 (Ensembl human gene)
3313 (Entrez Gene)
614 (G2Cdb plasticity & disease)
Literature
600548 (OMIM)
Marker Symbol
HGNC:5244 (HGNC)
Protein Sequence
P38646 (UniProt)

Synonyms (4)

  • GRP75
  • PBP74
  • mot-2
  • mthsp75

Literature (61)

Pubmed - other

  • Role of Magmas in protein transport and human mitochondria biogenesis.

    Sinha D, Joshi N, Chittoor B, Samji P and D'Silva P

    Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, India.

    Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Deltapam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

    Funded by: Wellcome Trust: WT081643MA

    Human molecular genetics 2010;19;7;1248-62

  • Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity.

    Pilzer D, Saar M, Koya K and Fishelson Z

    Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

    Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

    International journal of cancer 2010;126;6;1428-35

  • Mutational screening of the mortalin gene (HSPA9) in Parkinson's disease.

    De Mena L, Coto E, Sánchez-Ferrero E, Ribacoba R, Guisasola LM, Salvador C, Blázquez M and Alvarez V

    Genética Molecular, Hospital Central de Asturias-Maternidad, 33006, Oviedo, Spain.

    Mortalin is a mitochondrial chaperone of the heat shock protein 70 family. Mortalin plays a central role in mitochondrial biogenesis through its capacity to direct the import of nuclear-encoded proteins into the mitochondria. As mitochondrial dysfunction has been involved in Parkinson's disease (PD), changes in mortalin function and expression could manifest as a higher risk of developing PD. In agreement with this, mortalin expression was decreased in the mitochondrial fraction of neurons from the substantia nigra of PD patients. We hypothesised that DNA variants in the mortalin gene (HSPA9) could contribute to the risk of developing PD. We analysed the 17 HSPA9 coding exons in 330 PD patients and 250 controls. In addition to several polymorphisms, found in patients and controls, three variants were found in 3 patients but none of the controls: two missense (R126 > W and P509 > S) and a 17 bp insertion in intron 8 (predicted to affect RNA splicing). Our study suggests that putative mutations in the mortalin, although rare, could contribute to the risk of developing PD.

    Journal of neural transmission (Vienna, Austria : 1996) 2009;116;10;1289-93

  • From proliferative to neurological role of an hsp70 stress chaperone, mortalin.

    Deocaris CC, Kaul SC and Wadhwa R

    Institute of Health and Sports Science, University of Tsukuba, Ibaraki, 305-8574, Japan.

    Although the brain makes up approximately 2% of a person's body weight, it consumes more than 15% of total cardiac output and has a per capita caloric requirement of 10 times more than the rest of the body. Such continuous metabolic demand that supports the generation of action potentials in neuronal cells relies on the mitochondria, the main organelle for power generation. The phenomenon of mitochondrial biogenesis, although has long been a neglected theme in neurobiology, can be regarded as critical to brain physiology. The present review emphasizes the role of a key molecular player of mitochondrial biogenesis, the mortalin/mthsp70. Brain mortalin is discussed in relation to its aptitude to impact on mitochondrial function and homeostasis, to its interfacing energy metabolic functions with synaptic plasticity, and to its modulation of brain aging via the cellular senescence pathways. Recently, this chaperone has been implicated in Alzheimer's (AD) and Parkinson's (PD) diseases, with proteomic studies consistently identifying oxidatively-damaged mortalin as potential biomarker. Hence, it is possible that mitochondrial dysfunction coincides with the collapse in the mitochondrial chaperone network that aim not only to import, sort and maintain integrity of protein components within the mitochondria, but also to act as buffer to the molecular heterogeneity of damaged and aging mitochondrial proteins within a ROS-rich microenvironment. Inversely, it may also seem that vulnerability to mitochondrial dysfunction could be precipitated by malevolent (anti-chaperone) gain-of-function of a 'sick mortalin'.

    Biogerontology 2008;9;6;391-403

  • Oxidative stress, telomere length and biomarkers of physical aging in a cohort aged 79 years from the 1932 Scottish Mental Survey.

    Starr JM, Shiels PG, Harris SE, Pattie A, Pearce MS, Relton CL and Deary IJ

    MRC Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Royal Victoria Hospital, Edinburgh EH4 2DN, UK. jstarr@staffmail.ed.ac.uk

    Telomere shortening is a biomarker of cellular senescence and is associated with a wide range of age-related disease. Oxidative stress is also associated with physiological aging and several age-related diseases. Non-human studies suggest that variants in oxidative stress genes may contribute to both telomere shortening and biological aging. We sought to test whether oxidative stress-related gene polymorphisms contribute to variance in both telomere length and physical biomarkers of aging in humans. Telomere lengths were calculated for 190 (82 men, 108 women) participants aged 79 years and associations with 384 SNPs, from 141 oxidative stress genes, identified 9 significant SNPS, of which those from 5 genes (GSTZ1, MSRA, NDUFA3, NDUFA8, VIM) had robust associations with physical aging biomarkers, respiratory function or grip strength. Replication of associations in a sample of 318 (120 males, 198 females) participants aged 50 years confirmed significant associations for two of the five SNPs (MSRA rs4841322, p=0.008; NDUFA8 rs6822, p=0.048) on telomere length. These data indicate that oxidative stress genes may be involved in pathways that lead to both telomere shortening and physiological aging in humans. Oxidative stress may explain, at least in part, associations between telomere shortening and physiological aging.

    Funded by: Biotechnology and Biological Sciences Research Council: S18386; Chief Scientist Office: CZB/4/505, ETM/55; Medical Research Council; Wellcome Trust

    Mechanisms of ageing and development 2008;129;12;745-51

  • Identification of GRP75 as an independent favorable prognostic marker of neuroblastoma by a proteomics analysis.

    Hsu WM, Lee H, Juan HF, Shih YY, Wang BJ, Pan CY, Jeng YM, Chang HH, Lu MY, Lin KH, Lai HS, Chen WJ, Tsay YG, Liao YF and Hsieh FJ

    Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, 7 Chung-Shan South Road, Taipei, Taiwan.

    Purpose: Neuroblastoma (NB) is a heterogeneous neoplasm. Detailed biological discrimination is critical for the effective treatment of this disease. Because the tumor behavior of NB is closely associated with the histologic state of differentiation, we thus aimed to identify novel differentiation-associated markers of NB with prognostic implication.

    A human NB cell line SH-SY5Y was used as a model system to explore potential biomarkers for the differentiation of NB by proteomic analyses. Seventy-two NB tumor tissues were subsequently investigated by immunohistochemistry to validate the correlations between the expression of a novel prognostic marker, various clinicopathologic and biological factors, and patient survival.

