70-kDa Heat Shock Protein Family Research Articles

HSPA6 is Correlated With the Malignant Progression and Immune Microenvironment of Gliomas.

Gliomas are primary intracranial space lesions with a high mortality rate. Current treatments for glioma are very limited. Recently, immunotargeted therapy of the glioma microenvironment has been developed. Members of the 70 kDa heat shock protein (HSP70) family are involved in the development of many tumors and immunity. HSPA6 protein belongs to the HSP70 family; However, the biological function of this protein in gliomas has yet to be evaluated. In the present study, a range of analyses, involving protein networks, survival, clinical correlation, and function, revealed that the expression of HSPA6 was negatively correlated with clinical prognosis and closely associated with immunity, invasion, and angiogenesis. Quantitative protein analysis confirmed that HSPA6 was expressed at high levels in patients with glioblastoma. Vitro experiments further verified that HSPA6 enhanced the malignant progression of glioma cells by promoting proliferation, invasion and anti-apoptosis. We also found that HSPA6 was closely correlated with genomic variations and tumor microenvironment. Collectively, we demonstrated that HSPA6 may represent a new therapeutic target to improve the prognosis of patients with gliomas.

Paternal factors in recurrent pregnancy loss: an insight through analysis of non-synonymous single-nucleotide polymorphism in human testis-specific chaperone HSPA2 gene.

Heat shock protein A2 (HSPA2) is a testis-specific molecular chaperone of the 70 kDa heat shock protein (HSP70) family and reported to play a key role in spermatogenesis as well as in the remodelling of the sperm surface during capacitation. It is established that mice lacking HSPA2 gene are infertile and spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. However, its role in post fertilization events is not fully understood. Owing to the importance of HSPA2 in male reproduction, the present study is undertaken to reveal the association between genetic mutation and phenotypic variation in recurrent pregnancy loss (RPL) patients through an in silico prediction analysis. In this study, we used different computational tools and servers such as SIFT, PolyPhen2, PROVEAN, nsSNPAnalyzer, and SNPs & GO to analyse the functional consequences of the nsSNPs in human HSPA2 gene. The most damaging amino acid variants generated were subjected to I-Mutant 2.0 and ConSurf. Post-translational modifications such as phosphorylation mediated by these deleterious nsSNPs were analysed using NetPhos 2.0, and gene-gene interaction study was conducted using GeneMANIA. Finally, in-depth studies of the nsSNPs were studied through Project HOPE. The findings of the study revealed 18 nsSNPs to be deleterious using a combinatorial bioinformatic approach. Further functional analysis suggests that screening of nsSNP variants of HSPA2 that tend to be conserved and has potential to undergo phosphorylation at critical positions (rs764410231, rs200951589, rs756852956) may be useful for predicting outcome in altered reproductive outcome. The physicochemical alterations and its impact on the structural and functional conformity were determined by Project HOPE. Gene-gene interaction depicts its close association with antioxidant enzyme(SOD1) strongly supporting an inefficient oxidative scavenging regulatory mechanism in the spermatozoa of RPL patients as reported earlier. The present study has thus identified high-risk deleterious nsSNPs of HSPA2 gene and would be beneficial in the diagnosis and prognosis of the paternal effects in RPL patients.

Proteome-wide identification of HSP70/HSC70 chaperone clients in human cells.

The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant.

The ATPase domain of members of the 70 kDa heat shock protein (Hsp70) family shows a high degree of sequence, structural, and functional homology across species. A broadly conserved residue within the Hsp70 ATPase domain that captured our attention is an unpaired cysteine, positioned proximal to the site of nucleotide binding. Prior studies of several Hsp70 family members show this cysteine is not required for Hsp70 ATPase activity, yet select amino acid replacements of the cysteine can dramatically alter ATP hydrolysis. Moreover, post-translational modification of the cysteine has been reported to limit ATP hydrolysis for several Hsp70s. To better understand the underlying mechanism for how perturbation of this noncatalytic residue modulates Hsp70 function, we determined the structure for a cysteine-to-tryptophan mutation in the constitutively expressed, mammalian Hsp70 family member Hsc70. Our work reveals that the steric hindrance produced by a cysteine-to-tryptophan mutation disrupts the hydrogen-bond network within the active site, resulting in a loss of proper catalytic magnesium coordination. We propose that a similarly altered active site is likely observed upon post-translational oxidation. We speculate that the subtle changes we detect in the hydrogen-bonding network may relate to the previously reported observation that cysteine oxidation can influence Hsp70 interdomain communication.

Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure-activity relationship studies led to the discovery of compound 30, which exhibits 540 nm affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cell signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers.

Resorcinol-Based Grp94-Selective Inhibitors.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum resident of the 90 kDa heat shock protein (Hsp90) family and represents a promising therapeutic target for the treatment of several diseases. Grp94 is the most unique member of the 90 kDa heat shock protein family due to a five amino acid insertion into its primary sequence, which creates hydrophobic subpockets exclusive to Grp94 that can be utilized for selective inhibition. The first resorcinol-based Grp94-selective inhibitor to take advantage of the hydrophobic S2 subpocket has been developed and shown to manifest low nanomolar affinity and ∼10-fold selectivity for Grp94. Furthermore, these Grp94-selective inhibitors manifest low micromolar GI50 values against multiple myeloma cells, supporting Grp94 as an emerging target for the treatment of this disease.

Heat Shock Protein A2 (HSPA2): Regulatory Roles in Germ Cell Development and Sperm Function.

Among the numerous families of heat shock protein (HSP) that have been implicated in the regulation of reproductive system development and function, those belonging to the 70kDa HSP family have emerged as being indispensable for male fertility. In particular, the testis-enriched heat shock 70kDa protein 2 (HSPA2) has been shown to be critical for the progression of germ cell differentiation during spermatogenesis in the mouse model. Beyond this developmentally important window, mounting evidence has also implicated HSPA2 in the functional transformation of the human sperm cell during their ascent of the female reproductive tract. Specifically, HSPA2 appears to coordinate the remodelling of specialised sperm domains overlying the anterior region of the sperm head compatible with their principle role in oocyte recognition. The fact that levels of the HSPA2 protein in mature spermatozoa tightly correlate with the efficacy of oocyte binding highlight its utility as a powerful prognostic biomarker of male fertility. In this chapter, we consider the unique structural and biochemical characteristics of HSPA2 that enable this heat shock protein to fulfil its prominent roles in orchestrating the morphological differentiation of male germ cells during spermatogenesis as well as their functional transformation during post-testicularsperm maturation.

Testis-enriched heat shock protein A2 (HSPA2): Adaptive advantages of the birds with internal testes over the mammals with testicular descent.

The molecular chaperone heat shock protein A2 (HSPA2), a member of the 70 kDa heat shock protein (HSP70) family, plays an important role in spermatogenesis and male fertility. Although HSPA2 is evolutionarily highly conserved across the metazoan lineages, the observation of striking differences in temperature-sensitive expressions, testicular physiology, spermatogenesis, as well as its role in male fertility indicates that avian and mammalian HSPA2 may exhibit distinct evolutionary trajectory. The present study reports that while mammalian HSPA2 is constrained by intense purifying selection, avian HSPA2 has been subjected to positive selection. The majority of the positively selected amino acid residues fall on the α-helix and β-sheets of the peptide-binding domain located at the carboxyl-terminal region of the avian HSPA2. The detection of positively selected sites at the helix and β-sheets, which are less tolerant to molecular adaptation, indicates an important functional consequence and contribution to the structural and functional diversification of the avian HSPA2. Collectively, avian HSPA2 may have an adaptive advantage over the mammals in response to heat stress, and therefore, mammals with testicular descent may be at a greater risk in the event of scrotal temperature rise.

The regulatory roles of NADPH oxidase, intra- and extra-cellular HSP70in pancreatic islet function, dysfunction and diabetes.

The 70kDa heat-shock protein (HSP70) family is important for a dynamic range of cellular processes that include protection against cell stress, modulation of cell signalling, gene expression, protein synthesis, protein folding and inflammation. Within this family, the inducible 72kDa and the cognate 73kDa forms are found at the highest level. HSP70 has dual functions depending on location. For example, intracellular HSP70 (iHSP70) is anti-inflammatory whereas extracellular HSP70 (eHSP70) has a pro-inflammatory function, resulting in local and systemic inflammation. We have recently identified a divergence in the levels of eHSP70 and iHSP70in subjects with diabetes compared with healthy subjects and also reported that eHSP70 was correlated with insulin resistance and pancreatic β-cell dysfunction/death. In the present review, we describe possible mechanisms by which HSP70 participates in cell function/dysfunction, including the activation of NADPH oxidase isoforms leading to oxidative stress, focusing on the possible role of HSPs and signalling in pancreatic islet α- and β-cell physiological function in health and Type2 diabetes mellitus.

