Hsp70 Family Research Articles

Single-Cell Atlas Reveals the Hemocyte Subpopulations and Stress Responses in Asian Giant Softshell Turtle during Hibernation.

Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In the present study, we collected hemocytes from Pelochelys cantorii and compared the molecular signature of these cells between the active state and hibernation period based on single-cell RNA sequencing (scRNA-seq) analysis. We found six cell types and identified a list of new marker genes for each cell subpopulation. Moreover, several heat shock genes, including the Hsp40 family chaperone gene (DNAJ) and HSP temperature-responsive genes (HSPs), were upregulated during the hibernation period, which predicted these genes may play crucial roles in the stress response during hibernation. Additionally, compared to hemocytes in the active state, several upregulated differentially expressed immune-related genes, such as stat1, traf3, and socs6, were identified in hemocytes during the hibernation period, thus indicating the important immune function of hemocytes. Therefore, our findings provide a unified classification of P. cantorii hemocytes and identify the genes related to the stress response, thereby providing a better understanding of the adaptive mechanisms of hibernation.

Expression of Hsp70 in the red swamp crayfish, Procambarus clarkii in response to immune challenge

A heat shock protein 70 (Hsp70) was identified from Procambarus clarkii (named as PcHsp70). The open reading frame of the PcHsp70 cDNA was 1917 bp, encoding a 638 aa protein with signature sequences of the Hsp70 family. Tissue distribution results showed that PcHsp70 has relatively high expression in immune-related tissues such as hepatopancreas, gill, and hemocytes. The expressions of PcHsp70 in hepatopancreas and gill were significantly induced by Gram-positive bacterium, Gram-negative bacterium, and white spot syndrome virus stimulation, indicating that PcHsp70 may be involved in the immune response to pathogen invasion. The research will provide a foundation for understanding the resistance and stress mechanism of Hsp70 in P. clarkii.

Computational analysis of heat shock protein (HSP) in Citrus X Sinensis

Heat shock proteins (HSPs) are molecular chaperones and one of the cell’s most important regulatory proteins present in all species. HSPs are a multigene family classified into six families according to their molecular weight range between 8KDa to 110KDa: HSP100, HSP90, HSP70, HSP60, small heat shock proteins (sHSPs), and ubiquitin. Citrus X Sinensis is very economical, and widely grown in the world. It is one of the best sources of nutrition, vitamin C, fiber, thiamine, folate, and antioxidants. It has a variety of health benefits i.e., protects against cell damage, boosts the immune system, heals wounds, etc. For highlighting the aspect of HSPs of citrus in computing protein, this work has great significance. Present studies are based on the identification and characterization of heat shock proteins in Citrus sinuses. In silico research was performed; Arabidopsis thaliana protein sequences of HSP60, HSP70 and HSP90 were used as a reference to identify these molecular chaperones in Citrus sinuses through Blast Phytozome. Furthermore, physiochemical properties, multiple sequence alignment, Multiple Expectation Maximization for Motif Elicitation (MEME) and phylogeny were constructed for 44 sequences of HSP60, 22 sequences of HSP70 and 23 for HSP90. The best expression of HSPs was found in Endoplasmic Reticulum. The findings of the present studies provide a good understanding of the HSP60, HSP70 and HSP90 families' function and expression site in the cell of Citrus sinuses. It also provides the basic knowledge for further investigations of the putative structure of dynamic proteins.

DnaJ, a heat shock protein 40 family member, is essential for the survival and virulence of plant pathogenic Pseudomonas cichorii JBC1

Bacterial plant pathogens must cope with various environmental conditions and defenses from their hosts for colonization and infection. Heat shock proteins (HSPs) play critical roles in a variety of cellular processes, such as the maintenance of cellular homeostasis in response to environmental stress. However, the significance of HSP40 family protein DnaJ in virulence of plant pathogenic bacteria has not yet been explored. To elucidate the function of DnaJ in Pseudomonas cichorii JBC1 (PcJBC1) virulence, we generated dnaJ-deficient (JBC1ΔdnaJ) mutant using CRISPR-CAS9. The disease severity by JBC1ΔdnaJ was significantly reduced compared with wild-type (WT) and dnaJ-complemented (JBC1ΔdnaJ + pdnaJ) strain. The defect of DnaJ suppressed siderophore production, extracellular DNA (eDNA) release, biofilm formation, and swarming motility and made the strain sensitive to stresses such as heat and H2O2. The supplementation of eDNA recovered the amount of biofilm formation by JBC1ΔdnaJ. Our results indicate that DnaJ is a key player in the survival and colonization of bacterial plant pathogens on plant surfaces as well as bacterial responses to abiotic and biotic stresses, which are determinative to cause disease. These findings can broaden our understanding of plant and bacterial pathogen interactions.

