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Our previous study had shown that member 13 (Hspa13) of heat shock protein family A (Hsp70) promotes plasma cell (PC) production and antibody secretion. To further explore Hspa13 expression and function, we combined single-cell RNA-sequencing and antigen receptor lineage (BCR) analysis to characterize sheep red cell‒primed splenocytes. The single-cell transcriptional profiles revealed that Hspa13 is specifically and highly expressed in PCs. These results suggest that Hspa13 is a novel PC-specific marker. In terms of its function, we found that the CD19cre-mediated conditional knock-out (cKO) of Hspa13 reduced the expression of Ebi3 and IL-10 in PCs. Ebi3 and IL-10 are important factors in IL-4‒secreting type 2 helper T cell (Th2) activation and differentiation. As expected, we found that the Hspa13 cKO reduced IL‒4-expressing follicular helper T (Tfh2) cells. Finally, the single-cell antigen receptor analysis demonstrated that the Hspa13 cKO reduced the Aicda-mediated antibody class-switching recombination (CSR) and somatic hypermutation (SHM) in germinal centers (GCs) B cells. Altogether, the single-cell atlas of splenocytes revealed a critical indirect role for the novel PC-specific marker Hspa13 in CSR and SHM in GC B cells by promoting Ebi3 and IL-10 expression in PCs to induce IL-4-expressing Tfh2 cells. Further exploration of Hspa13 expression and function will provide valuable clues for how to use Hspa13 in the treatment of autoimmune diseases.

Prions, misfolded proteins that aggregate, cause an array of progressively deteriorating conditions to which, currently, there are no effective treatments. The presently accepted model indicates that the soluble non-prion forms of prion-forming proteins, such as the well-studied SUP35, do not exist in large aggregated molecular complexes. Here, we show using analytical ultracentrifugation with fluorescent detection that the non-prion form of SUP35 exists in a range of discretely sized soluble complexes (19S, 28S, 39S, 57S, and 70S-200S). Similar to the [PSI+] aggregated complexes, each of these [psi-] complexes associates at stoichiometric levels with a large variety of molecular chaperones: HSP70 proteins comprise the major component. Another yeast prion-forming protein, RNQ1 (known to promote the production of the prion SUP35 state), is also present in SUP35 complexes. These results establish that the non-prion SUP35, like its prion form, is predisposed to form large molecular complexes containing chaperones and other prion-forming proteins. These results agree with our previous studies on the huntingtin protein. That the normal forms for aggregation-prone proteins may preexist in large molecular complexes has important ramifications for the progression of diseases involving protein aggregation.

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.

Heat shock protein 90 (Hsp90) and Hsp70 are chaperones implicated in different inflammatory disorders, given their property to impact innate and adaptive immune responses. Here, we determined the so far unknown role of extracellular Hsp70 in epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-mediated blistering dermatosis. The in vivo pathophysiological relevance of extracellular Hsp70 was demonstrated in an anti-type VII collagen antibody transfer-induced EBA mouse model in which elevated blood levels of this chaperone were recorded. We found that Hsp70-treated mice had a more intense clinical disease severity compared to controls that were paralleled by increased levels of cutaneous matrix metalloproteinase 9 and plasma hydrogen peroxide. The latter finding was confirmed in an independent reactive oxygen species release assay using EBA-specific immune complexes combined with recombinant Hsp70. Finally, cell culture experiments using human naive peripheral blood mononuclear cells (PBMC) revealed that extracellular Hsp70 stimulated the secretion of the T cell-derived pro-inflammatory cytokines IL-6 and IL-8. This work extends knowledge about the role of Hsps in autoimmune bullous diseases, suggesting that extracellular Hsp70 represents a pathophysiological factor and potential treatment target in EBA.

In the current study, a novel derivative of sulfamethoxazole (a sulfonamide containing anti-biotic) named ZM-093 (IUPAC name: (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide) was synthesized via common diazotization-coupling reactions from sulfamethoxazole and subsequently characterized through NMR/FT-IR spectroscopy. After evaluation, the compound was geometrically optimized at the DFT level of theory with BL3YP method and 6/31++G (d,p) basis set and from the optimized structure, several molecular descriptors important in the biological reactivity of the compound, such as global reactivity parameters, molecular electrostatic potential, average local ionization energy, and drug-likeness features of the compound were computationally analyzed. The experimental in vitro investigations of the interaction between ZM-093 and heat shock protein 70 (HSP70), a protein that is highly expressed in several types of cancers, exhibited a significant inhibitory effect against the chaperone activity of HSP70 for the titled compound (P-value < 0.01) and the comparison between the experimental studies with the mentioned computational analysis, as well as molecular docking, illustrated that ZM-093 may inhibit HSP70 through binding to its substrate-binding domain. Finally, by taking all the previous results into account, a new method for assessing the inhibitory activity of ligand to HSP70 is introduced based on protonography, a recently developed method that is dependent on the catalytic activity of carbonic anhydrase on polyacrylamide gel electrophoresis.

Background The tick Dermacentor silvarum Olenev (Acari: Ixodidae) is a vital vector tick species mainly distributed in the north of China and overwinters in the unfed adult stage. The knowledge of the mechanism that underlies its molecular adaptation against cold is limited. In the present study, genes of hsp70 and hsp90 cDNA, named Dshsp70 and Dshsp90, and tubulin were cloned and characterized from D. silvarum, and their functions in cold stress were further evaluated. Methods The genome of the heat shock proteins and tubulin of D. silvarum were sequenced and analyzed using bioinformatics methods. Each group of 20 ticks were injected in triplicate with Dshsp90-, Dshsp70-, and tubulin-derived dsRNA, whereas the control group was injected with GFP dsRNA. Then, the total RNA was extracted and cDNA was synthesized and subjected to RT-qPCR. After the confirmation of knockdown, the ticks were incubated for 24 h and were exposed to − 20 °C lethal temperature (LT50), and then the mortality was calculated. Results Results indicated that Dshsp70 and Dshsp90 contained an open reading frame of 345 and 2190 nucleotides that encoded 114 and 729 amino acid residues, respectively. The transcript Dshsp70 showed 90% similarity with that identified from Dermacentor variabilis, whereas Dshsp90 showed 85% similarity with that identified from Ixodes scapularis. Multiple sequence alignment indicates that the deduced amino acid sequences of D. silvarum Hsp90, Hsp70, and tubulin show very high sequence identity to their corresponding sequences in other species. Hsp90 and Hsp70 display highly conserved and signature amino acid sequences with well-conserved MEEVD motif at the C-terminal in Hsp90 and a variable C-terminal region with a V/IEEVD-motif in Hsp70 that bind to numerous co-chaperones. RNA interference revealed that the mortality of D. silvarum was significantly increased after injection of dsRNA of Dshsp70 (P = 0.0298) and tubulin (P = 0.0448), whereas no significant increases were observed after the interference of Dshsp90 (P = 0.0709). Conclusions The above results suggested that Dshsp70 and tubulin play an essential role in the low-temperature adaptation of ticks. The results of this study can contribute to the understanding of the survival and acclimatization of overwintering ticks. Graphical abstract

GRP94 (glucose-regulated protein 94) is a well-studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C-terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER-associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule.