The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition.

One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI), recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2), as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF) success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

Structural Investigation of HSP70-HSP90 and HSP90-TDF Interactions

Tumor Differentiation Factor (TDF) is a pituitary protein, which is secreted into the blood stream and targets breast and prostate. The end effect of TDF on these tissues is differentiation of breast and prostate cells. However, it is not yet clear how TDF induces cell differentiation. Studies in our laboratory determined that the potential TDF receptor candidates are: HSPA8, a member of the 70 kDa heat shock protein family and HSP90 protein. Our previous studies also indicated that TDF may have an inducible receptor, composed of both HSP70 and HSP90, and that TDF signaling depends on the interaction of these proteins. Here we provide additional insights about the proposed interaction between HSP70 and HSP90 and about the HSP90-TDF interaction.

MicroRNAs regulate the chaperone network in cerebral ischemia.

The highly evolutionarily conserved 70 kDa heat shock protein (HSP70) family was first understood for its role in protein folding and response to stress. Subsequently, additional functions have been identified for it in regulation of organelle interaction, of the inflammatory response, and of cell death and survival. Overexpression of HSP70 family members is associated with increased resistance to and improved recovery from cerebral ischemia. MicroRNAs (miRNAs) are important posttranscriptional regulators that interact with multiple target messenger RNAs (mRNA) coordinately regulating target genes, including chaperones. The members of the HSP70 family are now appreciated to work together as networks to facilitate organelle communication and regulate inflammatory signaling and cell survival after cerebral ischemia. This review will focus on the new concept of the role of the chaperone network in the organelle network and its novel regulation by miRNA.

Stress-induced HSP70 from Musca domestica plays a functionally significant role in the immune system

As important molecular chaperones, members of the 70kDa heat shock protein (HSP70) family play essential roles in stress tolerance and innate immunity in organisms. The full-length complementary DNA (cDNA) of a novel inducible HSP70, named as MdHSP70, was isolated from Musca domestica. The cDNA clone consisted of 2411 bp with a 1956 bp open reading frame which encodes 651 amino acids. Using real-time quantitative polymerase chain reaction (qPCR), we investigated the transcriptional profile of the gene under heat shock, cadmium stress and in response to bacteria. Increased expression of MdHSP70 was observed in response to both heat shock and Cd stress. The expression of MdHSP70 was significantly induced by Escherichia coli or Staphylococcus aureus stimulation. Larvae were fed bacteria expressing dsRNA targeting the MdHSP70 gene. Our results showed high mortality in larvae treated with dsRNA of MdHSP70 at heat shock, Cd stress and bacterial invasion, suggesting that MdHSP70 is potentially involved in the stress and immune responses of the house fly and perhaps contributes to protection against cellular injury.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL‐10 dependent pathway

Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen- and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

Basic characterization of 90 kDa heat shock protein genes HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 expressed in Japanese quail (Coturnix japonica)

In the current study, we describe four novel members of the 90 kDa heat shock protein (HSP90) family expressed in Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1, exhibited more than 94% similarity to their related genes in chicken. The putative proteins encoded by these quail genes contained motifs considered essential for HSP90 gene function. In addition, the predicted proteins were more similar to HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 proteins expressed in vertebrates than they were to other members of the HSP90 family. Exon numbers of CjHSP90AA1 (11), CjHSP90AB1 (12) or CjTRAP1 (18) are the same as the chicken and mammalian orthologs. Furthermore, gene order in the regions surrounding CjHSP90AB1 and CjTRAP1 has been preserved, providing evidence that the genomic regions were orthologous to HSP90-containing regions in the chicken genome. The promoter regions of the genes also contained conserved motifs identified in related genes of chicken. However, the nucleotide sequences of the 5'-flanking region of these genes were highly polymorphic. We also found that CjHSP90AA1 exhibited a robust response to heat shock treatment. Taken together, the data suggest that CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1 encode orthologs of HSP90AA1, HSP90AB1, HSP90B1 and TRAP1, respectively.