HSPA4 regulated glioma progression via activation of AKT signaling pathway.

Glioma is still an incurable disease with high invasiveness. Heat shock 70kDa protein 4 (HSPA4) is a member of the HSP110 family, and is associated with the development and progression of various cancers. In the current study, we assessed the expression of HSPA4 in clinical samples, and found that HSPA4 was up-regulated in glioma tissues and correlated with tumor recurrence and grade. Survival analyses demonstrated that glioma patients with high HSPA4 expression had lower overall survival and disease-free survival times. In vitro knockdown of HSPA4 inhibited glioma cell proliferation, mediated cell cycle arrest at G2 phase and apoptosis, and reduced the migration ability. In vivo, the growth of HSPA4-knockdown xenografts was markedly suppressed compared to the tumors formed by HSPA4-positive control cells. Additionally, Gene set enrichment analyses disclosed that HSPA4 was associated with the PI3K/Akt signaling pathway. The regulatory effect of the AKT activator SC79 on cell proliferation and apoptosis was suppressed by HSPA4 knockdown, indicating that HSPA4 is capable of promoting glioma development. In summary, these data showed that HSPA4 is likely to play a pivotal role in the progression of glioma, and consequently may be a promising therapeutic target for glioma therapy.

HSP90: A potential drug target in inflammatory skin diseases

AbstractHeat shock protein 90 (HSP90) is a chaperone protein belonging to the HSP family. HSP90 modulates clients by stabilising, folding, maturing, and tagging clients for proteasomal degradation. Several clients of HSP90 are involved in cancer development and HSP90 is required for some oncoproteins, making HSP90 a potential target in anticancer treatment. Recently HSP90 has also been suggested as a potential drug target in inflammatory skin diseases based on both in vitro and in vivo studies in animal models and human clinical trials. The role of HSP90 in blistering skin diseases has been studied in vitro, ex vivo, and in vivo using animal models. Human clinical studies investigating HSP90 inhibition in psoriasis and hidradenitis suppurativa have either been conducted or are ongoing and in vitro and ex vivo studies have investigated the role of HSP90 in systemic lupus erythematosus and diffuse cutaneous systemic sclerosis. Here, we review the current literature investigating the role of HSP90 in these diseases.

RNA-Seq Transcriptome Analysis and Evolution of OsEBS, a Gene Involved in Enhanced Spikelet Number per Panicle in Rice.

Spikelet number per panicle (SNP) is one of the most important yield components in rice. Rice ENHANCING BIOMASS AND SPIKELET NUMBER (OsEBS), a gene involved in improved SNP and yield, has been cloned from an accession of Dongxiang wild rice. However, the mechanism of OsEBS increasing rice SNP is poorly understood. In this study, the RNA-Seq technology was used to analyze the transcriptome of wildtype Guichao 2 and OsEBS over-expression line B102 at the heading stage, and analysis of the evolution of OsEBS was also conducted. A total of 5369 differentially expressed genes (DEGs) were identified between Guichao2 and B102, most of which were down-regulated in B102. Analysis of the expression of endogenous hormone-related genes revealed that 63 auxin-related genes were significantly down-regulated in B102. Gene Ontogeny (GO) enrichment analysis showed that the 63 DEGs were mainly enriched in eight GO terms, including auxin-activated signaling pathway, auxin polar transport, auxin transport, basipetal auxin transport, and amino acid transmembrane transport, most of which were directly or indirectly related to polar auxin transport. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis further verified that the down-regulated genes related to polar auxin transport had important effects on increased SNP. Analysis of the evolution of OsEBS found that OsEBS was involved in the differentiation of indica and japonica, and the differentiation of OsEBS supported the multi-origin model of rice domestication. Indica (XI) subspecies harbored higher nucleotide diversity than japonica (GJ) subspecies in the OsEBS region, and XI experienced strong balancing selection during evolution, while selection in GJ was neutral. The degree of genetic differentiation between GJ and Bas subspecies was the smallest, while it was the highest between GJ and Aus. Phylogenetic analysis of the Hsp70 family in O. sativa, Brachypodium distachyon, and Arabidopsis thaliana indicated that changes in the sequences of OsEBS were accelerated during evolution. Accelerated evolution and domain loss in OsEBS resulted in neofunctionalization. The results obtained from this study provide an important theoretical basis for high-yield rice breeding.

