Molecular Chaperone Hsp90 Research Articles

Possible Role of Peroxynitrite in the Responses Induced by Fusicoccin in Plant Cultured Cells.

Fusicoccin (FC) is a well-known phytotoxin able to induce in Acer pseudoplatanus L. (sycamore) cultured cells, a set of responses similar to those induced by stress conditions. In this work, the possible involvement of peroxynitrite (ONOO−) in FC-induced stress responses was studied measuring both in the presence and in the absence of 2,6,8-trihydroxypurine (urate), a specific ONOO− scavenger: (1) cell death; (2) specific DNA fragmentation; (3) lipid peroxidation; (4) production of RNS and ROS; (5) activity of caspase-3-like proteases; and (6) release of cytochrome c from mitochondria, variations in the levels of molecular chaperones Hsp90 in the mitochondria and Hsp70 BiP in the endoplasmic reticulum (ER), and of regulatory 14-3-3 proteins in the cytosol. The obtained results indicate a role for ONOO− in the FC-induced responses. In particular, ONOO− seems involved in a PCD form showing apoptotic features such as specific DNA fragmentation, caspase-3-like protease activity, and cytochrome c release from mitochondria.

Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several μmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.

The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90.

The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP‐driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N‐terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD−MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD−MD arrangement with the accessibility of a client binding site on the NTD.

FKBP51‐Hsp90 complex as a novel therapeutic target for Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of dementia without effective treatments. FK506‐binding protein 51 (FKBP51) recently emerged as a possible therapeutic target for resolving both tau aggregation and insulin resistance in AD. FKBP51 in complex with its partner, molecular chaperone Hsp90, increases tau stability, thereby promoting tau aggregation [1, 2]. FKBP51 also regulates insulin resistance and altered glucose metabolism which represent early events that precede or accompany the development of AD [3, 4]. Our study aims to investigate the potential roles of FKBP51‐Hsp90 interaction inhibitors in AD.MethodsA large dataset of available compounds were investigated using AlphaScreen™ and in silico virtual screening techniques to evaluate their inhibition on FKBP51‐Hsp90 interactions. Two of the selected specific inhibitors, E8 and C674, were then applied to SH‐SY5Y cells to test their effects on neurite outgrowth, overexpressed tau levels and glucose uptake. Furthermore, insulin signaling pathways were compared between vehicle‐treated vs inhibitor‐treated AD specimens.ResultsCompounds E8 and C674 inhibit FKBP51‐Hsp90 interactions at sub‐micromolar concentrations. E8 and C674 enhance neurite elongation, reduce overexpressed tau levels, stimulate glucose uptake and regulate insulin resistance.ConclusionWe showed that inhibiting specific FKBP51‐Hsp90 interactions with small molecules provides novel therapeutic targets for AD.

The Hsp70-Hsp90 co-chaperone Hop/Stip1 shifts the proteostatic balance from folding towards degradation

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation.

Interaction profile of a mixed-culture fermentation of Issatchenkia orientalis and Saccharomyces cerevisiae by transcriptome sequencing

PurposeMixed fermentation with Saccharomyces cerevisiae and non-Saccharomyces yeasts has become an oenlogical tool to improve wines’ organoleptic properties. However, the maximum utilization of this method is dependent upon understanding the influence of mixed cultures on the physiology of S.cerevisiae and non-Saccharomyces yeasts.Design/methodology/approachIn this study, the supernatants from 48 h mixed-culture fermentation were added to the pure cultures of Issatchenkia orientalis and Saccharomyces, respectively. And the authors used RNA sequencing to determine the transcriptome change of I.orientalis and S.cerevisiae in a mixed culture.FindingsThe results showed that multiple genes associated with cell growth and death were differentially expressed. Genes related to biosynthesis of amino acids were enriched among those upregulated in the mixed-fermentation supernatant. Meanwhile, the differential expression level of genes encoding enzymes essential for formation of aroma compounds was found in the single and in the mixed fermentation. The high expression level of molecular chaperones Hsp70, Hsp90 and Hsp110 suggests that metabolites of mixed-culture fermentation may lead to aggregation of misfolded proteins. Moreover, upregulation of ethanol dehydrogenase I ADH1 in the mixed-culture fermentations was highlighted.Originality/valueThis is the first time that RNA-seq was used to analyze changes in the transcriptome of mixed cultures. According to the results the authors’ manuscript provided, an integrated view into the adaptive responses of S.cerevisiae and non-Saccharomyces yeasts to the mixed-culture fermentation is benefit for the potential application of S.cerevisiae and non-Saccharomyces yeasts in fruit wine brewing.