    Results: Using two-dimensional differential gel electrophoresis, we found a total of 24 spots of proteins in SH-SY5Y cells whose expression was enhanced following differentiation. Glucose-regulated protein 75 (GRP75) was unambiguously identified as one of the five proteins that were dramatically up-regulated following differentiation. Immunohistochemical analyses of 72 NB tumor tissues further revealed that positive GRP75 immunostaining is strongly correlated with differentiated histologies (P < 0.001), mass-screened tumors (P = 0.016), and early clinical stages (P < 0.001) but inversely correlated with MYCN amplification (P = 0.010). Univariate and multivariate survival analyses showed that GRP75 expression is an independent favorable prognostic factor.

    Conclusions: The present findings clearly showed that our proteomics-based novel experimental paradigm could be a powerful tool to uncover novel biomarkers associated with the differentiation of NB. Our data also substantiate an essential role of GRP75 in the differentiation of NB.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;19;6237-45

  • Toward a confocal subcellular atlas of the human proteome.

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

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

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

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

  • The layered structure of human mitochondrial DNA nucleoids.

    Bogenhagen DF, Rousseau D and Burke S

    Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA. dan@pharm.sunysb.edu

    Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.

    Funded by: NIEHS NIH HHS: R01-ES12039

    The Journal of biological chemistry 2008;283;6;3665-75

  • Association of mortalin (HSPA9) with liver cancer metastasis and prediction for early tumor recurrence.

    Yi X, Luk JM, Lee NP, Peng J, Leng X, Guan XY, Lau GK, Beretta L and Fan ST

    Department of Surgery, Peking University People's Hospital, Beijing, China.

    Hepatocellular carcinoma (HCC) is well known for poor prognosis and short survival because of high recurrence rate even after curative surgery. Today there is no available biomarker or biochemical test to indicate HCC recurrence, and this study aims to identify protein markers that can discriminate postoperative patients with early recurrence (ER), i.e. disease relapsed within the first year. In this study, 103 hepatitis B-related HCC patients were recruited, and 68 of them were used for ER-related biomarker discovery study. Proteomic expression patterns of matched tumor and adjacent non-tumor tissues from these patients plus 16 normal liver tissues were delineated by the two-dimensional gel electrophoresis differential profiling method. Significant protein spots were evaluated by hierarchical clustering analysis. SSP4612 that yielded the highest receiver operating characteristic (ROC) curve value for the ER subgroup of HCC was subsequently identified by tandem mass spectrometry, and the corresponding expression patterns were further confirmed by quantitative PCR, Western blot, and immunohistochemistry. Correlation analysis with clinicopathological data was also examined. Proteomic profiling analysis revealed overexpression of mortalin (gene HSPA9) in HCC when compared with the non-tumor and normal liver tissues (area under the curve (AUC) = 0.821). Furthermore, elevated mortalin level was also detected in the ER subgroup of HCC versus the recurrence-free state (where no cancer recurs for >1 year) (AUC = 0.833, sensitivity = 90.9%, specificity = 71.4%). Metastatic HCC cell lines also exhibited higher levels of mortalin and HSPA9 mRNA. Clinically, mortalin overexpression in HCC was closely associated with advanced tumor stages and venous infiltration, having implications for increased malignancy and aggressive behavior. Mortalin (HSPA9) is associated with HCC metastasis and thus suggested as a tumor marker for predicting early recurrence, which may have immediate clinical applications for cancer surveillance after curative surgery.

    Molecular & cellular proteomics : MCP 2008;7;2;315-25

  • Mortalin: a protein associated with progression of Parkinson disease?

    Shi M, Jin J, Wang Y, Beyer RP, Kitsou E, Albin RL, Gearing M, Pan C and Zhang J

    Department of Pathology, University of Washington, School of Medicine, Seattle, Washington, USA.

    Parkinson disease (PD) is a progressive neurodegenerative disorder that is considered to affect the brainstem at its early stages and other brain regions, including the limbic system and isocortex, in advanced stages. It has been suggested that PD progression is characterized pathologically by the spreading of Lewy body deposition. To identify novel proteins involved in PD progression, we prepared subcellular fractions from the frontal cortex of pathologically verified PD patients at different stages of disease and Lewy body deposition and from age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique called isobaric tagging for relative and absolute quantification in conjunction with mass spectrometry. Approximately 200 proteins were found to display significant differences in their relative abundance between PD patients at various stages and controls. Gene ontology analysis indicated that these altered proteins belonged to many categories (e.g. mitochondrial function and neurotransmission) that were likely critically involved in the pathogenesis of PD. Of those, mortalin, a mitochondrial protein, was decreased in the advanced PD cases and was further validated to be decreased using independent techniques. These results suggest a role for mortalin in PD progression.

    Funded by: NIA NIH HHS: AG025688, AG08671, R01AG025327; NICHD NIH HHS: P30HD02274; NIEHS NIH HHS: R01ES012703

    Journal of neuropathology and experimental neurology 2008;67;2

  • Mortalin is regulated by APOE in hippocampus of AD patients and by human APOE in TR mice.

    Osorio C, Sullivan PM, He DN, Mace BE, Ervin JF, Strittmatter WJ and Alzate O

    Neuroproteomics Laboratory, Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA. osorio@neuro.duke.edu

    Mortalin is a chaperone protein associated with cell survival, stress response, intracellular trafficking, control of cell proliferation, mitochondrial biogenesis, and cell fate determination. Human APOE targeted replacement (TR) mice have been used to elucidate the role of APOE4 in Alzheimer's disease (AD), since these animals express the APOE4 gene without the classical pathological signatures of AD. Using proteomics we found that mortalin isoforms are differentially expressed in the hippocampus of APOE4 TR mice compared with the APOE3 (control) TR mice. We also observed that these mortalin isoforms are differentially phosphorylated. Then we studied mortalin expression in patients with AD (genotypes APOE 3/3 and APOE 4/4) compared with patients without AD (genotype APOE 3/3). We observed that mortalin isoforms are also differentially expressed in the hippocampi of patients with AD, and that the expression of these mortalin isoforms is regulated by the APOE genotype. We propose that the differential regulation of mortalin in AD and by the APOE genotype is a cellular defense mechanism responding to increases in oxidative stress.

    Funded by: NIA NIH HHS: P50 AG05128

    Neurobiology of aging 2007;28;12;1853-62

  • Subtractive hybridisation screen identifies genes regulated by glucose deprivation in human neuroblastoma cells.