The roles of molecular chaperones in regulating cell metabolism.

Fluctuations in nutrient and biomass availability, often as a result of disease, impart metabolic challenges that must be overcome in order to sustain cell survival and promote proliferation. Cells adapt to these environmental changes and stresses by adjusting their metabolic networks through a series of regulatory mechanisms. Our understanding of these rewiring events has largely been focused on those genetic transformations that alter protein expression and the biochemical mechanisms that change protein behavior, such as post-translational modifications and metabolite-based allosteric modulators. Mounting evidence suggests that a class of proteome surveillance proteins called molecular chaperones also can influence metabolic processes. Here, we summarize several ways the Hsp90 and Hsp70 chaperone families act on human metabolic enzymes and their supramolecular assemblies to change enzymatic activities and metabolite flux. We further highlight how these chaperones can assist in the translocation and degradation of metabolic enzymes. Collectively, these studies provide a new view for how metabolic processes are regulated to meet cellular demand and inspire new avenues for therapeutic intervention.

Albumosomes formed by cytoplasmic pre-folding albumin maintain mitochondrial homeostasis and inhibit nonalcoholic fatty liver disease

Hepatic mitochondrial dysfunction contributes to the progression of nonalcoholic fatty liver disease (NAFLD). However, the factors that maintain mitochondrial homeostasis, especially in hepatocytes, are largely unknown. Hepatocytes synthesize various high-level plasma proteins, among which albumin is most abundant. In this study, we found that pre-folding albumin in the cytoplasm is completely different from folded albumin in the serum. Mechanistically, endogenous pre-folding albumin undergoes phase transition in the cytoplasm to form a shell-like spherical structure, which we call the “albumosome”. Albumosomes interact with and trap pre-folding carnitine palmitoyltransferase 2 (CPT2) in the cytoplasm. Albumosomes control the excessive sorting of CPT2 to the mitochondria under high-fat-diet-induced stress conditions; in this way, albumosomes maintain mitochondrial homeostasis from exhaustion. Physiologically, albumosomes accumulate in hepatocytes during murine aging and protect the livers of aged mice from mitochondrial damage and fat deposition. Morphologically, mature albumosomes have a mean diameter of 4μm and are surrounded by heat shock protein Hsp90 and Hsp70 family proteins, forming a larger shell. The Hsp90 inhibitor 17-AAG promotes hepatic albumosomal accumulation in vitro and in vivo, through which suppressing the progression of NAFLD in mice.

Integrated transcriptome and proteome analysis reveals molecular responses of soybean anther under high-temperature stress.

It is well documented that high temperature (HT) severely affects the development of soybean male reproductive organs. However, the molecular mechanism of thermo-tolerance in soybean remains unclear. To explore the candidate genes and regulatory mechanism of soybean response to HT stress and flower development, here, the anthers of two previously identified HT-tolerant (JD21) and HT-sensitive (HD14) varieties were analyzed by RNA-seq. In total, 219 (172 upregulated and 47 downregulated), 660 (405 upregulated and 255 downregulated), and 4,854 (2,662 upregulated and 2,192 downregulated) differentially expressed genes (DEGs) were identified between JD21 anthers treated with HT stress vs. JD21 anthers in the natural field conditions (TJA vs. CJA), HD14 anthers treated with HT stress vs. HD14 anthers in the natural field conditions (THA vs. CHA), and JD21 vs. HD14 anthers treated with HT stress (TJA vs. THA), respectively. The results showed that there were more DEGs upregulated in JD21; this might be the reason why JD21 was more HT-resistant than the HT-sensitive variety HD14. GO annotation and KEGG enriched analysis showed that many DEGs are mainly involved in defense response, response to biological stimuli, auxin-activated signaling pathway, plant hormone signal transduction, MAPK signaling pathway-plant, starch and sucrose metabolism, etc. The conjoint analysis of RNA-seq and previous iTRAQ results found that there were 1, 24, and 54 common DEGs/DAPs showing the same expression pattern and 1, 2, and 13 common DEGs/DAPs showing the opposite pattern between TJA vs. CJA, THA vs. CHA, and TJA vs. THA at the protein and gene level, respectively, among which HSPs, transcription factor, GSTU, and other DEGs/DAPs participated in the response to HT stress and flower development. Notably, the qRT-PCR analysis and physiological index change results coincided with the sequencing results of RNA-seq and iTRAQ. In conclusion, the HT-tolerant cultivar performed better under stress than the HT-sensitive cultivar through modulation of HSP family proteins and transcription factors, and by keeping key metabolic pathways such as plant hormone signal transduction normal. This study provided important data and some key candidate genes to better study the effect and molecular basis of HT on anther in soybean at a transcription and translation level.

Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

Effects of heat stress on physiological parameters, biochemical parameters and expression of heat stress protein gene in Lateolabraxmaculatus

With global warming and the increasing frequency of extreme heat, we have to consider the heat tolerance of fish to sudden high temperatures. This study investigated the effects of high temperature (32 °C) on physiology and biochemistry and heat shock proteins (HSPs) gene of spotted sea bass (Lateolabrax maculatus). The spotted sea bass (14.7 ± 1.54 g) temporarily cultured at 26 °C was directly transferred to the 32 °C high temperature group, and the gill morphology, liver antioxidant activity, respiratory metabolism related enzyme activity and the expression of five HSP70 family members genes were measured at 3, 6, 9, 12, 24, 48, 72, and 96 h. The results showed that 32 °C had damage effect on gill tissue and antioxidant system, and the damage degree increased with high temperature. Respiratory rate and malondialdehyde increased gradually with the continuous heat stress. Superoxide dismutase and total antioxidant capacity increased briefly and then decreased continuously. Succinate dehydrogenase decreased to the lowest value at 24 h and then continued to increase. Lactate dehydrogenase decreased continuously; the expression of HSP70 increased rapidly and then decreased. These results indicated that the antioxidant system and HSP70 were activated under heat stress condition and provided protection to the body, but with the continuous high temperature, the protective effect was limited, and the fish body was irreversibly damaged. It is necessary to pay close attention to the temperature change in the production practice of spotted sea bass to reduce the influence caused by high temperature.

A first-in-class inhibitor of Hsp110 molecular chaperones of pathogenic fungi

Proteins of the Hsp110 family are molecular chaperones that play important roles in protein homeostasis in eukaryotes. The pathogenic fungus Candida albicans, which causes infections in humans, has a single Hsp110, termed Msi3. Here, we provide proof-of-principle evidence supporting fungal Hsp110s as targets for the development of new antifungal drugs. We identify a pyrazolo[3,4-b] pyridine derivative, termed HLQ2H (or 2H), that inhibits the biochemical and chaperone activities of Msi3, as well as the growth and viability of C. albicans. Moreover, the fungicidal activity of 2H correlates with its inhibition of in vivo protein folding. We propose 2H and related compounds as promising leads for development of new antifungals and as pharmacological tools for the study of the molecular mechanisms and functions of Hsp110s.

Mechanisms of Protein Quality Control in the Endoplasmic Reticulum by a Coordinated Hsp40-Hsp70-Hsp90 System

The Hsp40, Hsp70, and Hsp90 chaperone families are ancient, highly conserved, and critical to cellular protein homeostasis. Hsp40 chaperones can transfer their protein clients to Hsp70, and Hsp70 can transfer clients to Hsp90, but the functional benefits of these transfers are unclear. Recent structural and mechanistic work has opened up the possibility of uncovering how Hsp40, Hsp70, and Hsp90 work together as unified system. In this review, we compile mechanistic data on the ER J-domain protein 3 (ERdj3) (an Hsp40), BiP (an Hsp70), and Grp94 (an Hsp90) chaperones within the endoplasmic reticulum; what is known about how these chaperones work together; and gaps in this understanding. Using calculations, we examine how client transfer could impact the solubilization of aggregates, the folding of soluble proteins, and the triage decisions by which proteins are targeted for degradation. The proposed roles of client transfer among Hsp40-Hsp70-Hsp90 chaperones are new hypotheses, and we discuss potential experimental tests of these ideas.

The ribosome quality control factor Asc1 determines the fate of HSP70 mRNA on and off the ribosome.