High blood glucose has long been established as a risk factor for tumor metastasis, yet the molecular mechanisms underlying this association have not been elucidated. Here we describe that hyperglycemia promotes tumor metastasis via increased platelet activity. Administration of glucose, but not fructose, reprogrammed the metabolism of megakaryocytes to indirectly prime platelets into a prometastatic phenotype with increased adherence to tumor cells. In megakaryocytes, a glucose metabolism-related gene array identified the mitochondrial molecular chaperone glucose-regulated protein 75 (GRP75) as a trigger for platelet activation and aggregation by stimulating the Ca2+-PKCα pathway. Genetic depletion of Glut1 in megakaryocytes blocked MYC-induced GRP75 expression. Pharmacologic blockade of platelet GRP75 compromised tumor-induced platelet activation and reduced metastasis. Moreover, in a pilot clinical study, drinking a 5% glucose solution elevated platelet GRP75 expression and activated platelets in healthy volunteers. Platelets from these volunteers promoted tumor metastasis in a platelet-adoptive transfer mouse model. Together, under hyperglycemic conditions, MYC-induced upregulation of GRP75 in megakaryocytes increases platelet activation via the Ca2+-PKCα pathway to promote cancer metastasis, providing a potential new therapeutic target for preventing metastasis. Significance: This study provides mechanistic insights into a glucose–megakaryocyte–platelet axis that promotes metastasis and proposes an antimetastatic therapeutic approach by targeting the mitochondrial protein GRP75.

The role of molecular chaperones, such as heat shock protein 70 (Hsp70), is not typically studied as a function of biological sex, but by addressing this gap we might improve our understanding of proteinopathic disorders that predominate in one sex. Therefore, we exposed male or female primary hippocampal cultures to preformed α-synuclein fibrils in a model of early-stage Lewy pathology. We first discovered that two mechanistically distinct inhibitors of Hsp70 function increased phospho-α-synuclein(+) inclusions more robustly in male-derived neurons. Because Hsp70 is released into extracellular compartments and may restrict cell-to-cell transmission/amplification of α-synucleinopathy, we then tested the effects of low-endotoxin, exogenous Hsp70 (eHsp70) in primary hippocampal cultures. eHsp70 was taken up by and reduced α-synuclein(+) inclusions in cells of both sexes, but pharmacological suppression of Hsp70 function attenuated the inhibitory effect of eHsp70 on perinuclear inclusions only in male neurons. In 20-month-old male mice infused with α-synuclein fibrils in the olfactory bulb, daily intranasal eHsp70 delivery also reduced inclusion numbers and the time to locate buried food. eHsp70 penetrated the limbic system and spinal cord of male mice within 3 h but was cleared within 72 h. Unexpectedly, no evidence of eHsp70 uptake from nose into brain was observed in females. A trend towards higher expression of inducible Hsp70—but not constitutive Hsp70 or Hsp40—was observed in amygdala tissues from male subjects with Lewy body disorders compared to unaffected male controls, supporting the importance of this chaperone in human disease. Women expressed higher amygdalar Hsp70 levels compared to men, regardless of disease status. Together, these data provide a new link between biological sex and a key chaperone that orchestrates proteostasis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01114-6.

Sevoflurane postconditioning (SPostC) has been proved effective in cardioprotection against myocardial ischemia/reperfusion injury. It was also reported that heat shock protein 70 (HSP70) could be induced by sevoflurane, which played a crucial role in hypoxic/reoxygenation (HR) injury of cardiomyocytes. However, the mechanism by which sevoflurane protects cardiomyocytes via HSP70 is still not understood. Here, we aimed to investigate the related mechanisms of SPostC inducing HSP70 expression to reduce the HR injury of cardiomyocytes. After the HR cardiomyocytes model was established, the cells transfected with siRNA for HSP70 (siHSP70) or not were treated with sevoflurane during reoxygenation. The lactate dehydrogenase (LDH) level was detected by colorimetry while cell viability and apoptosis were detected by MTT and flow cytometry. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect HSP70, apoptosis-, cell cycle-associated factors, iNOS, and Cox-2 expressions. Enzyme-linked immuno sorbent assay (ELISA) was used to measure malondialdehyde (MDA) and superoxide dismutase (SOD). SPostC decreased apoptosis, cell injury, oxidative stress and inflammation and increased viability of HR-induced cardiomyocytes. In addition, SPostC downregulated Bax and cleaved caspase-3 levels, while SPostC upregulated Bcl-2, CDK-4, Cyclin D1, and HSP70 levels. SiHSP70 had the opposite effect that SPostC had on HR-induced cardiomyocytes. Moreover, siHSP70 further reversed the effect of SPostC on apoptosis, cell injury, oxidative stress, inflammation, viability and the expressions of HSP70, apoptosis-, and cell cycle-associated factors in HR-induced cardiomyocytes. In conclusion, this study demonstrates that SPostC can reduce the HR injury of cardiomyocytes by inducing HSP70 expression.

The biocide chlorpyrifos (CPF) was described to increase breast cancer risk in humans, to produce breast cancer in animals, and to induce cell proliferation in MCF-7 and MDA-MB-231 cells after 1 and 14 days of treatment. The entire mechanisms related to these CPF actions remain unknown. CPF induced cell proliferation in MCF-7 and MDA-MB-231 cells after 1 and 14 days of treatment by AhR activation through the PGE2/Wnt/β-catenin pathway and HSP90 and HSP70 overexpression. Our results reveal new information on CPF toxic mechanisms induced in human breast cancer cell lines, which could assist in elucidating its involvement in breast cancer.

Abscisic acid (ABA) transport plays a crucial role in seed germination under unfavourable conditions such as cold stress. Both heat shock protein 70 (HSP70) and voltage-dependent anion channel (VDAC) protein are involved in cold stress responses in Arabidopsis. However, their roles in seed germination with regard to ABA signaling remain unknown. Here we demonstrated that Arabidopsis HSP70-16 and VDAC3 jointly suppress seed germination under cold stress conditions. At 4°C, both HSP70-16 and VDAC3 facilitated the efflux of ABA from the endosperm to the embryo and thus inhibited seed germination. HSP70-16 interacted with VDAC3 on the plasma membrane and in the nucleus, and the interplay between HSP70-16 and VDAC3 activated the opening of the VDAC3 ion channel. Our work established a novel function of HSP70-16 in seed germination under cold stress and a possible association of VDAC3 activity with ABA transportation from endosperm to embryo under cold stress conditions. This study reveals that HSP70-16 interacts with VDAC3 and facilitates the opening of the VDAC3 ion channel, which influences ABA efflux from endosperm to embryo, thus negatively regulates seed germination under cold stress.