Heat Shock Protein HSP60 in Left Ventricular Cardiomyocytes of Hypertensive Rats with and without Insulin-Dependent Diabetes Mellitus.

In cardiomyocytes, high molecular ATP-dependent HSP70 and HSP90 play an important role in protecting the myocardium from abnormal proteins that appear, in particular, due to activation of oxidative stress. Molecular chaperone HSP60 is of particular importance for cardiomyocytes as it is responsible for assembly of mitochondrial matrix proteins. We studied the peculiarities of expression of HSP60 in left ventricular cardiomyocytes in hypertension, insulin-dependent diabetes mellitus, and their combination. The experiment was performed on 38-week-old male Wistar-Kyoto and SHR (spontaneously hypertensive) rats aged 38-57 weeks. Insulin-dependent diabetes mellitus was modeled by a single parenteral administration of 65 mg/kg streptozotocin. Expression of HSP60 in left ventricular cardiomyocytes was evaluated by immunohistochemical methods. It was found that hypertension, diabetes mellitus, and their combination are associated with a significant decrease in the content of HSP60 in left ventricular cardiomyocytes in comparison with the control. This finding can be considered as a pathogenetic mechanism of myocardial damage induced by hypertension and diabetes mellitus.

TRAP1 enhances Warburg metabolism through modulation of PFK1 expression/activity and favors resistance to EGFR inhibitors in human colorectal carcinomas.

Metabolic rewiring is a mechanism of adaptation to unfavorable environmental conditions and tumor progression. TRAP1 is an HSP90 molecular chaperone upregulated in human colorectal carcinomas (CRCs) and responsible for downregulation of oxidative phosphorylation (OXPHOS) and adaptation to metabolic stress. The mechanism by which TRAP1 regulates glycolytic metabolism and the relevance of this regulation in resistance to EGFR inhibitors were investigated in patient-derived CRC spheres, human CRC cells, samples, and patients. A linear correlation was observed between TRAP1 levels and 18 F-fluoro-2-deoxy-glucose (18 F-FDG) uptake upon PET scan or GLUT1 expression in human CRCs. Consistently, TRAP1 enhances GLUT1 expression, glucose uptake, and lactate production and downregulates OXPHOS in CRC patient-derived spheroids and cell lines. Mechanistically, TRAP1 maximizes lactate production to balance low OXPHOS through the regulation of the glycolytic enzyme phosphofructokinase-1 (PFK1); this depends on the interaction between TRAP1 and PFK1, which favors PFK1 glycolytic activity and prevents its ubiquitination/degradation. By contrast, TRAP1/PFK1 interaction is lost in conditions of enhanced OXPHOS, which results in loss of TRAP1 regulation of PFK1 activity and lactate production. Notably, TRAP1 regulation of glycolysis is involved in resistance of RAS-wild-type CRCs to EGFR monoclonals. Indeed, either TRAP1 upregulation or high glycolytic metabolism impairs cetuximab activity invitro, whereas TRAP1 targeting and/or inhibition of glycolytic pathway enhances cell response to cetuximab. Finally, a linear correlation between 18 F-FDG PET uptake and poor response to cetuximab in first-line therapy in human metastatic CRCs was observed. These results suggest that TRAP1 is a key determinant of CRC metabolic rewiring and favors resistance to EGFR inhibitors through regulation of glycolytic metabolism.