Cells survive harsh environmental conditions by potently upregulating molecular chaperones such as heat shock proteins (HSPs), particularly the inducible members of the HSP70 family. The life cycle of HSP70 mRNA in the cytoplasm is unique-it is translated during stress when most cellular mRNA translation is repressed and rapidly degraded upon recovery. Contrary to its 5' untranslated region's role in maximizing translation, we discovered that the HSP70 coding sequence (CDS) suppresses its translation via the ribosome quality control (RQC) mechanism. The CDS of the most inducible Saccharomyces cerevisiae HSP70 gene, SSA4, is uniquely enriched with low-frequency codons that promote ribosome stalling during heat stress. Stalled ribosomes are recognized by the RQC components Asc1p and Hel2p and two novel RQC components, the ribosomal proteins Rps28Ap and Rps19Bp. Surprisingly, RQC does not signal SSA4 mRNA degradation via No-Go-Decay. Instead, Asc1p destabilizes SSA4 mRNA during recovery from heat stress by a mechanism independent of ribosome binding and SSA4 codon optimality. Therefore, Asc1p operates in two pathways that converge to regulate the SSA4 mRNA life cycle during stress and recovery. Our research identifies Asc1p as a critical regulator of the stress response and RQC as the mechanism tuning HSP70 synthesis.

Benzothiazole Substitution Analogs of Rhodacyanine Hsp70 Inhibitors Modulate Tau Accumulation.

The accumulation and aggregation of the microtubule-associated protein tau (tau) into intracellular neuronal tangles are a hallmark of a range of progressive neurodegenerative tauopathies, including Alzheimer's disease (AD), frontotemporal dementia, Pick's disease, and progressive supranuclear palsy. The aberrant phosphorylation of tau is associated with tau aggregates in AD. Members of the heat shock protein 70 kDa (Hsp70) family of chaperones bind directly to tau and modulate tau clearance and aggregation. Small molecules that inhibit the Hsp70 family of chaperones have been shown to reduce the accumulation of tau, including phosphorylated tau. Here, eight analogs of the rhodacyanine inhibitor, JG-98, were synthesized and evaluated. Like JG-98, many of the compounds inhibited ATPase activity of the cytosolic heat shock cognate 70 protein (Hsc70) and reduced total, aggregated, and phosphorylated tau accumulation in cultured cells. Three compounds, representing divergent clogP values, were evaluated for in vivo blood-brain barrier penetration and tau reduction in an ex vivo brain slice model. AL69, the compound with the lowest clogP and the lowest membrane retention in a parallel artificial membrane permeability assay (PAMPA), reduced phosphorylated tau accumulation. Our results suggest that benzothiazole substitutions of JG-98 that increase hydrophilicity may increase the efficacy of these Hsp70 inhibitors to reduce phosphorylated tau.

Molecular Chaperones' Potential against Defective Proteostasis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neuronal degenerative condition identified via a build-up of mutant aberrantly folded proteins. The native folding of polypeptides is mediated by molecular chaperones, preventing their pathogenic aggregation. The mutant protein expression in ALS is linked with the entrapment and depletion of chaperone capacity. The lack of a thorough understanding of chaperones' involvement in ALS pathogenesis presents a significant challenge in its treatment. Here, we review how the accumulation of the ALS-linked mutant FUS, TDP-43, SOD1, and C9orf72 proteins damage cellular homeostasis mechanisms leading to neuronal loss. Further, we discuss how the HSP70 and DNAJ family co-chaperones can act as potential targets for reducing misfolded protein accumulation in ALS. Moreover, small HSPB1 and HSPB8 chaperones can facilitate neuroprotection and prevent stress-associated misfolded protein apoptosis. Designing therapeutic strategies by pharmacologically enhancing cellular chaperone capacity to reduce mutant protein proteotoxic effects on ALS pathomechanisms can be a considerable advancement. Chaperones, apart from directly interacting with misfolded proteins for protein quality control, can also filter their toxicity by initiating strong stress-response pathways, modulating transcriptional expression profiles, and promoting anti-apoptotic functions. Overall, these properties of chaperones make them an attractive target for gaining fundamental insights into misfolded protein disorders and designing more effective therapies against ALS.

