Clinical Implications of HSC70 Expression in Clear Cell Renal Cell Carcinoma.

Purpose: The role of heat shock protein 70 (HSC70) in the progression of clear cell renal cell carcinoma (ccRCC) is unclear. This study explored the effect of the HSC70 on the survival of ccRCC patients. Methods: Immunohistochemical analysis was performed to determine HSC70 expression in samples obtained from 121 ccRCC patients with at least 5 years of follow-up. We also analyzed the association between HSC70 expression and clinicopathological characteristics. Furthermore, the association of overall survival (OS) with HSC70 expression was analyzed using Kaplan-Meier curves. Finally, we used the Oncomine and CCLE databases to determine the effects of HSC70 mRNA expression on ccRCC. Results: HSC70 expression was associated with distant metastasis and death of ccRCC patients. HSC70 was expressed in the nucleus and/or cytoplasm of ccRCC cells. The incidence of distant organ metastasis and death was higher in patients with HSC70 expression than in those without it. Survival analysis revealed that patients with HSC70 expression had significantly shorter OS. Oncomine analyses also showed that the HSC70 mRNA was significantly upregulated in ccRCC tissues. Conclusions: HSC70 expression was related to adverse prognosis, and patients with HSC70 expression had a worse prognosis than those without HSC70 expression. HSC70 may thus serve as a potential therapeutic target for ccRCC.

Hsc70 is a Component of Bacterially Generated Actin-Rich Structures: An Immunolocalization Study.

Enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium, and Listeria monocytogenes usurp the actin cytoskeleton for their attachment, internalization and transport within and amongst infected cells. To try to gain a greater understanding of the molecular components utilized by these microbes during their infections we previously concentrated actin-rich structures generated during EPEC infections (called pedestals) and identified the heat shock cognate 70 protein (Hsc70) as a potential candidate. This multifunctional protein classically acts as a chaperone for the proper folding of a variety of proteins and is involved in uncoating clathrin from coated pits. Here we demonstrated that Hsc70 is recruited to actin structures generated during EPEC, Listeria and Salmonella infections, but not to the same location as clathrin. Anat Rec, 301:2095-2102, 2018. © 2018 Wiley Periodicals, Inc.

Mechanisms of intracellular defense and activity of free radical oxidation in rat myocardium in the dynamics of chronic fluorine intoxication.

The mechanisms of intracellular defense and activity of free radical oxidation in the myocardium were studied in the dynamics of chronic fluorine intoxication. At the early stages of fluorine intoxication (day 3-week 3), the concentrations of defense proteins HIF-1α, HSC73, and HOx-2 and activity of the main metabolic enzymes increased, which promoted maintenance of cardiomyocyte structure and function at the normal physiological level. At late stages of fluorine intoxication (weeks 6 and 9), metabolic changes in the myocardium attest to high strain of the adaptive mechanisms.

Comprehensive review on the HSC70 functions, interactions with related molecules and involvement in clinical diseases and therapeutic potential.

Heat shock cognate protein 70 (HSC70) is a constitutively expressed molecular chaperone which belongs to the heat shock protein 70 (HSP70) family. HSC70 shares some of the structural and functional similarity with HSP70. HSC70 also has different properties compared with HSP70 and other heat shock family members. HSC70 performs its full functions by the cooperation of co-chaperones. It interacts with many other molecules as well and regulates various cellular functions. It is also involved in various diseases and may become a biomarker for diagnosis and potential therapeutic targets for design, discovery, and development of novel drugs to treat various diseases. In this article, we provide a comprehensive review on HSC70 from the literatures including the basic general information such as classification, structure and cellular location, genetics and function, as well as its protein association and interaction with other proteins. In addition, we also discussed the relationship of HSC70 and related clinical diseases such as cancer, cardiovascular, neurological, hepatic and many other diseases and possible therapeutic potential and highlight the progress and prospects of research in this field. Understanding the functions of HSC70 and its interaction with other molecules will help us to reveal other novel properties of this protein. Scientists may be able to utilize this protein as a biomarker and therapeutic target to make significant advancement in scientific research and clinical setting in the future.

Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis.