Transcriptome analysis reveals molecular mechanisms responsive to acute cold stress in the tropical stenothermal fish tiger barb (Puntius tetrazona)

BackgroundTropical stenothermal fish exhibit special tolerance and response to cold stress. However current knowledge of the molecular mechanisms response to cold stress in aquatic ectotherms is largely drawn from eurythermal or extreme stenothermal species. The tiger barb Puntius tetrazona is a tropical stenothermal fish, with great popularity in aquarium trade and research.ResultsTo investigate the response mechanism of P. tetrazona to low temperature, fish were exposed to increasing levels of acute cold stress. Histopathological analysis showed that the brain, gill, liver and muscle tissues appeared serious damage after cold stress (13 °C). Brain, gill, liver and muscle tissues from control (CTRL) groups (27 °C) and COLD stress groups (13 °C) of eight-month fish (gender-neutral) were sampled and assessed for transcriptomic profiling by high-throughput sequencing. 83.0 Gb of raw data were generated, filtered and assembled for de novo transcriptome assembly. According to the transcriptome reference, we obtained 392,878 transcripts and 238,878 unigenes, of which 89.29% of the latter were annotated. There were 23,743 differently expressed genes (DEGs) been filtered from four pairs of tissues (brain, gill, liver and muscle) between these cold stress and control groups. These DEGs were mainly involved in circadian entrainment, circadian rhythm, biosynthesis of steroid and fatty acid. There were 64 shared DEGs between the four pairs of groups, and five were related to ubiquitylation/deubiquitylation. Our results suggested that ubiquitin-mediated protein degradation might be necessary for tropical stenothermal fish coping with acute cold stress. Also, the significant cold-induced expression of heat shock 70 kDa protein (HSP70) and cold-induced RNA-binding protein (CIRBP) was verified. These results suggested that the expression of the molecular chaperones HSP70 and CIRBP in P. tetrazona might play a critical role in coping with acute cold stress.ConclusionsThis is the first transcriptome analysis of P. tetrazona using RNA-Seq technology. Novel findings about tropical stenothermal fish under cold stress (such as HSP70 and CIRBP genes) are presented here. This study contributes new insights into the molecular mechanisms of tropical stenothermal species response to acute cold stress.

TRAP1 Regulates Wnt/β-Catenin Pathway through LRP5/6 Receptors Expression Modulation.

Wnt/β-Catenin signaling is involved in embryonic development, regeneration, and cellular differentiation and is responsible for cancer stemness maintenance. The HSP90 molecular chaperone TRAP1 is upregulated in 60–70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/β-Catenin pathway and preventing β-Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/β-Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/β-Catenin signaling was highlighted by a TCF/LEF Cignal Reporter Assay in Wnt-off HEK293T and CRC HCT116 cell lines. Of note, this regulation occurs through the modulation of Wnt ligand receptors LRP5 and LRP6 that are both downregulated in TRAP1-silenced cell lines. However, while LRP5 mRNA is significantly downregulated upon TRAP1 silencing, LRP6 mRNA is unchanged, suggesting independent mechanisms of regulation by TRAP1. Indeed, LRP5 is regulated upon promoter methylation in CRC cell lines and human CRCs, whereas LRP6 is controlled at post-translational level by protein ubiquitination/degradation. Consistently, human CRCs with high TRAP1 expression are characterized by the co-upregulation of active β-Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/β-Catenin signaling is modulated at multiple levels by TRAP1.

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions.

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Dissecting Molecular Principles of the Hsp90 Chaperone Regulation by Allosteric Modulators Using a Hierarchical Simulation Approach and Network Modeling of Allosteric Interactions: Conformational Selection Dictates the Diversity of Protein Responses and Ligand-Specific Functional Mechanisms.