Genome-wide analysis reveals Hsf1 maintains high transcript abundance of target genes controlled by strong constitutive promoter in Saccharomyces cerevisiae

BackgroundIn synthetic biology, the strength of promoter elements is the basis for precise regulation of target gene transcription levels, which in turn increases the yield of the target product. However, the results of many researches proved that excessive transcription levels of target genes actually reduced the yield of the target product. This phenomenon has been found in studies using different microorganisms as chassis cells, thus, it becomes a bottleneck problem to improve the yield of the target product.ResultsIn this study, promoters PGK1p and TDH3p with different strengths were used to regulate the transcription level of alcohol acetyl transferase encoding gene ATF1. The results demonstrated that the strong promoter TDH3p decreased the production of ethyl acetate. The results of Real-time PCR proved that the transcription level of ATF1 decreased rapidly under the control of TDH3p, and the unfolded protein reaction was activated, which may be the reason for the abnormal production caused by the strong promoter. RNA-sequencing analysis showed that the overexpression of differential gene HSP30 increased the transcriptional abundance of ATF1 gene and production of ethyl acetate. Interestingly, deletion of the heat shock protein family (e.g., Hsp26, Hsp78, Hsp82) decreased the production of ethyl acetate, suggesting that the Hsp family was also involved in the regulation of ATF1 gene transcription. Furthermore, the results proved that the Hsf1, an upstream transcription factor of Hsps, had a positive effect on alleviating the unfolded protein response and that overexpression of Hsf1 reprogramed the pattern of ATF1 gene transcript levels. The combined overexpression of Hsf1 and Hsps further increased the production of ethyl acetate. In addition, kinase Rim15 may be involved in this regulatory pathway. Finally, the regulation effect of Hsf1 on recombinant strains constructed by other promoters was verified, which confirmed the universality of the strategy.ConclusionsOur results elucidated the mechanism by which Rim15–Hsf1–Hsps pathway reconstructed the repression of high transcription level stress and increased the production of target products, thereby providing new insights and application strategies for the construction of recombinant strains in synthetic biology.

Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.

HtpG Is a Metal-Dependent Chaperone Which Assists the DnaK/DnaJ/GrpE Chaperone System of Mycobacterium tuberculosis via Direct Association with DnaJ2.

Protein folding is a crucial process in maintaining protein homeostasis, also known as proteostasis, in the cell. The requirement for the assistance of molecular chaperones in the appropriate folding of several proteins has already called into question the previously held view of spontaneous protein folding. These chaperones are highly ubiquitous cellular proteins, which not only help in mediating the proper folding of other nascent polypeptides but are also involved in refolding of the misfolded or the aggregated proteins. Hsp90 family proteins such as high-temperature protein G (HtpG) are abundant and ubiquitously expressed in both eukaryotic and prokaryotic cells. Although HtpG is known as an ATP-dependent chaperone protein in most organisms, function of this protein remains obscured in mycobacterial pathogens. Here, we aim to investigate significance of HtpG as a chaperone in the physiology of Mycobacterium tuberculosis. We report that M. tuberculosis HtpG (mHtpG) is a metal-dependent ATPase which exhibits chaperonin activity towards denatured proteins in coordination with the DnaK/DnaJ/GrpE chaperone system via direct association with DnaJ2. Increased expression of DnaJ1, DnaJ2, ClpX, and ClpC1 in a ΔhtpG mutant strain further suggests cooperativity of mHtpG with various chaperones and proteostasis machinery in M. tuberculosis. IMPORTANCE M. tuberculosis is exposed to variety of extracellular stressful conditions and has evolved mechanisms to endure and adapt to the adverse conditions for survival. mHtpG, despite being dispensable for M. tuberculosis growth under in vitro conditions, exhibits a strong and direct association with DnaJ2 cochaperone and assists the mycobacterial DnaK/DnaJ/GrpE (KJE) chaperone system. These findings suggest the potential role of mHtpG in stress management of the pathogen. Mycobacterial chaperones are responsible for folding of nascent protein as well as reactivation of protein aggregates. M. tuberculosis shows differential adaptive response subject to the availability of mHtpG. While its presence facilitates improved protein refolding via stimulation of the KJE chaperone activity, in the absence of mHtpG, M. tuberculosis enhances expression of DnaJ1/J2 cochaperones as well as Clp protease machinery for maintenance of proteostasis. Overall, this study provides a framework for future investigation to better decipher the mycobacterial proteostasis network in the light of stress adaptability and/or survival.