    Kobayashi K, Xin Y, Ymer SI, Werther GA and Russo VC

    Centre for Hormone Research, Murdoch Children's Research Institute, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, 3052 Parkville, Australia.

    Glucose is the major source of energy for the brain and inadequate glucose supply causes damage of neuronal cells. In this study we employed the human neuroblastoma cell line SH-SY5Y, as an in vitro model for neuronal cells, to identify genes regulated by glucose deprivation. Using subtractive hybridisation screen, validated by Northern analysis, we identify for the first time specific targets of the glucopenic response. These genes are involved in key cellular process including gene transcription, protein synthesis, mitochondrial metabolism, neuronal development, neuroprotection and neuronal apoptosis. Our findings suggest that the fate of neuronal cells undergoing glucose starvation relies on complex gene interactions. Modulation of the expression of these genes in vivo will enable determination of the precise role of each gene and possibly identify key elements and potential therapeutic targets of the glucopenic response.

    Brain research 2007;1170;129-39

  • Mortalin is a novel mediator of erythropoietin signaling.

    Ohtsuka R, Abe Y, Fujii T, Yamamoto M, Nishimura J, Takayanagi R and Muta K

    Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

    Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late-stage erythroid progenitor cells. However, the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanisms by which EPO affects the growth of erythroid progenitor cells, we analyzed proteins obtained from purified human erythroid colony-forming cells (ECFCs) cultured with or without EPO, and one of the proteins apparently related with EPO stimuli was identified as mortalin (mthsp70/PBP74/Grp75/mot-2), which is a member of the heat shock protein 70 family of chaperones. The amount of mortalin mRNA in ECFCs increased in an EPO dose-dependent manner, and ECFC growth was dependent on the amount of mortalin. Furthermore, expression of mortalin in ECFCs was suppressed by a phosphatidylinositol 3-kinase inhibitor. Finally, we analyzed gene expression patterns in ECFCs cultured with or without EPO after treatment with mortalin small interfering RNA (siRNA) using a DNA microarray. When ECFCs treated with mortalin siRNA were cultured with EPO, the expression of several genes overlapped with the profile seen in control ECFCs cultured without EPO. Our data suggest that mortalin is involved in the mediation of EPO signaling and plays an important role in stimulating the growth of erythroid progenitor cells.

    European journal of haematology 2007;79;2;114-25

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

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

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

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

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

    Molecular cell 2007;27;2;262-74

  • A genetic association analysis of cognitive ability and cognitive ageing using 325 markers for 109 genes associated with oxidative stress or cognition.

    Harris SE, Fox H, Wright AF, Hayward C, Starr JM, Whalley LJ and Deary IJ

    Department of Psychology, University of Edinburgh, Edinburgh, UK. Sarah.Harris@hgu.mrc.ac.uk <Sarah.Harris@hgu.mrc.ac.uk&gt;

    Background: Non-pathological cognitive ageing is a distressing condition affecting an increasing number of people in our 'ageing society'. Oxidative stress is hypothesised to have a major role in cellular ageing, including brain ageing.

    Results: Associations between cognitive ageing and 325 single nucleotide polymorphisms (SNPs), located in 109 genes implicated in oxidative stress and/or cognition, were examined in a unique cohort of relatively healthy older people, on whom we have cognitive ability scores at ages 11 and 79 years (LBC1921). SNPs showing a significant positive association were then genotyped in a second cohort for whom we have cognitive ability scores at the ages of 11 and 64 years (ABC1936). An intronic SNP in the APP gene (rs2830102) was significantly associated with cognitive ageing in both LBC1921 and a combined LBC1921/ABC1936 analysis (p < 0.01), but not in ABC1936 alone.

    Conclusion: This study suggests a possible role for APP in normal cognitive ageing, in addition to its role in Alzheimer's disease.

    Funded by: Medical Research Council: MRC_MC_U127561128

    BMC genetics 2007;8;43

  • Physical and functional interaction between mortalin and Mps1 kinase.

    Kanai M, Ma Z, Izumi H, Kim SH, Mattison CP, Winey M and Fukasawa K

    Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.

    Mortalin is a member of Hsp70 chaperoning protein family involved in various cellular functions. Through the search of the kinases that mortalin physically interact with, we identified Mps1 as such a kinase. Mps1 kinase has been implicated in the regulation of centrosome duplication and mitotic checkpoint response. Mortalin binds to Mps1, and is phosphorylated by Mps1 on Thr62 and Ser65. The phosphorylated mortalin then super-activates Mps1 in a feedback manner. Mortalin has been previously shown to localize to centrosomes, and to be involved in the regulation of centrosome duplication. We found that centrosomal localization of mortalin depends on the presence of Mps1. Moreover, Mps1-associated acceleration of centrosome duplication depends on the presence of mortalin and super-activation by the Thr62/Ser65 phosphorylated mortalin.

    Funded by: NCI NIH HHS: CA90522, CA95925

    Genes to cells : devoted to molecular & cellular mechanisms 2007;12;6;797-810

  • Involvement of mortalin in cellular senescence from the perspective of its mitochondrial import, chaperone, and oxidative stress management functions.

    Yaguchi T, Aida S, Kaul SC and Wadhwa R

    Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305 8562, Japan. renu-wadhwa@aist.go.jp

    Mortalin (mtHSP70/GRP75) is a heat uninducible member of hsp70 family of proteins. Some of the established features of mortalin include its various subcellular sites, multiple binding partners, and differential subcellular distribution in normal and immortal cells. Overexpression of mortalin leads to extended life span in nematode and normal human cells. On the other hand, it serves as a major target for oxidation and was shown to be involved in old age pathologies including Parkinson's and Alzheimer's disease. Since mortalin interacts with many proteins, its modifications in response to stress and damage caused by intracellular oxidation are likely to generate pleiotropic effects. For example, (a) inefficient import of mitochondrial proteins by mortalin-Tim complexes may result into inefficient mitochondrial genesis, energy generation, and functional decline and (b) inefficient chaperoning of proteins can result into a garbage catastrophe.

    Annals of the New York Academy of Sciences 2007;1100;306-11

  • Mortalin controls centrosome duplication via modulating centrosomal localization of p53.

    Ma Z, Izumi H, Kanai M, Kabuyama Y, Ahn NG and Fukasawa K

    Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521, USA.