Bacterial production and functional characterization of the Fab fragment of the murine IgG1/ monoclonal antibody cmHsp70.1, a reagent for tumour diagnostics

Hsp70, the major stress-inducible member of the 70 kDa heat shock protein family, is frequently exposed on the plasma membrane of human tumours and, even more pronounced, on metastases but not detectable on normal tissues. The mouse monoclonal antibody cmHsp70.1, which recognizes a peptide epitope in the C-terminal substrate binding domain of both human and murine Hsp70, provides a promising reagent for the monitoring of Hsp70-positive tumours during cancer therapy. Here, we describe the variable domain sequences of the antibody produced by the hybridoma cell line and attempts to secrete the corresponding recombinant Fab fragment in Escherichia coli. Initially, the yield of soluble functional Fab fragment that could be purified from the periplasmic cell extract was extremely low, even when preparing different chimeric versions with constant domains of human or murine origin or with the light chain constant domain belonging to the kappa or lambda class. Surprisingly, this yield could be raised dramatically by more than a factor 100 in the presence of the folding helper plasmid pTUM4, which overexpresses two periplasmic disulphide oxido-reductases as well as two chaperones with proline-cis/trans-isomerase activity. Thus, more than 15 mg functional recombinant Fab fragment could be purified per litre E.coli culture from a bench top fermenter. This Fab fragment showed high and specific Hsp70 binding activity in ELISA and SPR measurements, revealing a dissociation constant of 35 nM. Notably, the Fab fragment sensitively recognizes the membrane-associated Hsp70 on tumour cell lines both in immunofluorescence microscopy and flow cytometry, thus showing potential for tumour detection in vitro and in vivo.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4+IL-17+, CD4+IFN-γ+ and CD4+Foxp3+ cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4+IFN-γ+ and CD4+IL-17+ T cells, corroborating with higher levels of IFN-γ. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Expression of the 70 kDa Heat shock protein family in Alpine freshwater chironomids (Diptera, Chironomidae) under natural conditions

Chironomidae represent up to 100% of the fauna of Alpine streams. Because they survive stress conditions such as extremely low temperature (annual mean <4 °C), these animals represent a good organism model to analyze the relationship between adaptation to cold and expression of stress proteins such as the 70 kDa Heat shock protein family. Fourth instar larvae of ten species of coldstenothermal chironomids (Pseudodiamesa branickii, Diamesa latitarsis, D. laticauda, D. cinerella, D. insignipes, D. zernyi, D. vaillanti, Orthocladius (O.) frigidus, O. (Euorthocladius) thienemanni and Paratrichocladius nivalis) were collected in a glacier-fed stream in NE Italy at two stations (1300 and 2600 m a.s.l.) and in two seasons (summer 2005 and spring 2006). Immunodetection and quantification of the relative levels of Hsp70 family were performed via Western blot analysis. Significantly different levels of Hsp70 were detected among species. The highest amounts were recorded in P. nivalis and D. insignipes, the lowest in P. branickii. Within the genus Diamesa, lower levels of Hsp70 were observed in the most cold-stenothermal species than in the less coldstenothermal ones. These differences may be explained by different species autoecology. The results provide information on biochemical strategies of alpine midges to face cold temperatures under natural conditions and new insights into their possible response to global warming.

The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

Design, Synthesis, and StructureActivity Relationships for Chimeric Inhibitors of Hsp90

Inhibition of the 90 kDa heat shock protein (Hsp90) family of molecular chaperones represents a promising new chemotherapeutic approach toward the treatment of several cancers. Previous studies have demonstrated that the natural products, radicicol and geldanamycin, are potent inhibitors of the Hsp90 N-terminal ATP binding site. The cocrystal structures of these molecules bound to Hsp90 have been determined, and through molecular modeling and superimposition of these ligands, hybrids of radicicol and geldanamycin have been designed. A series of macrocylic chimeras of radicicol and geldanamycin and the corresponding seco-agents have been prepared and evaluated for both antiproliferative activity and their ability to induce Hsp90-dependent client protein degradation.