Few studies have focused on the expression of heat shock proteins (HSPs) after chronic heat stress. The objective of this study was to investigate the effect of chronic high temperature-humidity index treatment on the expressions of HSP60, HSP70, HSP90, HSPA2 and HSC70, in the Rex rabbit testis and the expressions of these proteins after recovery from the chronic heat shock. Thirty mature male rabbits of the same age were randomly divided into three groups: control, heat stress, and recovery. The western blot results showed that the expressional levels of HSP60, HSP90, and HSC70 increased significantly and HSPA2 was elevated slightly after a 9-week heat treatment. HSP70 was absent in the control testis and had a high level of expression after heat stress. All of these proteins partially reverted back to normal levels after a 9-week recovery. The immunohistochemical results indicated that the expression patterns of HSP60, HSP90, HSPA2, and HSC70 did not change.

[Screen and identify of differential proteins expressed in the placenta of Down's syndrome].

OBJECTIVE: To discuss protein marks expressed differentially in placenta of Down's syndrome by means of proteomics. METHODS: We collected placenta of 18 patients (from March 2009 to December 2009 at Beijing Obstetrics and Gynecology Hospital), and divided them into two groups, one was 10 patients with fetal Down's syndrome, the other was normal pregnancies (normal chromosome) with other diseases. We separated proteins expressed in placentas of two groups by two-dimensional difference gel electrophoresis (2D-DIGE), and then analyzed the differential protein spots by software Decyder 6.5, then, spots differentially expressed by 1.5 fold or more were analyzed by matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). In the end, the differential expressional levels of partially identified proteins were validated by western blot analysis. RESULTS: (1) Differential proteins of two groups protein spots of placentas separated by 2D-DIGE were analyzed by software Decyder 6.5 (these colored lights scattered in the image were protein spots), a total of 56 spots out of 352 were differentially expressed (P < 0.05) in two groups. We analyzed 17 protein spots (12 protein spots were over-expressed and 5 protein spots were down-expressed) differentially expressed by 1.5 fold or more by MALDI-TOF-MS. (2) Protein matching after searching protein database, 17 protein spots turn out to be 10 proteins. Four kinds [superoxide dismutase 1 (SOD1), peroxiredoxin 6 (PRDX6), heat shock protein 27 (HSP27), endoplasmic reticulum protein 29 (ERP29)] of them were validated by western blot analysis, the group of fetal Down's syndrome were 0.74 ± 0.12, 0.29 ± 0.10, 0.53 ± 0.16, 0.20 ± 0.09, the group of normal pregnancies were 0.51 ± 0.08, 0.34 ± 0.16, 0.18 ± 0.07, 0.35 ± 0.09, the results confirmed the observed changes in proteomics. CONCLUSIONS: Compared with normal pregnancies, there were differential proteins expressed in placenta of Down's syndrome. This approach might provide new screening markers in use for prediction of Down's syndrome, however, further study should be done to make these 4 proteins (SOD1, HSP27, ERP29, PRDX6) be new screening markers.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.

Proteomic analysis of the membrane palmitoylated protein-4 (MPP4)-associated protein complex in the retina.

Membrane palmitoylated protein-4 (MPP4) is a retina-specific scaffolding protein of the membrane-associated guanylate kinase family that has been implicated in organizing presynaptic protein complexes in the photoreceptor ribbon synapse. To isolate the components of this complex we applied a proteomic approach based on immunoaffinity chromatography with a monoclonal anti-MPP4 antibody followed by two-dimensional electrophoresis and mass spectrometry. Among the identified molecules were previously reported proteins of the MPP4 scaffolding complex including adaptor proteins Veli3 and Psd95. Here we demonstrate a selective association between MPP4 and the Psd95-beta isoform that is mediated by interaction of their N-terminal L27 domains. In addition, we have identified recoverin and Hsc70 as novel associated proteins of the MPP4 multiprotein complex in the retina. This study demonstrates the utility of anti-MPP4 antibody precipitation for the elucidation of the MPP4-associated protein complex, which is essential in understanding its precise role in signal transmission at the photoreceptor synapse.

Altered expression of HSPA5, HSPA8 and PARK7 in spinocerebellar ataxia type 17 identified by 2-dimensional fluorescence difference in gel electrophoresis.