Conformational plasticity of the Hsp90 molecular chaperones underlies the diversity of functional mechanisms that these versatile molecular machines employ to coordinate their vast protein clientele in the cellular environment. Despite a steady progress in studies of the Hsp90 machinery, a great deal remains unknown about molecular principles and ligand-specific functional mechanisms of the Hsp90 regulation by allosteric modulators that attracted significant attention because of their therapeutic potential. Due to structural complexity and dynamic nature of the Hsp90 responses to allosteric modulators, the atomistic details about the mode of action of these small molecules continue to be fairly scarce and controversial. In this work, we employ an integrative strategy that encompassed atomistic simulations of the Hsp90 proteins and hierarchical modeling of Hsp90-ligand binding with network analysis to explore functional mechanisms of the Hsp90 regulation by a panel of allosteric modulators (novobiocin, KU-135, KU-174, and KU-32) with different models of action. The results show that functional mechanisms of allosteric modulation in the Hsp90 proteins may be driven by conformational selection principles in which ligands elicit pre-existing states of the unbound chaperone to drive ligand-specific protein responses and distinct scenarios of Hsp90 regulation. We found that novobiocin can selectively sequester an ensemble of open chaperone conformations and inhibit the progression of the functional cycle through a cascade of cumulative dynamic changes. In contrast, KU-32 displayed unique preferences toward partially closed dynamic states, inducing robust allosteric signaling and stimulation of the ATPase cycle. The proposed model of the Hsp90 regulation by allosteric modulators reconciled diverse experimental data and showed that allosteric modulators may operate via targeted exploitation of dynamic landscapes eliciting vastly different protein responses and diverse mechanisms of action.

ATP-Driven Nonequilibrium Activation of Kinase Clients by the Molecular Chaperone Hsp90

The molecular chaperone 90-kDa heat-shock protein (Hsp90) assists the late-stage folding and activation of diverse types of protein substrates (called clients), including many kinases. Previous studies have established that the Hsp90 homodimer undergoes an ATP-driven cycle through open and closed conformations. Here, I propose a model of client activation by Hsp90 that predicts that this cycle enables Hsp90 to use ATP energy to drive a client out of thermodynamic equilibrium toward its active conformation. My model assumes that an Hsp90-bound client can transition between a deactivating conformation and an activating conformation. It suggests that the cochaperone Cdc37 aids Hsp90 to activate kinase clients by differentiating between these two intermediate conformations. My model makes experimentally testable predictions, including how modulating the stepwise kinetics of the Hsp90 cycle—for example, by various cochaperones—affects the activation of different clients. My model may inform client-specific and cell-type-specific therapeutic intervention of Hsp90-mediated protein activation.

Abstract 1232: Synergistic roles played by autophagy and Hsp70 in cancer cell survival

Abstract To avoid cell death, cancer cells adapt to extreme conditions. Chaperone upregulation, modulation of the unfolded protein response (UPR), and induction of protein degradation pathways are critical for cancer cell survival and metastasis in some cancers. Thus, it is not surprising that the molecular chaperone Hsp70 is upregulated in many cancers. For example, increased Hsp70 abundance correlates with metastasis formation and poor prognosis in breast cancer patients. We previously demonstrated that inhibition of Hsp70, by treating cells with a specific inhibitor known as MAL3-101, induces rhabdomyosarcoma cancer cell death due to induction of a UPR-mediated apoptotic response (Sabinis et al., 2016). Moreover, by taking advantage of a MAL3-101-resistant cell line, we established that protein degradation pathways act as a compensatory mechanism in MAL3-101 resistant rhabdomyosarcoma cells. In particular, we found that autophagy inhibition re-sensitized resistant cells to Hsp70 inhibition, suggesting that autophagy is a key compensatory mechanism for Hsp70 inhibition. Autophagy was further induced by MAL3-101 treatment in these cells, as evidenced by both autophagy related gene mRNA and protein accumulation as well as by autophagic-like structures detected by electron microscopy (Sannino et al., 2018). These data highlight a pro-survival role for autophagy induction upon exposure to an Hsp70 inhibitor in cancer, and provide a link between Hsp70, proteasome activity, the UPR, and autophagy in rhabdomyosarcoma. We next asked if other cancers might be dependent on Hsp70 activity and investigated the benefit of combined treatment with autophagy and/or proteasome inhibitors together with MAL3-101 in breast cancer cells. Our recent studies suggest that diverse breast cancer cell lines are either sensitive or resistant to Hsp70 inhibition. MAL3-101 resistant breast cancer cells have higher autophagy levels compared to MAL3-101 sensitive lines. Moreover, autophagy is upregulated upon Hsp70 inhibition in resistant breast cancer cells, again highlighting the potential benefit of combined treatment with autophagy and Hsp70 inhibitors. Overall, our work positions autophagy as a critical compensatory mechanism when molecular chaperone function is overwhelmed and misfolded proteins accumulate in cancer cells. Further investigations will reveal the potential for Hsp70 inhibitors and the role of protein degradation pathways in vivo in breast cancer. Citation Format: Sara Sannino, Jeffrey L. Brodsky. Synergistic roles played by autophagy and Hsp70 in cancer cell survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1232.