The warming climate is causing livestock to experience heat stress at an increasing frequency. Holstein cows are particularly susceptible to heat stress because of their high metabolic rate. Heat stress negatively affects immune function, particularly with respect to the cell-mediated immune response, which leads to increased susceptibility to disease. Cattle identified as having enhanced immune response have lower incidence of disease. Therefore, the objective of this study was to evaluate the impact of in vitro heat challenge on blood mononuclear cells from dairy cattle, that had previously been ranked for immune response, in terms of heat shock protein 70 concentration, nitric oxide production, and cell proliferation. Bovine blood mononuclear cells, from Holstein dairy cattle previously ranked for immune response based on their estimated breeding values, were subjected to three heat treatments: thermoneutral, heat stress 1 and heat stress 2. Cells of each treatment were evaluated for heat shock protein 70, cell proliferation and nitric oxide production. Blood mononuclear cells from dairy cattle classified as high immune responders, based on their estimated breeding values for antibody and cell-mediated responses, produced a significantly greater concentration of heat shock protein 70 under most heat stress treatments compared to average and low responders, and greater cell-proliferation across all treatments. Similarly, a trend was observed where high responders displayed greater nitric oxide production compared to average and low responders across heat treatments. Overall, these results suggest that blood mononuclear cells from high immune responder dairy cows are more thermotolerant compared to average and low immune responders, Comment: 37 pages, 3 figures, submitted to BMC Journal of Veterinary Research

The chaperone heat shock protein 70 (Hsp70) is conserved from bacteria to humans and is crucial for avoiding protein misfolding under stress. Bim functions, mainly as one of the B-cell lymphoma 2 (Bcl-2) family proapoptotic members, were identified to be a cochaperone of Hsp70. Herein, we reported that ectopic Bim could constitute the interactions with intrinsic Hsp70 and translate its positive cochaperone activity in vitro to the yeast growth promotion and help Hsp70 to fold its client Ras-like protein. With the help of a specific Hsp70/Bim disruptor, we illustrated that Hsp70/Bim dimers rescue yeast from heat shock. In an organism lacks apoptotic Bcl-2 factors, the proapoptotic Bim in mammalian cells exhibits prosurvival functions.

Activated and promoter-bound heat-shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.

The present study was performed to evaluate the toxic effects of feed-born zearalenone (ZEN) on antioxidative status, immunity, transcriptomic responses of European seabass, and the modulating roles of dietary garlic and/or chitosan powders. Fish (30.7 ± 0.6 g) were randomly arranged in five experimental groups (in triplicates), whereas the first group was fed on the control diet only without any supplements (control), and the second group was fed on the basal diet contaminated with ZEN (0.725 mg/kg diet). Three other groups were fed on ZEN-contaminated diets and simultaneously supplemented with garlic powder (GP) (30 g/kg diet) (ZEN + GP), chitosan powder (CH) (10 g/kg diet) (ZEN + CH), and a mixture of GP and CH (ZEN + GP + CH). Fish were fed on the experimental diets thrice a day for 4 weeks. Two-way ANOVA revealed a gradual decline in serum superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in the ZEN group reaching their lowest levels at the 4th week. Contrariwise, malondialdehyde levels were markedly higher in the ZEN group reaching their highest values at the end of the experiment. A significant decline of total immunoglobulins (P 0.05) was observed in the serum of the ZEN group, especially after the 4th week. Moreover, significant down-regulation of interleukin-4 (IL-4) and interleukin 1 beta (IL-1β) genes (P 0.05) alongside significant up-regulation of tumor necrosis factor-alpha (TNF-α) and heat shock protein 70 (HSP70) genes (P 0.05) in the liver and anterior kidney of ZEN-intoxicated group. Interestingly, dietary supplementation with GP and CH significantly attenuated ZEN-induced oxidative stress, immunosuppression, and modulated transcriptomic responses of ZEN-exposed fish. Moreover, combined dietary supplementation of both feed additives resulted in better effects than each one alone.

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

Hsp70 molecular chaperones facilitate protein disaggregation and proper folding through iterative cycles of polypeptide binding and release that are allosterically coupled to ATP binding and hydrolysis. Hsp70s are ubiquitous and highly conserved across all of life; they bind ATP at an N-terminal nucleotide-binding domain (NBD) and client peptides in the substrate-binding domain (SBD). The NBD and SBD are connected by a highly conserved linker segment that is integrated into the NBD when ATP is bound but is flexible when the NBD is nucleotide-free or bound with ADP. Allosteric coupling is lost when the linker is flexible, and the freed SBD binds peptide clients with high affinity. It was recently discovered that Hsp70–ATP is in an equilibrium between a restraining state (R) with little affinity for peptides and a low ATPase activity, and a stimulating state (S) that binds peptides efficiently, but with rapid kinetics, and has a relatively high ATPase activity. While attempting to characterize the S state, crystal structures of DnaK–ATP were obtained that demonstrate intrinsic Hsp70 plasticity that affects binding interactions with substrate peptides. These structures provide insights into intermediate states along transition pathways in the Hsp70 chaperone cycle.

The kinetic patterns of CpG methylation of the cis-regulatory region of heat stress-related genes on exposed to heat stress (at 42 °C) between the Sahiwal and Frieswal cattle was compared in the present study. Using an in vitro whole blood culture model, cells were continuously exposed to heat stress (at 42 °C) for 6 h. Methylation levels of five genes, viz., GPX1, HSP70, HSP90, c-FOS, and JUN were estimated by SyberGreen-based quantitative methylation-specific PCR (qMSP) assay. CpG methylation kinetics at different time points of heat stress (0.5, 1, 2, 4, 6 h) were analyzed using mixed ANOVA. The initial methylation level, estimated at 37 °C, of HSP70 was significantly high in the Sahiwal breed. A significant (p

His4r is the only known variant of histone H4 in Drosophila. It is encoded by the His4r single-copy gene that is located outside of the histone gene cluster and expressed in a different pattern than H4, although the encoded polypeptides are identical. We generated a null mutant (His4rΔ42) which is homozygous viable and fertile without any apparent morphological defects. Heterozygous His4rΔ42 is a mild suppressor of position-effect variegation, suggesting that His4r has a role in the formation or maintenance of condensed chromatin. Under standard conditions loss of His4r has a modest effect on gene expression. Upon heat-stress the induction of the Heat shock protein (HSP) genes Hsp27 and Hsp68 is stronger in His4rΔ42 mutants with concordantly increased survival rate. Analysis of chromatin accessibility after heat shock at a Hsp27 regulatory region showed less condensed chromatin in the absence of His4r while there was no difference at the gene body. Interestingly, preconditioning before heat shock led to increased chromatin accessibility, HSP gene transcription and survival rate in control flies while it did not cause notable changes in His4rΔ42. Thus, our results suggest that His4r might play a role in fine tuning chromatin structure at inducible gene promoters upon environmental stress conditions.