    Abnormal amplification of centrosomes, commonly found in human cancer, is the major cause of mitotic defects and chromosome instability in cancer cells. Like DNA, centrosomes duplicate once in each cell cycle, hence the defect in the mechanism that ensures centrosome duplication to occur once and only once in each cell cycle results in abnormal amplification of centrosomes and mitotic defects. Centrosomes are non-membranous organelles, and undergo dynamic changes in its constituents during the centrosome duplication cycle. Through a comparative mass spectrometric analysis of unduplicated and duplicated centrosomes, we identified mortalin, a member of heat shock protein family, as a protein that associates preferentially with duplicated centrosomes. Further analysis revealed that mortalin localized to centrosomes in late G1 before centrosome duplication, remained at centrosomes during S and G2, and dissociated from centrosomes during mitosis. Overexpression of mortalin overrides the p53-dependent suppression of centrosome duplication, and mortalin-driven centrosome duplication requires physical interaction between mortalin and p53. Moreover, mortalin promotes dissociation of p53 from centrosomes through physical interaction. The p53 mutant that lacks the ability to bind to mortalin remains at centrosomes, and suppresses centrosome duplication in a transactivation function-independent manner. Thus, our present findings not only identify mortalin as an upstream molecule of p53 but also provide evidence for the involvement of centrosomally localized p53 in the regulation of centrosome duplication.

    Funded by: NCI NIH HHS: CA90522, CA95925

    Oncogene 2006;25;39;5377-90

  • Upregulation of mortalin/mthsp70/Grp75 contributes to human carcinogenesis.

    Wadhwa R, Takano S, Kaur K, Deocaris CC, Pereira-Smith OM, Reddel RR and Kaul SC

    Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.

    Mortalin, also known as mthsp70/GRP75/PBP74, interacts with the tumor suppressor protein p53 and inactivates its transcriptional activation and apoptotic functions. Here, we examined the level of mortalin expression in a large variety of tumor tissues, tumor-derived and in vitro immortalized human cells. It was elevated in many human tumors, and in all of the tumor-derived and in vitro immortalized cells. In human embryonic fibroblasts immortalized with an expression plasmid for hTERT, the telomerase catalytic subunit, with or without human papillomavirus E6 and E7 genes, we found that subclones with spontaneously increased mortalin expression levels became anchorage-independent and acquired the ability to form tumors in nude mice. Furthermore, overexpression of mortalin was sufficient to increase the malignancy of breast carcinoma cells. The study demonstrates that upregulation of mortalin contributes significantly to tumorigenesis, and thus is a good candidate target for cancer therapy.

    International journal of cancer 2006;118;12;2973-80

  • Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules.

    Kuwabara H, Yoneda M, Hayasaki H, Nakamura T and Mori H

    Department of Pathology, Osaka Medical College, 2-7, Takatsuki, Osaka 569-8686, Japan. pa2020@art.osaka-med.ac.jp

    The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM.

    Biochemical and biophysical research communications 2006;339;3;971-6

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

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

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

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

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

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

  • Activation of wild type p53 function by its mortalin-binding, cytoplasmically localizing carboxyl terminus peptides.

    Kaul SC, Aida S, Yaguchi T, Kaur K and Wadhwa R

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

    The Hsp70 family member mortalin (mot-2/mthsp70/GRP75) binds to a carboxyl terminus region of the tumor suppressor protein p53. By in vivo co-immunoprecipitation of mot-2 with p53 and its deletion mutants, we earlier mapped the mot-2-binding site of p53 to its carboxyl terminus 312-352 amino acid residues. In the present study we attempted to disrupt mot-2-p53 interactions by overexpression of short p53 carboxyl-terminal peptides. We report that p53 carboxyl-terminal peptides (amino acid residues 312-390, 312-352, 323-390, and 323-352) localize in the cytoplasm, whereas 312-322, 337-390, 337-352, and 352-390 locate mostly in the nucleus. Most interestingly, the cytoplasmically localizing p53 peptides harboring the residues 323-337 activated the endogenous p53 function by displacing it from p53-mortalin complexes and relocating it to the nucleus. Such activation of p53 function was sufficient to cause growth arrest of human osteosarcoma and breast carcinoma cells.

    The Journal of biological chemistry 2005;280;47;39373-9

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

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

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

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

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

    Nature 2005;437;7062;1173-8

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

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

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

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

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

  • 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

  • Nucleolar proteome dynamics.

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

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

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

    Funded by: Wellcome Trust: 073980

    Nature 2005;433;7021;77-83

  • Tid1, the human homologue of a Drosophila tumor suppressor, reduces the malignant activity of ErbB-2 in carcinoma cells.

    Kim SW, Chao TH, Xiang R, Lo JF, Campbell MJ, Fearns C and Lee JD

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

    The ErbB-2/HER-2 receptor tyrosine kinase is overexpressed in a wide range of solid human tumors. The ErbB-2 gene product is a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, and its cytoplasmic domain is responsible for sending the mitogenic signals into cells. We discovered that this domain of ErbB-2 interacts with Tid1 protein, the human counterpart of the Drosophila tumor suppressor Tid56, whose null mutation causes lethal tumorigenesis during the larval stage. Tid1 also is known as a cochaperone of heat shock protein 70 (HSP70) and binds to HSP70 through its conserved DnaJ domain. We found that increased expression of Tid1 in human mammary carcinomas overexpressing ErbB-2 suppresses the expression level of ErbB-2 and attenuates the resultant ErbB-2-dependent oncogenic extracellular signal-regulated kinase 1/2 and big mitogen-activated protein kinase 1 signaling pathways leading to programmed cell death (PCD). A functional DnaJ domain of Tid1 also is required for its inhibition of ErbB-2 expression and the consequent PCD of carcinoma cells resulting from increased Tid1 expression. Importantly, ErbB-2-dependent tumor progression in animals is inhibited by increased expression of Tid1 in tumor cells. Collectively, these results suggest that Tid1 modulates the uncontrolled proliferation of ErbB-2-overexpressing carcinoma cells by reducing ErbB-2 expression and as a result suppresses the ErbB-2-dependent cancerous signaling and tumor progression. Moreover, the cochaperonic and regulatory functions of Tid1 on HSP70 most likely play an essential role in this antitumor function of Tid1 in carcinoma cells.

    Funded by: NCI NIH HHS: CA079871

    Cancer research 2004;64;21;7732-9

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

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

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

    Funded by: PHS HHS: N01-C0-12400

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

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

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

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

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

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

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

    Funded by: NIDDK NIH HHS: DK44239

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

  • A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

    Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B and Superti-Furga G

    Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. tewis.bouwmeester@cellzome.com

    Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

    Nature cell biology 2004;6;2;97-105

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • gp120 neurotoxicity fails to induce heat shock defenses, while the over expression of hsp70 protects against gp120.