Recombinant Tumor Suppressor TSC1 Differentially Interacts with Escherichia coli DnaK and Human HSP70.

Tuberous sclerosis complex (TSC) is a neurological syndrome manifested by non-cancerous tumors in several organs. Mutations in either TSC1 or TSC2 tumor suppressor gene cause the disease. In the cell, TSC1 is known to form a heterodimer with TSC2 because of which an active complex is formed that negatively regulates the mTORC1 activity during cellular stress. Hence, mutation in TSC1 or TSC2 is manifested by excess proliferation of the cells leading to the development of numerous benign tumors. The TSC1 and TSC2 complex is known to interact with several protein-binding partners. One such significant interaction of this complex is with the molecular chaperone HSP70. The role of TSC1 in that interaction is still elusive. Here, we have expressed and purified TSC1 (302–420 residues) in a bacterial expression system and have shown that this region directly interacts with HSP70. We have shown that TSC1 increases the ATPase activity of Escherichia coli DnaK, a HSP70 homologue. On the contrary, TSC1 was found to show inhibitory activity toward human HSP70. Our result suggests that TSC1 (302–420 aa) shows differential interaction between the HSP70 homologues. This points toward the evolutionary significance of chaperoning system and the importance of eukaryotic tetratricopeptide repeat domain interaction motif -EEVD. Our study shows the evidence that TSC1 interacts with HSP70 and has a role to play in the chaperoning activity to maintain cellular homeostasis.

Harnessing the Proteostasis Network in Alcohol-associated Liver Disease

Alcohol-associated liver disease (ALD) accounts for significant mortality and morbidity in the USA. Prolonged alcohol exposure leads to increased reactive oxygen species and oxidative stress resulting in protein misfolding and/or aggregation. Cellular protein homeostasis network is an adaptive cellular response that regulates biogenesis or degradation of proteins with chaperones as central coordinators to maintain proteome integrity during stress. Two extensively studied organelle-specific transcriptional proteostasis pathways are the heat shock response (HSR) in the cytosol and unfolded protein response (UPR) in the endoplasmic reticulum (ER). Here, we discuss the pathophysiological role of HSR and UPR and their potential as therapeutic targets in ALD. The HSR and UPR pathways are major players in ALD pathogenesis. Acute/binge and chronic alcohol can activate the HSR to selectively induce downstream target chaperones based on the duration of alcohol exposure. Molecular chaperone HSP90 contributes to pro-inflammatory responses in ALD. Recent findings report HSP90 in extracellular vesicles in ALD suggesting their role in cellular communication and disease progression. Extensive studies have reported UPR activation due to ER stress resulting in hepatocyte cell death, inflammation, and lipogenesis contributing to liver injury. Recent studies report that ER stress plays a role in neutrophil-mediated liver injury in ALD. This review highlights the contribution of HSR and UPR in the pathogenesis of ALD. Acute/binge or chronic alcohol exposure perturbs proteostasis mediators which fail to maintain proteome integrity and disease ensues. Understanding mechanisms that regulate proteostasis pathways, HSR and UPR could identify novel disease modulators and guide the development of therapeutic targets in ALD.