The major clinical problem in human cancer is metastasis. Metastases are the cause of 90% of human cancer deaths. TAp63 is a critical suppressor of tumorigenesis and metastasis. ΔNp63 acts as a dominant-negative inhibitor to block the function of p53 and TAp63. Although several ubiquitin E3 ligases have been reported to regulate p63 stability, the mechanism of p63 regulation remains partially understood. Herein, we show that CHIP, an E3 ligase with a U-box domain, physically interacts with p63 and promotes p63 degradation. Notably, Hsp70 depletion by siRNA stabilizes TAp63 in H1299 cells and destabilizes ΔNp63 in SCC9 cells. Loss of Hsp70 results in a reduction in the TAp63-CHIP interaction in H1299 cells and an increase in the interaction between ΔNp63 and CHIP in SCC9 cells. Our results reveal that Hsp70 acts as a molecular switch to control CHIP-mediated ubiquitination and degradation of p63 isoforms. Furthermore, regulation of p63 by the Hsp70-CHIP axis contributes to the migration and invasion of tumor cells. Hence, our findings demonstrate that Hsp70 is a crucial regulator of CHIP-mediated ubiquitination and degradation of p63 isoforms and identify a new pathway for maintaining TAp63 or ΔNp63 stability in cancers.

Data de publicació electrónica: 01-01-2021 Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degradation and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissolution. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates observed in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiological and heat-shock triggered PQC. This work is supported by the Ministerio de Ciencia, Innovación y Universidades (Spain), PLAN E and FEDER (BFU2016-75116-P to M.C. and PGC2018-093920-B-I00 to E.H.). The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539) and by Unidad de Excelencia María de Maeztu, funded by the AEI (CEX2018-000792-M) (Spain). M.C. is funded by the Ramon y Cajal program (MINECO-RYC2013-12858). E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain).

Binary enterotoxins Clostridioides difficile CDT toxin, Clostridium botulinum C2 toxin, and Clostridium perfringens iota toxin consist of two separate protein components. The B-components facilitate receptor-mediated uptake into mammalian cells and form pores into endosomal membranes through which the enzymatic active A-components translocate into the cytosol. Here, the A-components ADP-ribosylate G-actin which leads to F-actin depolymerization followed by rounding of cells which causes clinical symptoms. The protein folding helper enzymes Hsp90, Hsp70, and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506 binding protein (FKBP) families are required for translocation of A-components of CDT, C2, and iota toxins from endosomes to the cytosol. Here, we demonstrated that simultaneous inhibition of these folding helpers by specific pharmacological inhibitors protects mammalian, including human, cells from intoxication with CDT, C2, and iota toxins, and that the inhibitor combination displayed an enhanced effect compared to application of the individual inhibitors. Moreover, combination of inhibitors allowed a concentration reduction of the individual compounds as well as decreasing of the incubation time with inhibitors to achieve a protective effect. These results potentially have implications for possible future therapeutic applications to relieve clinical symptoms caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs for their membrane translocation into the cytosol of target cells.

In order to explore the function of vitamin C (VC) and VC-Na in the relief of heat stress injury in chicken cardiomyocytes, 150 30-day-old specific-pathogen-free chickens were randomly divided into a control group (fed normal drinking water), a VC group (50 μg/mL VC in drinking water), and a VC-Na group (50 μg/mL VC-Na in drinking water). After 7 days of adaptation feeding, the chickens were subjected to heat stress at 40 ± 2 °C and 60%-70% humidity for 0, 1, 3, 5, and 10 h, respectively, and the sera and heart tissues of the chickens were collected immediately at the corresponding heat stress time points. The effects of VC and VC-Na supplementation on the relief of chicken myocardial cell injury following heat stress was studied by detecting the levels of LDH, CK, CK-MB, and total antioxidant capacity (T-AOC) in the sera, and through histopathological analysis and the expression of CRYAB, Hsp27, and Hsp70 in the myocardial cells. The results showed that supplementing with 50 μg/mL VC or VC-Na significantly reduced the levels of LDH, and CK-MB in serum as well as heat-stress-induced granular and vacuolar degeneration, myocardial fiber breakage, and cell necrosis, indicating effective resistance to heat-stress damage. Additionally, the levels of T-AOC in serum were increased in the VC and VC-Na groups, suggesting enhancing of antioxidant capacity. Furthermore, the expression of CRYAB were induced at 0, 3, 5, and 10 h (P 0.01) in both VC and VC-Na group, and that of Hsp70 were induced at 0 h (P 0.05) in VC group and at 0, 3, 5, 10 h (P 0.01) in VC-Na group. Thus, supplementing chicken diets with VC or VC-Na presented heat-stress damage resistance by enhancing antioxidant capacity and inducing expression of CRYAB and Hsp70.

The impact of water temperature on the physiology of Channa punctata (Bloch, 1793) was evaluated in the present study. Fish were acclimated at 25 ± 1 °C and then exposed at six different temperatures: 10, 15, 20, 25, 30, and 35 °C. C. punctata exposed at 10, 15, and 20 °C showed 30, 21, and 11% reduced food consumption, respectively compared to 25 °C. Significantly higher respiratory burst and myeloperoxidase activities were found in fish exposed at 20 and 25 °C after 12 h of exposure compared to other treatments. Nitric oxide synthase was significantly higher at 25 °C after 12 h and at 25 and 30 °C exposed fish after 7 days compared to others. The reduced glutathione level was significantly higher at 25 °C compared to other treatments after 7 days of exposure. The thiobarbituric acid reactive substances level was minimum at 25 °C. Significantly lower antioxidant enzymes, catalase, glutathione peroxidase, and glutathione S-transferase were found in gills of fish exposed at 25 °C compared to others in both samples. The highest antioxidant enzyme levels were found at 10 °C. Heat shock protein (Hsp) 70 levels were significantly lower in liver and muscle of fish exposed at 25 °C compared to other treatments. The Hsp level was significantly higher at 35 and 30 °C exposed fish compared to others after 12 h, and the level reduced after 7 days in these treatments. Thermal stress affects food consumption rate, immune system, antioxidant enzymes, and enzyme systems in fish. The elevated Hsp70 level serves as a biomarker of stress in C. punctata. Graphical Abstract.