    Lim MC, Brooke SM and Sapolsky RM

    Department of Biological Sciences, Stanford University MC 5020, Stanford, CA 94305-5020, USA.

    gp120, the coat glycoprotein of HIV, can damage CNS neurons. This appears to mostly involve an indirect pathway in which gp120 infects microglia, triggering the release of cytokines and glutamatergic excitotoxins which then damage neurons. A well-characterized response of cells to insults is to mobilize the heat stress response, a defense that has a number of protective consequences. We tested the capacity of gp120, at a dose well-documented to be neurotoxic, to activate the heat shock response in cultures from cortex and hippocampus, two brain regions sensitive to the neurotoxic effects of gp120. We found that gp120 failed to induce expression of hsp70, hsp25 or hsp90 in cortical or hippocampal cultures, under conditions where induction can be demonstrated in response to other insults. The failure of gp120 to induce a heat shock response is significant because we subsequently demonstrated that such an induction would have been beneficial. Specifically, over expression of hsp70 with a herpes viral amplicon vector protected cultured hippocampal neurons from gp120 neurotoxicity.

    Funded by: NIMH NIH HHS: R01 MH 53814

    Brain research bulletin 2003;61;2;183-8

  • Identification and purification from the plasma of Type 1 diabetic subjects of a proteolytically active Grp94Evidence that Grp94 is entirely responsible for plasma proteolytic activity.

    Pagetta A, Folda A, Brunati AM and Finotti P

    Department of Pharmacology and Anaesthesiology, University of Padova, Largo E. Meneghetti 2, 35131 Padua, Italy.

    The overall increase in proteolytic activity in diabetes is known to be associated with the development and progression of vascular complications. Our aim was to investigate in detail the molecular nature of this activity in the plasma of Type 1 diabetic subjects.

    Methods: Plasma of both diabetic and control subjects was subjected to various purification procedures (ion exchange and affinity chromatography, HPLC, immunoprecipitation, electrophoresis, immunoblot and mass analyses) to identify the proteins of interest. Biological activities were measured on specific substrates.

    Results: In diabetic but not normal plasma we identified the presence of two heat shock proteins, Grp94 (Glucose-regulated protein94) and HSP70. The higher-than-normal proteolytic activity of Grp94 was: (i) directed against casein, but not against endogenous plasma proteins; (ii) fully and specifically inhibited only by anti-Grp94 polyclonal antibodies; and (iii) coupled with low-level ATPase activity. In addition, ATP binding to Grp94 was able to modulate proteolytic activity. We found that Grp94 in plasma circulates only as high molecular mass homo- and hetero-complexes, the latter mostly formed with IgG to which Grp94 is also linked by tenacious binding. Proteolytically-active Grp94 was purified by immunoprecipitation, which co-immunoprecipitated alpha(1)antitrypsin.

    Our results show the unexpected extracellular location and characteristic biological function of Grp94 even at a late stage of disease. These findings have physiopathological relevance for predicting activation of both autoimmune and inflammatory processes potentially associated with vascular complications.

    Diabetologia 2003;46;7;996-1006

  • Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts.

    Kaul SC, Yaguchi T, Taira K, Reddel RR and Wadhwa R

    National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. s-kaul@aist.go.jp

    The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.

    Experimental cell research 2003;286;1;96-101

  • Mortalin-MPD (mevalonate pyrophosphate decarboxylase) interactions and their role in control of cellular proliferation.

    Wadhwa R, Yaguchi T, Hasan MK, Taira K and Kaul SC

    National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

    Mortalin (mot-2/GRP75/PBP74/mthsp70) is a member of the hsp70 family of proteins and is differentially distributed in normal and immortal cells. It was shown to be involved in pathways to cell senescence and immortalization. To elucidate its functional aspects, a yeast interactive screen for mortalin (mot-2) binding proteins was performed. Mevalonate pyrophosphate decarboxylase (MPD) was identified as one of the mortalin binding partners. The interactions were confirmed in mammalian cells by two-hybrid assay and in vivo coimmunoprecipitation. MPD is known to furnish prenyl groups required for prenylation, protein modification that is essential for the activity of many proteins including p21(Ras) (Ras). We have examined the effect of MPD-mot-2 interactions on the level and activity of p21(Ras) and its downstream effectors, p44 and p42 MAP kinases (ERK1/ERK2), in Ras-Raf pathway. An overexpression of mot-2 resulted in reduced level of Ras and phosphorylated ERK2. These were rescued by co-expression of MPD from an exogenous promoter demonstrating a functional link between mot-2, MPD, and Ras. Ras and its oncogenic forms act as key players in controlling proliferation of normal and cancerous cells. Assigning mot-2 upstream of p21(Ras) offers an important mechanism for influence over cell proliferation.

    Biochemical and biophysical research communications 2003;302;4;735-42

  • 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

  • Mortalin: present and prospective.

    Kaul SC, Taira K, Pereira-Smith OM and Wadhwa R

    Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

    Mortalin, also known as mthsp70/PBP74/GRP75, resides in multiple subcellular sites including mitochondria, ER, plasma membrane, cytoplasmic vesicles and cytosol. It is differentially distributed in normal and cancerous cells; the latter, when reverted back to normal phenotype, also show change in mortalin staining pattern similar to normal cells. Depending on its different subcellular niche and binding partner therein, mortalin is expected to perform multiple functions relevant to cell survival, control of proliferation and stress response.

    Experimental gerontology 2002;37;10-11;1157-64

  • Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex1 and the heat-shock protein PBP74.

    Schwarzer C, Barnikol-Watanabe S, Thinnes FP and Hilschmann N

    Max-Planck-Institute for Experimental Medicine, Department of Immunochemistry, Hermann-Rein Street 3, 37075 Göttingen, Germany.