Virulence of HtpG<sup>+</sup> and HtpG<sup>−</sup> strains of <i>Yersinia pestis</i> for Mice and Guinea Pigs

HtpG (high-temperature protein G) is a bacterial homologue of the highly conserved molecular chaperone Hsp90 of eukaryotes, which plays an important role in protection against stress in many bacterial species. The role of the htpG gene encoding the synthesis of high-temperature prokaryotic G protein in the pathogenesis of bacterial infections is still unclear.The aim of this work is to study the functional importance of HtpG in the pathogenesis of plague.Materials and methods. Isogenic Yersinia pestis sets based on attenuated and virulent strains differing in the presence of the functional htpG gene (YPO3119) were generated with the help of site-directed mutagenesis. The HtpG amino acid sequence was analyzed using the BLAST program. The properties of the resulting mutant strains were evaluated using microbiological and biological methods.Results and discussion. The bioinformatics analysis showed high conservativeness of the HtpG protein within the Y. pestis species (100% identity), as well as 99 % identity with the Y. pseudotuberculosis protein and 96 % identity – Y. enterocolitica protein. Y. pestis htpG knock-out mutants showed increase of susceptibility to temperature and oxidative stress like mutants of the other bacterial species. However, the mutant was not sensitive to osmotic stress and human serum complement. The loss of the ability to synthesize HtpG by plague microbe did not affect the virulence and average life duration of mice and guinea pigs challenged subcutaneously. It means that htpG gene is not a good molecular target for the treatment and/or immunoprophylaxis of plague.

The molecular chaperone Hsp90 regulates heterochromatin assembly through stabilizing multiple complexes in fission yeast.

In the fission yeast Schizosaccharomyces pombe, both RNAi machinery and RNAi-independent factors mediate transcriptional and posttranscriptional silencing and heterochromatin formation. Here, we show that the silencing of reporter genes at major native heterochromatic loci (centromeres, telomeres, mating-type locus and rDNA regions) and an artificially induced heterochromatin locus is alleviated in a fission yeast hsp90 mutant, hsp90-G84C Also, H3K9me2 enrichment at heterochromatin regions, especially at the mating-type locus and subtelomeres, is compromised, suggesting heterochromatin assembly defects. We further discovered that Hsp90 is required for stabilization or assembly of the RNA-induced transcriptional silencing (RITS) and Argonaute siRNA chaperone (ARC) RNAi effector complexes, the RNAi-independent factor Fft3, the shelterin complex subunit Poz1 and the Snf2/HDAC-containing repressor complex (SHREC). Our ChIP data suggest that Hsp90 regulates the efficient recruitment of the methyltransferase/ubiquitin ligase complex CLRC by shelterin to chromosome ends and targeting of the SHREC and Fft3 to mating type locus and/or rDNA region. Finally, our genetic analyses demonstrated that increased heterochromatin spreading restores silencing at subtelomeres in the hsp90-G84C mutant. Thus, this work uncovers a conserved factor critical for promoting RNAi-dependent and -independent heterochromatin assembly and gene silencing through stabilizing multiple effectors and effector complexes.

Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana.

Females of the brine shrimp Artemia franciscana produce either free-swimming nauplii via ovoviviparous pathway of reproduction or encysted embryos, known as cysts, via oviparous pathway, in which biological processes are arrested. While previous study has shown a crucial role of ATP-dependent molecular chaperone, heat shock protein 70 (Hsp70) in protecting A. franciscana nauplii against various abiotic and abiotic stressors, the function of this protein in diapausing embryos and cyst development, however, remains unknown. RNA interference (RNAi) was applied in this study to examine the role of Hsp70 in cyst development and stress tolerance, with the latter performed by desiccation and freezing, a common method used for diapause termination in Artemia cysts. Hsp70 knockdown was apparent in cysts released from females that were injected with Hsp70 dsRNA. The loss of Hsp70 affected neither the development nor morphology of the cysts. The time between fertilization and cyst release from Artemia females injected with Hsp70 dsRNA was delayed slightly, but the differences were not significant when compared to the controls. However, the hatching percentage of cysts which lacks Hsp70 were reduced following desiccation and freezing. Taken together, these results indicated that Hsp70 possibly plays a role in the stress tolerance but not in the development of diapause-destined embryos of Artemia. This research makes fundamental contributions to our understanding of the role molecular chaperone Hsp70 plays in Artemia, an excellent model organism for diapause studies of the crustaceans.