Intratumoral hypoxia occurs in 90% of solid tumors and is associated with a poor prognosis for patients. Cancer cells respond to hypoxic microenvironments by activating the transcription factors, hypoxia-inducible factor 1 (HIF1) and HIF2. Here, we studied the unique gene expression patterns of 31 different breast cancer cell lines exposed to hypoxic conditions. The EGFR, a member of the ErbB (avian erythroblastosis oncogene B) family of receptors that play a role in cell proliferation, invasion, metastasis, and apoptosis, was induced in seven of the 31 breast cancer cell lines by hypoxia. A functional hypoxia response element (HRE) was identified, which is activated upon HIF1 binding to intron 18 of the EGFR gene in cell lines in which EGFR was induced by hypoxia. CpG methylation of the EGFR HRE prevented induction under hypoxic conditions. The HRE of EGFR was methylated in normal breast tissue and some breast cancer cell lines, and could be reversed by treatment with DNA methyltransferase inhibitors. Induction of EGFR under hypoxia led to an increase in AKT, ERK, and Rb phosphorylation as well as increased levels of cyclin D1, A, B1, and E2F, and repression of p21 in an HIF1α-dependent manner, leading to cell proliferation and migration. Also, increased EGFR expression sensitized cells to EGFR inhibitors. Collectively, our data suggest that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors currently used in the clinic. Significance: Hypoxia sensitizes breast cancer cells to EGFR inhibitors in an HIF1α- and a methylation-specific manner, suggesting patients with hypoxic tumors may benefit from EGFR inhibitors already available in the clinic.

The deposition of highly ordered fibrillar-type aggregates into inclusion bodies is a hallmark of neurodegenerative diseases such as Parkinson’s disease. The high stability of such amyloid fibril aggregates makes them challenging substrates for the cellular protein quality-control machinery1,2. However, the human HSP70 chaperone and its co-chaperones DNAJB1 and HSP110 can dissolve preformed fibrils of the Parkinson’s disease-linked presynaptic protein α-synuclein in vitro3,4. The underlying mechanisms of this unique activity remain poorly understood. Here we use biochemical tools and nuclear magnetic resonance spectroscopy to determine the crucial steps of the disaggregation process of amyloid fibrils. We find that DNAJB1 specifically recognizes the oligomeric form of α-synuclein via multivalent interactions, and selectively targets HSP70 to fibrils. HSP70 and DNAJB1 interact with the fibril through exposed, flexible amino and carboxy termini of α-synuclein rather than the amyloid core itself. The synergistic action of DNAJB1 and HSP110 strongly accelerates disaggregation by facilitating the loading of several HSP70 molecules in a densely packed arrangement at the fibril surface, which is ideal for the generation of ‘entropic pulling’ forces. The cooperation of DNAJB1 and HSP110 in amyloid disaggregation goes beyond the classical substrate targeting and recycling functions that are attributed to these HSP70 co-chaperones and constitutes an active and essential contribution to the remodelling of the amyloid substrate. These mechanistic insights into the essential prerequisites for amyloid disaggregation may provide a basis for new therapeutic interventions in neurodegeneration. The molecular steps that lead to the disaggregation of amyloid fibrils are shown to involve the synergistic action of HSP70 and its co-chaperones DNAJB1 and HSP110.

The DNA fragmentation and failure in post-meiotic maturation of the spermatozoa because of testosterone withdrawal can affect the fertilization potential in varicocele (VCL) patients. To find out the exact mechanism of VCL-induced failure in histone-protamine replacement process and DNA fragmentation, the correlations between the levels of expression of HSP70-2a, HSP90, PCNA, TP1/2 and PCNA genes and the patterns of DNA methylation were investigated before and after testosterone administration in rats. In total, 40 mature male Wistar rats (10 in each group) were assigned between control (with no intervention), control-sham (undergone a simple laparotomy), VCL-induced (VCL-sole), and testosterone-treated VCL-induced (VCLT) groups. The HSP70-2a, HSP90, PCNA, TP1, and TP2 genes expressions and the patterns of global DNA methylation were determined in all groups. A statistically significant (p < 0.05) reduction were found in the HSP70-2a, HSP90, PCNA, TP1 and TP2 genes expressions in VCL-sole group. In VCLT group, testosterone was shown to significantly (p < 0.05) up-regulate the HSP70-2a, HSP90, PCNA, and TP2expression levels, but TP1 expression has not been changed. Furthermore, the VCLT group exhibited higher DNA methylation rates compared to VCL-sole animals. In conclusion, testosterone, by up-regulating the HSP70-2a and HSP90 expressions and maintaining the pre-existing HSP70-2a and HSP90 proteins levels, may be the reason for the significant increment in TP2 expression during post-meiotic stage and can boost the global methylation rates of DNA via up-regulating the PCNA expression, suggesting that administration of testosterone can mitigate the VCL-impaired histone-protamine replacement and DNA methylation rates and protect the cellular DNA content from VCL-induced oxidative stress.

Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to repress CREB-reporter gene activity. This repression is mitigated either by stabilizing osmolytes, which deplete diffuse mHtt or by urea, which negates protein–protein interaction. Our results show that stabilizing polyol osmolytes promote mHtt aggregation, alleviate CREB dysfunction, and promote survival under stress to support the hypothesis that lower molecular weight entities of disease protein are relevant pathogenic species in neurodegeneration.

Molecular chaperones can prevent and repair protein misfolding and aggregation to maintain protein homeostasis in cells. Hsp40 chaperones interact with unfolded client proteins via the dynamic multivalent interaction (DMI) mechanism with their multiple client-binding sites. Here we report that a type I Hsp40 chaperone from Streptococcus pneumonia (spHsp40) forms a concentration-independent polydispersity oligomer state in solution. The crystal structure of spHsp40 determined at 2.75 A revealed that each monomer has a type I Hsp40 structural fold containing a zinc finger domain and C-terminal domains I and II (CTD I and CTD II). Subsequent quaternary structure analysis using a PISA server generated two dimeric models. The interface mutational analysis suggests the conserved C-terminal dimeric motif as a basis for dimer formation and that the novel dimeric interaction between a client-binding site in CTD I and the zinc finger domain promotes the formation of the spHsp40 oligomeric state. In vitro functional analysis demonstrated that spHsp40 oligomer is fully active and possess the optimal activity in stimulating the ATPase activity of spHsp70. The oligomer state of type I Hsp40 and its formation might be important in understanding Hsp40 function and its interaction with client proteins.

The 3.25 Å resolution crystallographic structure of the substrate-binding domain of the erythrocyte-exported Hsp70 chaperone from the malaria parasite Plasmodium falciparum was determined. The parasite domain is shown to be highly similar to the equivalent domain of a human Hsp70 chaperone in structure and mode of substrate engagement., The malaria parasite Plasmodium falciparum extensively modifies erythrocytes that it invades by exporting a large complement of proteins to the host cell. Among these exported components is a single heat-shock 70 kDa class protein, PfHsp70-x, that supports the virulence and growth rate of the parasite during febrile episodes. The ATP-binding domain of PfHsp70-x has previously been resolved and showed the presence of potentially druggable epitopes that differ from those on human Hsp70 chaperones. Here, the crystallographic structure of the substrate-binding domain (SBD) of PfHsp70-x is presented in complex with a hydrophobic peptide. The PfHsp70-x SBD is shown to be highly similar to the counterpart from a human erythrocytic Hsp70 chaperone. The binding of substrate at the interface between β-sandwich and α-helical subdomains of this chaperone segment is also conserved between the malaria parasite and humans. It is hypothesized that the parasite may partly exploit human chaperones for intra-erythrocytic trafficking and maintenance of its exported proteome.

Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology. Significance: This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer.

Heat stress (HS) reaction is a stress response caused by adverse conditions. Currently, the incidence of reproductive malignancies particularly in males has been constantly increasing. This work investigated the effects of saponins derived from the stems and leaves of Panax ginseng (GSLS) on testicular injury induced by scrotal hyperthermia in mice. GSLS (150, 300 mg/kg) were administered intragastrically to mice for 14 days, then exposed to a single scrotal heat treatment at 43°C for 18 min on seventh day. HS induced a significant loss of multinucleate giant cells, desquamation of germ cells in destructive seminiferous tubules. Moreover, HS reduced the serum testosterone, testicular tissue superoxide dismutase activity and glutathione (GSH) content, while significantly enhanced the production of malondialdehyde (p .05). GSLS exhibited the protective potential against HS-induced injury not only by modulating Bcl-2 family and caspase protease family, but also by suppressing the protein levels of heme oxygenase-1 (HO-1), heat shock protein 70 (HSP70), hypoxia inducible factor-1α (HIF-1α) and activation of Mitogen-activated protein kinase (MAPK) signaling pathways (p .05). In conclusion, we clearly demonstrated that GSLS exhibited a significant protective effect against HS-induced testicular dysfunction, mainly the inhibition of oxidative stress associated apoptosis partly via regulation of the MAPK signaling pathway.

Sustaining a healthy proteome is a lifelong challenge for each individual cell of an organism. However, protein homeostasis or proteostasis is constantly jeopardized since damaged proteins accumulate under proteotoxic stress that originates from ever-changing metabolic, environmental, and pathological conditions. Proteostasis is achieved via a conserved network of quality control pathways that orchestrate the biogenesis of correctly folded proteins, prevent proteins from misfolding, and remove potentially harmful proteins by selective degradation. Nevertheless, the proteostasis network has a limited capacity and its collapse deteriorates cellular functionality and organismal viability, causing metabolic, oncological, or neurodegenerative disorders. While cell-autonomous quality control mechanisms have been described intensely, recent work on Caenorhabditis elegans has demonstrated the systemic coordination of proteostasis between distinct tissues of an organism. These findings indicate the existence of intricately balanced proteostasis networks important for integration and maintenance of the organismal proteome, opening a new door to define novel therapeutic targets for protein aggregation diseases. Here, we provide an overview of individual protein quality control pathways and the systemic coordination between central proteostatic nodes. We further provide insights into the dynamic regulation of cellular and organismal proteostasis mechanisms that integrate environmental and metabolic changes. The use of C. elegans as a model has pioneered our understanding of conserved quality control mechanisms important to safeguard the organismal proteome in health and disease.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role. Electronic supplementary material The online version of this article (10.1007/s12192-020-01134-9) contains supplementary material, which is available to authorized users.

Preeclampsia (PE) and fetal growth restriction (FGR) have abnormal placental implantation and endothelial dysfunction in common. However, their etiologies are not well understood. Both heat shock protein 70 (Hsp70) and nitric oxide (NO) are suggested to play a major role in the regulation of maternal and fetoplacental hemodynamics. In this study, the association of PE with FGR and Hsp70 or NO was analyzed.A total of 30 cases of PE, 25 cases of PE complicated with FGR and 50 cases of normal pregnant women were chose, and PE and normal animal models were constructed. Subsequently, the levels of Hsp70 and NO in serum and placental tissues of humans and animals were measured and compared. Further, rats were injected with pLV-NC-shRNA, pLV-Hsp70-shRNA, pLV-EFIa-NC, and pLV-EFIa-Hsp70, respectively, the weight of each conceptus, number of pups, fetal crown to tail length, total weight of the placenta/fetus unit, and the content of NO were analyzed.The expression of Hsp70 in serum and placental tissues of PE complicated with or without FGR group was increased, whereas the content of NO was decreased compared to the normal group. The fetal weight (FW) of the Hsp70 targeted suppression group was higher than the other two groups, whereas the placental weight (PW) was reversed. Also, NO synthase (NOS) expression was decreased in the Hsp70 over-expression group.We speculated that the enhancement of Hsp70 might be related to the development of PE combined with FGR through inhibiting the synthesis of NOS.

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH(4)) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH(4) triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.

Rattle-structured nanoparticles with movable cores, porous shells and hollow interiors have shown great effectiveness in drug delivery and cancer theranostics. Targeting autophagy and glucose have provided alternative strategies for cancer intervention therapy. Herein, rattle-structured polydopamine@mesoporous silica nanoparticles were prepared for in vivo photoacoustic (PA) imaging and augmented low-temperature photothermal therapy (PTT) via complementary autophagy inhibition and glucose metabolism. Methods: The multifunctional rattle-structured nanoparticles were designed with the nanocore of PDA and the nanoshell of hollow mesoporous silica (PDA@hm) via a four-step process. PDA@hm was then loaded with autophagy inhibitor chloroquine (CQ) and conjugated with glucose consumer glucose oxidase (GOx) (PDA@hm@CQ@GOx), forming a corona-like structure nanoparticle. Results: The CQ and GOx were loaded into the cavity and decorated onto the surface of PDA@hm, respectively. The GOx-mediated tumor starvation strategy would directly suppress the expression of HSP70 and HSP90, resulting in an enhanced low-temperature PTT induced by PDA nanocore. In addition, autophagy inhibition by the released CQ made up for the loss of low-temperature PTT and starvation efficiencies by PTT- and starvation-activated autophagy, realizing augmented therapy efficacy. Furthermore, the PDA nanocore in the PDA@hm@CQ@GOx could be also used for PA imaging. Conclusion: Such a "drugs" loaded rattle-structured nanoparticle could be used for augmented low-temperature PTT through complementarily regulating glucose metabolism and inhibiting autophagy and in vivo photoacoustic imaging.