BACKGROUND: Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous system and peripheral tissues. The underlying molecular changes of SCA17 are rarely explored. METHODS: To study the molecular mechanisms underlying SCA17, we generated stably induced isogenic 293 cells expressing normal TBP-Q(36) and expanded TBP-Q(61) and analyzed the expressed proteins using two-dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting. RESULTS: Upon induction with doxycycline, the expanded TBP-Q(61) formed aggregates with significant increase in the cell population at subG1 phase and cleaved caspase-3. Proteomics study identified a total of 16 proteins with expression changes greater than 1.5 fold. Among the 16 proteins, PARK7, GLRX3, HNRNPA1, GINS1, ENO1, HNRPK and NPM1 are increased, and SERPINA5, HSPA5, VCL, KHSRP, HSPA8, HNRPH1, IMMT, VCP and HNRNPL are decreased in cells expressing TBP-Q(61) compared with those expressing TBP-Q(36). The altered expression of HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses. CONCLUSIONS: The results illustrate the utility of proteomics to identify alterations of proteins which underlie pathogenesis of SCA17, and may serve as potential therapeutic targets.

Effects of HSPA8, an evolutionarily conserved oviductal protein, on boar and bull spermatozoa.

Previous studies have shown that a soluble protein fraction derived from preparations of apical plasma membrane (APM) of the oviductal epithelium enhances the in vitro survival of mammalian spermatozoa. Here, we show that the survival enhancing property of the soluble protein fraction seems to depend significantly upon heat shock 70 kDa protein 8 (HSPA8 previously known as HSPA10). The following findings in the present study enabled us to draw this conclusion: first, using proteomic analysis, we identified a subset of 70 kDa oviductal surface proteins that bound to spermatozoa, one of which was HSPA8. Second, pre-treatment of the soluble protein fraction with anti-HSPA8 antibody reduced the 24 h (at 39 degrees C) sperm survival enhancement effect normally induced by the presence of 200 microg/ml soluble APM proteins. Third, complementary experiments showed that substituting the soluble protein fraction with bovine recombinant HSPA8 (0.5-2 microg/ml) also elicited the sperm survival effect. Finally, we also tested the effect of bovine recombinant HSPA8 on bull spermatozoa and found similar, dose-responsive, sperm survival promoting effects. The conserved nature of HSPA8 between mammalian species suggests that this protein may represent a common biological mechanism for the maintenance of sperm survival in the oviduct.

Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.

Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.

Modulation of the hippocampal protein machinery in voluntary and treadmill exercising rats.

Information about protein expression studies in the brain of exercising and sedentary animals is limited. Cognitive functions change during exercise and the aim of this study was to investigate rat protein levels of the protein machinery in the hippocampus, the main cognitive brain area for spatial learning and memory, in exercising rats. Protein fluctuations may reflect functional variation during exercise. Male Sprague-Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats undertaking voluntary exercise from 5 months to 23 months and the third undertaking involuntary exercise on a treadmill from 5 months to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification assigning spots to proteins and determination of coomassie-densities was carried out. Heterogeneous nuclear ribonucleoprotein K, one protein variant of heat shock cognate 71 kDa protein and BAG family molecular chaperone regulator 5 showed differential protein levels in the three groups when a p-value of <0.005 was considered as statistically significant thus respecting multiple testing. The biological meaning of changed protein levels in hippocampus under different conditions of exercise is not known but warrants further investigation.

Downregulation of the constitutively expressed Hsc70 in diabetic myocardium is mediated by insulin deficiency.

The 70 kDa heat shock protein family plays important cardiac protective roles against myocardial injuries. Reduced myocardial protection is a common feature of diabetic myocardium. This study was carried out to define the changes in the 70 kDa heat shock protein family in the myocardium in the of streptozotocin-diabetes rats, and to explore the mechanisms through which diabetes alters the abundance of Hsp70/Hsc70 in cardiac muscle. In the diabetic myocardium, the abundance of Hsc70 was significantly reduced. The abundance of Hsp70 was low in cardiac muscle and was not induced in the diabetic myocardium. Unlike Hsp60, Hsp70 and Hsc70 did not augment insulin-like growth factor-I receptor signaling in cardiac muscle cells. In cultured cardiomyocytes, insulin directly increased the abundance of Hsc70, whereas insulin could not modulate Hsp70. Treating diabetic rats with insulin restored myocardial Hsc70 level, but phlorizin treatment failed to restore myocardial Hsc70. These in vivo and in vitro studies showed that downregulation of Hsc70 in diabetic myocardium was secondary to insulin deficiency. Thus, insulin played a major role in maintaining adequate expression of Hsc70 in cardiac muscle.

Proteomic analysis of anti-angiogenic effects by a combined treatment with vinblastine and rapamycin in an endothelial cell line.