    The voltage-dependent anion-selective channel 1 (VDAC1), i.e. eukaryotic porin, functions as a channel in membranous structures as described for the outer mitochondrial membrane, the cell membrane, endosomes, caveolae, the sarcoplasmatic reticulum, synaptosomes, and post-synaptic density fraction. The identification of VDAC1 interacting proteins may be a promising approach for better understanding the biological context and function of the channel protein. In this study human VDAC1 was used as a bait protein in a two-hybrid screening, which is based on the Sos recruitment system (SRS). hVDAC1 interacts with the dynein light chain Tctex-1 and the heat-shock protein peptide-binding protein 74 (PBP74)/mitochondrial heat-shock protein 70 (mtHSP70)/glucose-regulated protein 75 (GRP75)/mortalin in vivo. Both interactions were confirmed by overlay-assays using recombinant partner proteins and purified hVDAC1. Indirect immunofluorescence on HeLa cells indicates a co-localisation of hVDAC1 with the dynein light chain and the PBP74. In addition, HeLa cells were transfected transiently with enhanced green fluorescent protein (EGFP)-hVDAC1 fusion proteins, which also clearly co-localise with both proteins. The functional relevance of the identified protein interactions was analysed in planar lipid bilayer (PLB) experiments. In these experiments both recombinant binding partners altered the electrophysiological properties of hVDAC1. While rTctex-1 increases the voltage-dependence of hVDAC1 slightly, the rPBP74 drastically minimises the voltage-dependence, indicating a modulation of channel properties in each case. Since the identified proteins are known to be involved in the transport or processing of proteins, the results of this study represent additional evidence of membrane-associated trafficking of the voltage-dependent anion-selective channel 1.

    The international journal of biochemistry & cell biology 2002;34;9;1059-70

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

    Gurer C, Cimarelli A and Luban J

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

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

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

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

  • Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein.

    Wadhwa R, Yaguchi T, Hasan MK, Mitsui Y, Reddel RR and Kaul SC

    Chugai Research Institute for Medical Sciences, 153-2 Nagai, Niihari Mura, Ibaraki, 300-41, Japan.

    Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.

    Experimental cell research 2002;274;2;246-53

  • Implication of PBP74/mortalin/GRP75 in the radio-adaptive response.

    Carette J, Lehnert S and Chow TY

    Department of Oncology, McGill University, Montréal, Québec, Canada.

    Purpose: To investigate the relationship between expression of the human peptide-binding protein PBP74 and the occurrence of an adaptive response to ionizing radiation.

    Human tumour cell lines HT29 and MCF-7 were transfected with a PBP74 or PBP74 antisense construct. For demonstration of an adaptive response, cells lines were irradiated with a conditioning dose of 0.25 Gy cobalt-60 gamma-rays followed by a second dose of 4.0 Gy after an interval of 4.5 h. Response was measured in terms of clonogenic survival.

    Results: Transfection of a PBP74 plasmid caused transient overexpression of PBP74 mRNA in both cell lines. The optimal dose for the induction of PBP74 in the cell lines investigated was 0.1-0.25Gy and PBP74 induction occurred within 30 min of irradiation. For both cell lines, the adaptive response was repressed when cells were transfected with the anti-PBP plasmid. However, the converse, an enhancement of the adaptive response in cell lines transfected with the PBP74 construct, was seen only for HT29 cells under certain experimental conditions.

    Conclusions: The results support the view that while PBP74 is necessary to the adaptive response, it may not by itself be sufficient for the adaptive response to occur.

    International journal of radiation biology 2002;78;3;183-90

  • Directed proteomic analysis of the human nucleolus.

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

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

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

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

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

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

  • Human mortalin (HSPA9): a candidate for the myeloid leukemia tumor suppressor gene on 5q31.

    Xie H, Hu Z, Chyna B, Horrigan SK and Westbrook CA

    Department of Medicine, University of Illinois at Chicago, 60607-7170, USA.

    Human mortalin (HSPA9) was originally identified by its close homology to murine mortalins, which play important roles in cellular senescence. The two murine genes, mot-1 and mot-2, differ in only two amino acid residues, but have opposite functions in cellular immortalization. HSPA9 was recently localized to chromosome 5, band q31, a region that is frequently deleted in myeloid leukemias and myelodysplasia (MDS), making it a candidate tumor suppressor gene, which is consistent with the biological function of its murine homologue. To evaluate mortalin in this capacity, its expression in normal and leukemic cell lines was investigated, and its genomic structure was determined in order to facilitate mutation detection. RT-PCR and Northern blot analysis revealed a broad distribution in normal tissues and in leukemia cell lines, producing a single 2.8 kb transcript. Genomic characterization showed that the gene spans 18 kb, and consisted of 17 exons with boundaries that were almost identical to its murine counterpart. Using intron-based primers to flank each exon, sequence of the complete protein-coding regions was obtained for three AML cell lines, including two lines with chromosome 5 loss (KG-1 and HL-60) and one without (AML-193) compared to normal DNA. No mutations were identified although one conservative nucleotide sequence variant was observed in exon 16. We have shown that mortalin is highly conserved in genomic structure as well as sequence, and the designed primers will be suitable for future studies to detect mutations in clinical samples.

    Funded by: NCI NIH HHS: P01-CA-75606, R01-CA-72593

    Leukemia 2000;14;12;2128-34

  • Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex.

    Agostini I, Popov S, Li J, Dubrovsky L, Hao T and Bukrinsky M

    The Picower Institute for Medical Research, Manhasset, New York 11030, USA.

    Heat-shock proteins (Hsp's) are a family of molecular chaperones that contribute to protection from environmental stress. In this report, we demonstrate that a member of this family, Hsp70, facilitates nuclear import of HIV-1 preintegration complexes (PICs). The mechanism of this activity appears to be similar to the one used by Vpr, an HIV-1 protein regulating viral nuclear import and replication in macrophages. Indeed Hsp70 stimulated binding of HIV-1 matrix antigen to GST-karyopherin alpha fusion protein and rescued nuclear import of a Vpr-defective HIV-1 strain in vitro. Binding studies with truncated forms of GST-karyopherin alpha demonstrated that both Vpr and Hsp70 bind to a region in the amino-terminal part of the karyopherin alpha molecule. This region appears to be distinct from the binding sites for two other karyopherin alpha cargoes, basic-type NLS-containing proteins and transcription factor STAT-1. Vpr competed with Hsp70 for binding to karyopherin alpha. These results suggest the presence of a novel regulatory site on karyopherin alpha which is used by Hsp70 and Vpr to stimulate interaction between the HIV-1 PIC and karyopherin alpha and thus promote viral nuclear import.

    Funded by: NIAID NIH HHS: R01 AI 33776

    Experimental cell research 2000;259;2;398-403

  • Inactivation of p53 and life span extension of human diploid fibroblasts by mot-2.

    Kaula SC, Reddelb RR, Sugiharac T, Mitsuia Y and Wadhwac R

    National Institute of Bioscience and Human Technology, AIST, Tsukuba, Ibaraki, Japan.