Angiotensin II exerts a cardinal role in the pathogenesis of hypertension and renal injury via action of angiotensin II type 1 (AT(1)) receptors. Local renin-angiotensin system (RAS) activity is essential for the mechanisms mediating pathophysiological functions. Proximal tubular angiotensinogen and tubular AT(1) receptors are augmented by intrarenal angiotensin II. Caveolin 1 plays an important role as a regulatory molecule for the compartmentalization of redox signaling events through angiotensin II–induced NADPH oxidase activation in the kidney. A role for the renin-angiotensin system in the development and/or maintenance of hypertension has been demonstrated in spontaneously hypertensive rats (SHRs). Many effects of angiotensin II are dependent on the AT(1) stimulation of reactive oxygen species (ROS) production by NADPH oxidase. Angiotensin II upregulation stimulates oxidative stress in proximal tubules from SHR. The NADPH oxidase 4 (Nox4) is abundantly expressed in kidney proximal tubule cells. Induction of the stress response includes synthesis of heat shock protein 70, a molecular chaperone that has a critical role in the recovery of cells from stress and in cytoprotection, guarding cells from subsequent insults. HSP70 chaperones function in part by driving the molecular triage decision, which determines whether proteins enter the productive folding pathway or result in client substrate ubiquitination and proteasomal degradation. This review examines regulation of losartan-mediated antioxidative stress responses by the chaperone HSP70 in proximal tubule cells of spontaneously hypertensive rats.

Extracellular heat shock proteins (Hsp) influence the adaptive immune response and may ameliorate pathogenesis of autoimmune diseases. While some preclinical observations suggest that highly conserved bacterial and/or murine Hsp70 peptides have potential utility in treatment of rheumatoid arthritis (RA) via induction of T regulatory cells (Treg), the role of extracellular inducible human Hsp70 in adaptive immune processes requires further investigation. The present study evaluated Hsp70 influence on inflammatory cytokine-mediated modulation of T cell immunophenotype in ways that influence RA onset and severity. Initial experiments in the present investigation revealed that serum levels of Hsp70 are approximately 2-fold higher in RA patients versus healthy control subjects. To explore the effect of extracellular Hsp70 on key processes underlying the adaptive immune system, the effects of a highly pure, substrate-, and endotoxin-free human Hsp70 on polarization of the T helper cell subpopulations, including CD4+IL-17+ (Th17), CD4+FoxP3+ (Treg), CD4+IFN-γ+ (Th1), and CD4+IL-4+ (Th2), were studied in naïve human peripheral blood mononuclear cell (PBMC) cultures stimulated with anti-CD3/28 mAb. Major findings included an observation that while Hsp70 treatment increased Th17 frequencies and Th17/Treg ratio, the frequency of Th1 cells and the Th1/Th2 ratio were significantly decreased in the Hsp70-treated PBMC cultures. Moreover, data shown here provides preliminary suggestion that major contributing Hsp70-mediated immunomodulation includes interleukin 6 (IL-6) influence on Th17/Treg and Th1/Th2, since expression of this inflammatory cytokine is enhanced by in vitro Hsp70 treatment. These results are nevertheless preliminary and require further investigation to validate the above model.

The heat-shock factor Hsp70 and other molecular chaperones play a central role in nascent protein folding. Elucidating the task performed by individual chaperones within the complex cellular milieu, however, has been challenging. One strategy for addressing this goal has been to monitor protein biogenesis in the absence and presence of inhibitors of a specific chaperone, followed by analysis of folding outcomes under both conditions. In this way, the role of the chaperone of interest can be discerned. However, development of chaperone inhibitors, including well-known proline-rich antimicrobial peptides, has been fraught with undesirable side effects, including decreased protein expression yields. Here, we introduce KLR-70, a rationally designed cationic inhibitor of the Escherichia coli Hsp70 chaperone (also known as DnaK). KLR-70 is a 14-amino acid peptide bearing naturally occurring residues and engineered to interact with the DnaK substrate-binding domain. The interaction of KLR-70 with DnaK is enantioselective and is characterized by high affinity in a buffered solution. Importantly, KLR-70 does not significantly interact with the DnaJ and GroEL/ES chaperones, and it does not alter nascent protein biosynthesis yields across a wide concentration range. Some attenuation of the anti-DnaK activity of KLR-70, however, has been observed in the complex E. coli cell-free environment. Interestingly, the d enantiomer D-KLR-70, unlike its all-L KLR-70 counterpart, does not bind the DnaK and DnaJ chaperones, yet it strongly inhibits translation. This outcome suggests that the two enantiomers (KLR-70 and D-KLR-70) may serve as orthogonal inhibitors of chaperone binding and translation. In summary, KLR-70 is a novel chaperone inhibitor with high affinity and selectivity for bacterial Hsp70 and with considerable potential to help in parsing out the role of Hsp70 in nascent protein folding.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

The 70-kDa heat shock proteins (HSP70) may provide relevant information about the endothelial dysfunction in cardiovascular diseases. Located in the intracellular milieu (iHSP70), they are essential chaperones that inhibit nuclear factor kappa B activation, stimulate nitric oxide production and superoxide dismutase activity, and inhibit apoptosis. However, under stressful conditions, HSP70 can be released into the extracellular medium (eHSP70) and act as an inflammatory mediator. Although studies have reported the vasoprotective role of iHSP70, the evidence regarding eHSP70 is contradictory. eHSP70 can activate NFκB and activator protein-1, thus stimulating the release of inflammatory cytokines and production of reactive oxygen species. Due to the antagonistic nature of HSP70 according to its location, the eHSP70/iHSP70 ratio (Heck index) has been proposed as a better marker of inflammatory status; however, more studies are required to confirm this hypothesis. Therefore, this review summarises studies that, together, describe the role of HSP70 in endothelial dysfunction.

Resveratrol (Res) was recently reported to ameliorate hypoxia/reoxygenation (H/R)-caused oxidative stress in H9c2 cardiomyocytes through promoting the mitochondrial translocation of DJ-1 protein and subsequently preserving the activity of mitochondrial complex I. However, it is noteworthy that DJ-1 possesses no mitochondria-targeting sequence. Therefore, how Res induces DJ-1 mitochondrial translocation is an important and interesting question for further exploration. Glucose-regulated protein 75 (Grp75), whose N-terminus contains a 51-amino acid long mitochondrial-targeting signal peptide, is a cytoprotective chaperone that partakes in mitochondrial import of several proteins. Here, the contribution of Grp75 to mitochondrial import of DJ-1 by Res was investigated in a cellular model of H/R. Our results showed that Res upregulated the expression of DJ-1 protein, enhanced the interaction of DJ-1 and Grp75, and promoted DJ-1 translocation to mitochondria from cytosol in H9c2 cardiomyocytes undergoing H/R. Importantly, knockdown of Grp75 markedly reduced the interaction of DJ-1 with Grp75 and subsequent DJ-1 mitochondrial translocation induced by Res. Furthermore, Res pretreatment promoted the association of DJ-1 with ND1 and NDUFA4 subunits of complex I, preserved the activity of complex I, decreased mitochondria-derived reactive oxygen species production, and eventually ameliorated H/R-caused oxidative stress damage. Intriguingly, these effects were largely prevented also by small interfering RNA targeting Grp75. Overall, these results suggested that Grp75 interacts with DJ-1 to facilitate its translocation from cytosol to mitochondria, which is required for Res-mediated preservation of mitochondria complex I and cardioprotection from H/R-caused oxidative stress injury.