Angiogenesis controls the new blood supply routes into the tumor mass via the host endothelial cells (ECs). In this study, the EA.hy926 endothelial cell line has been treated with vinblastine (VBL) and rapamycin (RAP), both separately and in combination at low doses. Recently, we demonstrated the synergistic antiangiogenic effects of a combination of VBL and RAP at very low doses in vitro and in vivo. Herein, we confirm the ability of this combined treatment to statistically inhibit the proliferation of ECs, in a synergistic manner, by inducing apoptosis. The aim of this study was to substantiate these findings at the protein level. Differential proteomic analysis was performed on untreated control cells, treated with VBL, incubated with RAP, or subjected to a drug combination. Differentially expressed 113 polypeptide chains were visualized and 65 were identified via MALDI-TOF analysis. Some of the regulated proteins are involved in the processes of angiogenesis, proliferation, migration, and apoptosis. The down-modulation of ATP synthase, annexin A2, heat shock p70, glucose-6-phosphate dehydrogenase, vasodilator-stimulated phosphoprotein, proteasome 26S, tryptophanyl-tRNA synthetase, and stathmin/OP18, as well as the up-modulation of carbonyl reductase, Rho-GDI, and histone H1.0 correlates with the synergistic antiangiogenic activity of VBL and RAP.

Cell death and expression of heat-shock protein Hsc70 in the hyperthermic rat brain.

Stressful stimuli induce two distinct cellular reactions, the heat-shock (stress) response and programmed cell death. This study utilizes a dual procedure involving immunocytochemistry for heat-shock protein localization and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay for localization of cell death at the cellular level. Whole-body hyperthermia resulted in cell death in the adult rat thymus and testis with a robust signal seen in the testis. Distinct populations of constitutively expressed Hsc70-positive cells and TUNEL-positive cells were apparent. Cellular layers that exhibited high levels of Hsc70 were not triggered into cell death by the thermal stress. High expression of Hsc70 was observed in neuronal populations in the dentate gyrus, CA1 and CA2 region of the hippocampus and Purkinje neurons of the cerebellum. Hyperthermia-induced cell death was not observed in these neuronal cell types, suggesting that neuronal expression of constitutive Hsc70 may play roles in preprotecting neurons from stressful stimuli.

Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification.

Conformational lability of two molecular chaperones Hsc70 and gp96: effects of pH and temperature.

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.

[HSC70 protein of human blood mononuclears as a sign of adaptation under normobaric hypoxia training].

We investigated two isoforms of heat shock protein 70 kDa: HSP70 and HSC70, in the human blood mononuclears under normobaric hypoxia training. It was shown that hypoxia regimen does not lead to manifestation of stress but exerts activation of the organism. The obtained organism adaptation is achieved with a little cost that is confirmed by absence of HSP70 content increase. HSC70 content in the blood mononuclears was increased in most case up to 1.5-2.0 fold. HSC70 displays itself as a sign of adaptation. We connect the increase of HSC70 with mitochondria biogenesis which is given a leading importance under adaptation of aerobic organism cells to hypoxia.

Induction of HSP70 expression and recruitment of HSC70 and HSP70 in the nucleus reduce aggregation of a polyalanine expansion mutant of PABPN1 in HeLa cells.

Nuclear inclusions formed by the aggregation of a polyalanine expansion mutant of the nuclear poly(A)-binding protein (PABPN1) is a hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a dominant autosomal disease in which patients exhibit progressive difficulty of swallowing and eyelid elevation, starting around the age of 50. At present, there is no specific treatment to reduce the aggregate burden in patients. However, in cell culture models of OPMD, reduction of protein aggregation can be achieved by ectopic expression of HSP70. As gene transfer may not be the most effective means to elevate HSP70 levels, we tested four pharmacological agents for their ability to induce HSP70, recruit both HSP70 and HSC70 into the cell nucleus and reduce mutant PABPN1 aggregation in a HeLa cell culture model. We show here that exposure to moderate levels of ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin produced a robust stress response resulting in the induction of HSP70 in HeLa cells expressing the mutant PABPN1 as a green fluorescent protein (GFP) fusion protein. Both HSP70 and the constitutive chaperone HSC70 localized in the nucleus of cells treated with any one of the four agents. This stress response was similar to what was observed following hyperthermia. All four agents also caused a significant reduction in the cellular burden of protein aggregates, as was judged by confocal microscopy and solubility changes of the aggregates. A concomitant reduction of cell death in drug-treated mutant PABPN1 expressing cells was also observed.