    Normal human lung fibroblasts were transfected with expression plasmids encoding mot-2, an hsp70 family member that is associated with the immortal phenotype. After the empty vector-transfected controls had become senescent and positive for senescence-associated beta-galactosidase (SA-beta-gal), the mot-2-expressing cells continued to proliferate for an additional 12-18 population doublings and showed a young cell morphology and much lower SA-beta-gal activity. The tumor suppressor p53 was found to be transcriptionally inactivated in life span-extended cells. We have thus shown for the first time that overexpression of mot-2 in normal human cells is able to permit their temporary escape from senescence.

    FEBS letters 2000;474;2-3;159-64

  • Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription.

    O'Keeffe B, Fong Y, Chen D, Zhou S and Zhou Q

    Department of Molecular Biology, University of California at Berkeley, Berkeley, California 94720-3206, USA.

    Tat activation of HIV-1 transcription is mediated by human transcription elongation factor P-TEFb, which interacts with Tat and phosphorylates the C-terminal domain of RNA polymerase II. The catalytic subunit of the P-TEFb complex, Cdk9, has been shown to interact with cyclin T and several other proteins of unknown identity. Consequently, the exact subunit composition of active P-TEFb has not been determined. Here we report the affinity purification and identification of the Cdk9-associated proteins. In addition to forming a heterodimer with cyclin T1, Cdk9 interacted with the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperone-Cdk9 complexes. Although the Cdk9/cyclin T1 dimer was exceptionally stable and produced slowly in the cell, free and unprotected Cdk9 appeared to be degraded rapidly. Several lines of evidence indicate the heterodimer of Cdk9/cyclin T1 to be the mature, active form of P-TEFb responsible for phosphorylation of the C-terminal domain of RNA polymerase II interaction with the Tat activation domain, and mediation of Tat activation of HIV-1 transcription. Pharmacological inactivation of Hsp90/Cdc37 function by geldanamycin revealed an essential role for the chaperone-Cdk9 complexes in generation of Cdk9/cyclin T1. Our data suggest a previously unrecognized chaperone-dependent pathway involving the sequential actions of Hsp70 and Hsp90/Cdc37 in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation.

    Funded by: NIAID NIH HHS: AI-41757

    The Journal of biological chemistry 2000;275;1;279-87

  • Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin.

    Mizukoshi E, Suzuki M, Loupatov A, Uruno T, Hayashi H, Misono T, Kaul SC, Wadhwa R and Imamura T

    Biosignaling Department, National Institute of Bioscience and Human Technology, Higashi 1-1,Tsukuba, Ibaraki 305-8566, Japan.

    Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation. In the study reported here, we purified proteins that bind intracellular FGF-1. Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1. One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality. The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system. Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins. These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1.

    The Biochemical journal 1999;343 Pt 2;461-6

  • TID1, a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions.

    Syken J, De-Medina T and Münger K

    Department of Pathology and Harvard Center for Cancer Biology, Harvard Medical School, Boston, MA 02115, USA.

    Mitochondria have emerged as central regulators of apoptosis. Here, we show that TID1, a human homolog of the Drosophila tumor suppressor lethal (2) tumorous imaginal discs, l(2)tid, encodes two mitochondrial matrix proteins, designated hTid-1(L) and hTid-1(S). These splice variants are both highly conserved members of the DnaJ family of proteins, which regulate the activity of and confer substrate specificity to Hsp70 proteins. Both hTid-1(L) and hTid-1(S) coimmunoprecipitate with mitochondrial Hsp70. Expression of hTid-1(L) or hTid-1(S) have no apparent capacity to induce apoptosis but have opposing effects on apoptosis induced by exogenous stimuli. Expression of hTid-1(L) increases apoptosis induced by both the DNA-damaging agent mitomycin c and tumor necrosis factor alpha. This activity is J domain-dependent, because a J domain mutant of hTid-1(L) can dominantly suppress apoptosis. In sharp contrast, expression of hTid-1(S) suppresses apoptosis, whereas expression of a J domain mutant of hTid-1(S) increases apoptosis. Hence, we propose that TID1 gene products act to positively and negatively modulate apoptotic signal transduction or effector structures within the mitochondrial matrix.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;15;8499-504

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • A two-dimensional gel database of human colon carcinoma proteins.

    Ji H, Reid GE, Moritz RL, Eddes JS, Burgess AW and Simpson RJ

    Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Victoria, Australia.

    The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M(r)/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http:(/)/www.ludwig.edu.au.

    Electrophoresis 1997;18;3-4;605-13

  • Mouse and human chromosomal assignments of mortalin, a novel member of the murine hsp70 family of proteins.

    Kaul SC, Wadhwa R, Matsuda Y, Hensler PJ, Pereira-Smith OM, Komatsu Y and Mitsui Y

    National Institute of Bioscience and Human Technology, AIST, Ibaraki, Japan.

    Mortalin has been shown to exhibit differential distributions in cells with mortal and immortal phenotypes. In the present study, we report mot-2 cDNA cloning from RS-4 cells--an immortal clone from CD1-ICR mouse embryonic fibroblasts--and the chromosomal assignments of mortalin related genes to mouse chromosomes 18 and X by fluorescence in situ hybridization. Similar analysis assigned the gene to chromosome 5q31.1 in human.

    Funded by: NIA NIH HHS: P01 AG07123, R 37 AG05333, T32 AG00183

    FEBS letters 1995;361;2-3;269-72

  • The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2.

    Yang T, Kozopas KM and Craig RW

    Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.

    A family of genes related to the bcl-2 protooncogene has recently emerged. One member of this family, mcl-1, was cloned from a human myeloblastic leukemia cell line (ML-1) undergoing differentiation. The intracellular localization of mcl-1, as well as the kinetics of its expression during differentiation, have now been studied. These studies show that the intracellular distribution of mcl-1 overlaps with, but is not identical to, that of bcl-2: mcl-1 is similar to bcl-2 in that the mcl-1 protein has a prominent mitochondrial localization, and in that it associates with membranes through its carboxyl hydrophobic tail. mcl-1 differs from bcl-2, however, in its relative distribution among other (nonmitochondrial/heavy membrane) compartments, mcl-1 also being abundant in the light membrane fraction of immature ML-1 cells while bcl-2 is abundant in the nuclear fraction. Similarly, in differentiating ML-1 cells, the timing of expression of mcl-1 overlaps with, but is not identical to, that of bcl-2: the mcl-1 protein increases rapidly as cells initiate differentiation, and mcl-1 is a labile protein. In contrast, bcl-2 decreases gradually as cells complete differentiation. Overall, the mcl-1 and bcl-2 proteins have some properties in common and others tht are distinct. A burst of expression of mcl-1, prominently associated with mitochondria, complements the continued expression of bcl-2 in ML-1 cells differentiating along the monocyte/macrophage pathway.