Background This phase I study aimed to evaluate the safety, peptide-specific immune responses, and anti-tumor effects of a novel vaccination therapy comprising multi-HLA-binding heat shock protein (HSP) 70/glypican-3 (GPC3) peptides and a novel adjuvant combination of hLAG-3Ig and Poly-ICLC against metastatic gastrointestinal cancers. Methods HSP70/GPC3 peptides with high binding affinities for three HLA types (A*24:02, A*02:01, and A*02:06) were identified with our peptide prediction system. The peptides were intradermally administered with combined adjuvants on a weekly basis. This study was a phase I dose escalation clinical trial, which was carried out in a three patients’ cohort; in total, 11 patients were enrolled for the recommended dose. Results Seventeen patients received this vaccination therapy without dose-limiting toxicity. All treatment-related adverse events were of grades 1 to 2. Peptide-specific CTL induction by HSP70 and GPC3 proteins was observed in 11 (64.7%) and 13 (76.5%) cases, respectively, regardless of the HLA type. Serum tumor marker levels were decreased in 10 cases (58.8%). Immunological analysis using PBMCs indicated that patients receiving dose level 3 presented with significantly reduced T cell immunoglobulin and mucin-domain containing-3 (TIM3)-expressing CD4 + T cells after one course of treatment. PD-1 or TIM3-expressing CD4 + T cells and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT)-expressing CD8 + T cells in PBMCs before vaccination were negative predictive factors for survival. Conclusions This novel peptide vaccination therapy was safe for patients with metastatic gastrointestinal cancers.

Significance Heat shock protein 70 kDa (Hsp70) plays a central role in maintaining protein homeostasis. It cooperates with cochaperone Hsp40, which stimulates Hsp70 ATPase activity and presents protein substrates to Hsp70 to assist refolding. The mechanism by which Hsp40 regulates the intramolecular and intermolecular changes of Hsp70 is still largely unknown. Here, by bulk and single-molecule FRET, we report the conformational dynamics of Hsp70 and its regulation by Hsp40 as well as the kinetics of the multistep Hsp70–Hsp40 functional cycle. We show that Hsp40 modulates the conformations of ATP-bound Hsp70 to a domain-undocked ATPase-stimulated state, and facilitates the formation of a heterotetrameric Hsp70–Hsp40 complex. Our findings provide insights into the functional mechanism of this core chaperone machinery., Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

Research efforts of elucidating the molecular mechanisms governing heat shock response which imparts thermo-tolerance ability to indigenous breeds are very scanty. Therefore, a study was conducted with the primary objective to determine the impact of heat stress on the expression pattern of different heat shock response genes in the hepatic tissues of indigenous Salem Black goat. The study was conducted for a period of 45 days in twelve 1-year-old female Salem Black breed goats. The animals were randomly allocated into two groups of six animals each, C (n = 6; Salem Black control) and HS (n = 6; Salem Black heat stress). The C animals were maintained in the shed in comfort condition while HS animals were exposed outside to summer heat stress between 10:00 h to 16:00 h during experimental period. The animals were slaughtered at the end of study and their liver samples were collected for assessing the different heat shock response genes. Based on the results obtained from the study it was established that the heat shock protein 70 (HSP70), HSP90, super oxide dismutase (SOD), nitrous oxide synthase 1 (NOS1) genes were significantly (P

The present study aimed to investigate the protective role of sirtuin 1 (SIRT1) and oxygen regulated protein 150 (ORP150) in a rat COPD model by inducing changes in ER stress and apoptosis. We separated 48 Sprague Dawley (SD) rats into four groups randomly: the control group, resveratrol group, COPD group and the resveratrol intervention group. Rats were challenged with cigarette smoke and lipopolysaccharide with resveratrol (a selective activator of SIRT1). The lung functions of the rats were measured and recorded. The expression levels of SIRT1 and ORP150 in lung tissues were examined by western blot and RTq PCR. The expression levels of the ER stress apoptosis-associated protein were determined .The apoptotic level of lung tissues was analyzed. The results suggest that SIRT1 attenuated apoptosis and ER stress in the lung tissues of rats with COPD. During this process, a positive correlation was identified between SIRT1 and ORP150.

Μeagre (Argyrosomus regius) is a newly introduced species in the aquaculture sector characterized by high growth rates and good nutritional value. However, the optimum conditions such as water temperature for the rearing of this species are still under research. The aim of the present study was to investigate several biochemical responses in the heart, muscle, liver, and intestine of meagre reared under different water temperatures (17, 23, and 26 °C) in comparison to the initial acclimation temperature at 20 °C. To assess its cellular stress response, the molecular markers of Hsp70 and Hsp90 induced levels, p38 MAPK and p44/42 MAPK phosphorylated levels, ubiquitin conjugates, and Bcl-2 levels were determined. Moreover, to investigate its antioxidant defense, the activities of superoxide dismutase, catalase, and glutathione reductase were measured. The obtained results in meagre showed a distinct tissue-specific response regarding the variations observed in the studied parameters under different acclimation temperatures. Specifically, most examined tissues presented a decrease in the levels of the examined markers compared to 20 °C, while in the intestine, an increase of Hsp70 and p38 MAPK levels was observed at 23 and 26 °C. However, data on the final weight and length of the fish lead to the conclusion that high water temperatures such as 26 °C might be the best for the rearing of this species. Therefore, it is assumed that the observed differential molecular responses may act in a cell-protective way at all temperatures, irrespectively of the effect of temperature on growth parameters.

The Alzheimer’s disease pathology is associated with accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. The formation of initial nucleus triggers conformational changes in Tau and leads to its deposition. Hence, there is a need to eliminate these toxic proteins for proper functioning of neuronal cells. In this aspect, we screened the effect of basic limonoids such as gedunin, epoxyazadiradione, azadirone and azadiradione on inhibiting Tau aggregation as well as disintegration of induced Tau aggregates. It was observed that these basic limonoids effectively prevented aggregates formation by Tau and also exhibited the property of destabilizing matured Tau aggregates. The molecular docking analysis suggests that the basic limonoids interact with hexapeptide regions of aggregated Tau. Although these limonoids caused the conformational changes in Tau to β-sheet structure, the cytological studies indicate that basic limonoids rescued cell death. The dual role of limonoids in Tau aggregation inhibition and disintegration of matured aggregates suggests them to be potent molecules in overcoming Tau pathology. Further, their origin from a medicinally important plant neem, which known to possess remarkable biological activities was also found to play protective role in HEK293T cells. Basic limonoids were non-toxic to HEK293T cells and also aided in activation of HSF1 by inducing its accumulation in nucleus. Western blotting and immunofluorescence studies showed that HSF1 in downstream increased the transcription of Hsp70 thus, aggravating cytosolic Hsp70 levels that can channel clearance of aberrant Tau. All these results mark basic limonoids as potential therapeutic natural products.