    Funded by: NCI NIH HHS: CA54385, CA57359, R01 CA057359

    The Journal of cell biology 1995;128;6;1173-84

  • Cloning and subcellular localization of human mitochondrial hsp70.

    Bhattacharyya T, Karnezis AN, Murphy SP, Hoang T, Freeman BC, Phillips B and Morimoto RI

    Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60201.

    We report the cloning, nucleotide sequence, and localization of mitochondrial hsp70, a member of the human hsp70 multi-gene family. The human mthsp75 gene was cloned by screening an expression library with monoclonal antibody 3A3 that recognizes three members of the human hsp70 family (hsp70, hsc70, and a 75-kDa protein with characteristics identical to that previously established for mitochondrial hsp70). The identity of the 75-kDa protein was confirmed by subcellular fraction of HeLa cells and the demonstration that the 3A3-reactive 75-kDa protein co-fractionates with mitochondrial localized proteins. The nucleotide sequence of the respective cDNA clone revealed an open reading frame of 679 amino acids with extensive sequence identity with members of the human hsp70 family. The derived amino-terminal pre-sequence shares features common to other mitochondrial targeting sequences. The identity of the cDNA was unequivocally established by introduction of an epitope-tag at the carboxyl terminus of the cloned gene, transfection and analysis by immunofluorescence. The tagged 75-kDa protein localizes to mitochondria, thus providing conclusive evidence that it corresponds to the human mitochondrial hsp70, referred to here as mthsp75.

    Funded by: NIGMS NIH HHS: GM38109, GM47150

    The Journal of biological chemistry 1995;270;4;1705-10

  • The addition of 5'-coding information to a 3'-directed cDNA library improves analysis of gene expression.

    Matoba R, Okubo K, Hori N, Fukushima A and Matsubara K

    Institute for Molecular and Cellular Biology, Osaka University, Japan.

    Large-scale sequencing of a 3'-cDNA library permits one to analyse gene expression profiles in various tissues. However, many such sequences lack enough information about the encoded proteins. To overcome this problem, we tested a new library, consisting of a 3'-directed cDNA sequence fused to a to a 5' sequence of about 300 bp. Such 'joint molecules' of about 600 bp were amplified by PCR and directly sequenced. About 40% of these joint molecules included the 5' and 3' terminal portions of the mRNA, and most of the remaining clones contained the middle portion and 3' end of the mRNA. The upstream sequences contained sufficient information with which to search for similarity, ORFs, motifs and hydropathy, thus allowing the mRNAs to be categorized and their functions predicted. The rapid categorization of the cDNAs will help to sort those clones that merit further analysis.

    Gene 1994;146;2;199-207

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Human immunodeficiency virus type 1 interaction with the membrane of CD4+ cells induces the synthesis and nuclear translocation of 70K heat shock protein.

    Furlini G, Vignoli M, Re MC, Gibellini D, Ramazzotti E, Zauli G and La Placa M

    Institute of Microbiology, University of Bologna, St Orsola General Hospital, Italy.

    In the last few years a growing body of experimental evidence has indicated that the interaction of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein (gp120) with the membrane of CD4+ cells may deliver negative signals, eventually leading to programmed cell death (apoptosis) of either mature CD4+ lymphocytes or CD34+ haematopoietic progenitor cells, in the absence of cell infection with HIV-1. However, information on the possible activation of the classical signal transduction pathway through gp120 engagement of cell surface CD4 is contradictory. Heat shock proteins (hsp) or 'stress' proteins' are involved in protecting cells from the deleterious effects of heat and other stresses and perform various cell roles. In mammalian cells there is evidence that hsp70 is involved in the transport of proteins to lysosomes, mitochondria and the nucleus. The results obtained in our study demonstrate that early (3 h) after the exposure of permissive CD4+ cells to HIV-1 (or to purified recombinant gp120) a peak of increased synthesis and nuclear translocation of a 70K hsp (and possibly other proteins) is observed. These data indicate that gp120 possesses the capacity to trigger a cascade of events through a transmembrane signalling activity.

    The Journal of general virology 1994;75 ( Pt 1);193-9

  • Human liver protein map: update 1993.

    Hughes GJ, Frutiger S, Paquet N, Pasquali C, Sanchez JC, Tissot JD, Bairoch A, Appel RD and Hochstrasser DF

    Medical Biochemistry Department, Geneva University.

    This publication updates the reference human liver protein map. By microsequencing, 27 spots or 34 polypeptide chains were identified. The most abundant polypeptides detected on the silver stained liver map were key elements in major hepatic biochemical pathways. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing the 1993 reference human liver SWISS-2DPAGE database. SWISS-2DPAGE and the SWISS-PROT protein sequence databases are closely linked together through the use of common accession numbers.

    Electrophoresis 1993;14;11;1216-22

  • Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family.

    Domanico SZ, DeNagel DC, Dahlseid JN, Green JM and Pierce SK

    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.

    We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen processing and are serologically related to the 70-kDa heat shock protein (hsp70) family. Here we report the cloning and sequencing of the cDNA encoding PBP74 in mice and in humans, accomplished by using amino acid sequence information obtained from the purified protein. We show that PBP74 is highly homologous to members of the hsp70 family but, significantly, is not identical to any known member of this family. Inspection of the cDNA nucleotide sequence indicates that it encodes a 46-residue N-terminal peptide which is not present in the mature protein. Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein. The presequence does not conform to known motifs for organelle-targeting sequences, and at present, its function is not known. By confocal microscopy, PBP74 was localized to cytoplasmic vesicles but not to the nucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74. By RNA filter hybridization analysis, PBP74 mRNAs are detected in all cell types tested. Exposure of cells to heat shock does not result in an increase in the mRNA levels of PBP74, unlike the dramatic increase observed for the stress-inducible hsp70 mRNA. Thus, PBP74 appears to be a constitutive, new member of the hsp70 family.

    Funded by: NHLBI NIH HHS: HL45168; NIAID NIH HHS: AI18939, AI27957

    Molecular and cellular biology 1993;13;6;3598-610

  • Human liver protein map: a reference database established by microsequencing and gel comparison.

    Hochstrasser DF, Frutiger S, Paquet N, Bairoch A, Ravier F, Pasquali C, Sanchez JC, Tissot JD, Bjellqvist B, Vargas R et al.

    Medicine Department, Geneva University, Switzerland.

    This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

    Electrophoresis 1992;13;12;992-1001

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000010 G2C Homo sapiens Human mitochondria Human orthologues of mouse mitochondria adapted from Collins et al (2006) 91
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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