In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

Global warming poses severe threats to agricultural production, including soybean. One of the major mechanisms for organisms to combat heat stress is through heat shock proteins (HSPs) that stabilize protein structures at above-optimum temperatures, by assisting in the folding of nascent, misfolded, or unfolded proteins. The HSP40 subgroups, or the J-domain proteins, functions as co-chaperones. They capture proteins that require folding or refolding and pass them on to HSP70 for processing. In this study, we have identified a type-I HSP40 gene in soybean, GmDNJ1, with high basal expression under normal growth conditions and also highly inducible under abiotic stresses, especially heat. Gmdnj1-knockout mutants had diminished growth in normal conditions, and when under heat stress, exhibited more severe browning, reduced chlorophyll contents, higher reactive oxygen species (ROS) contents, and higher induction of heat stress-responsive transcription factors and ROS-scavenging enzyme-encoding genes. Under both normal and heat-stress conditions, the mutant lines accumulated more aggregated proteins involved in protein catabolism, sugar metabolism, and membrane transportation, in both roots and leaves. In summary, GmDNJ1 plays crucial roles in the overall plant growth and heat tolerance in soybean, probably through the surveillance of misfolded proteins for refolding to maintain the full capacity of cellular functions.

Heat shock protein 40 (Hsp40) acts as a co-chaperone with Hsp70 to promote protein folding, protein transport and degradation. The human Hsp40 family contains more than 40 members, some of which can exist as phosphoproteins in the cell. However, little is known about the protein kinases responsible for their phosphorylation and the functional relevance of this post-translational modification remains elusive. Here we show that Hsp40/DnaJB1 is an in vitro and in vivo substrate for the mitogen-activated protein kinase-activated protein kinase 5 (MK5). MK5 and Hsp40/DnaJB1 form complexes in cells and this interaction is accomplished by the C-terminal regions of both proteins. MK5 can phosphorylate Hsp40/DnaJB1 at several residues in vitro. Studies with specific phosphoantibodies indicate that MK5 phosphorylates Hsp40/DnaJB1 in vivo at Ser-149 or/and Ser-151 and Ser-171 in the C-terminal domain of Hsp40/DnaJB1. MK5 modestly stimulates the ATP hydrolyse activity of Hsp40/Hsp70 complex and enhances the repression of heat shock factor 1 driven transcription by Hsp40/DnaJB1.

Plant and animal cells possess a ubiquitous protein known as heat shock proteins (HSPs). Hsps were originally described in relation to heat shock and against abiotic and biotic stresses. Heat shock protein was classified in other crops on the bases of single classes or all classes but in Citrus sinensis Hsps groups, classes, subfamilies and members were not classified and characterized up to our knowledge.

The effects of early heat conditioning on the acute heat stress response in broilers were investigated via the growth performance, dopamine, serotonin, and corticosterone and the expression of heat shock proteins (HSP) and heat shock factors. One-day-old chicks (n = 144) were divided into 3 groups in a 35-d experiment (48 chicks per each group). Group 1 (C) was treated with an optimum temperature, group 2 (CH) was treated with 40°C ± 1°C on day 35 (5 h), and group 3 (HH) was treated with 40°C ± 1°C on day 5 (24 h) and day 35 (5 h). On day 7, the body weight gain was lower (P < 0.05) in HH than in C and CH. On day 35, the heat-treated groups (CH and HH) had lower weight gains than the C group (P < 0.05), whereas the feed conversion ratio was lower in HH (P < 0.05). Serum corticosterone was higher in CH than in C, but HH and C did not differ (P < 0.05). Liver HSP70 protein expression was higher in CH than HH and C (P < 0.05), which did not differ, and HSP40 protein expression was higher in CH than C (P < 0.05). These results suggest that early heat conditioning may reduce acute heat stress on broiler.

How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.

Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes. Thus, tagging Hsps with fluorescent proteins can interfere with their chaperone activity. To overcome this limitation, we have exploited bicistronic constructs for the concurrent expression of a non-tagged Hsp and fluorescent reporter from a single mRNA in cells. We used the Hsp-encoding bicistronic constructs in a cell-based model of protein aggregation, using a destabilised (mutant) form of firefly luciferase (mFluc) that forms inclusion bodies in cells. Expression of Hsp40, Hsp70, or Hsp40 and Hsp70 in cells expressing mFluc decreased the formation of inclusion bodies by 25-46% compared to controls. Moreover, there was a concentration-dependent decrease in the proportion of cells with inclusions when Hsp70, or Hsp40 and Hsp70 were co-expressed with mFluc in cells. The Hsp-encoding bicistronic constructs enable transfection efficiencies and concentration-dependent effects of Hsp expression to be determined using fluorescence based techniques, without the need to tag the Hsp with a fluorescent protein.

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.

Cells and tissues are continuously subject to environmental insults, such as heat shock and oxidative stress, which cause the accumulation of cytotoxic, aggregated proteins. We previously found that Fas Apoptosis Inhibitory Molecule (FAIM) protects cells from stress-induced cell death by preventing abnormal generation of protein aggregates similar to the effect of small heat shock proteins (HSPs). Protein aggregates are often associated with neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we sought to determine how FAIM protein dynamics change during cellular stress and how FAIM prevents the formation of amyloid-β aggregates/fibrils, one of the pathological hallmarks of AD. Here, we found that the majority of FAIM protein shifts to the detergent-insoluble fraction in response to cellular stress. A similar shift to the insoluble fraction was also observed in small heat shock protein (sHSP) family molecules, such as HSP27, after stress. We further demonstrate that FAIM is recruited to sHSP-containing complexes after cellular stress induction. These data suggest that FAIM might prevent protein aggregation in concert with sHSPs. In fact, we observed the additional effect of FAIM and HSP27 on the prevention of protein aggregates using an in vitro amyloid-β aggregation model system. Our work provides new insights into the interrelationships among FAIM, sHSPs, and amyloid-β aggregation.

We characterized Schistosoma japonicum HSP40 (Sjp40) and HSP90α (Sjp90α) in this study. Western blot analysis revealed both are present in soluble egg antigens and egg secretory proteins, implicating them in triggering the host immune response after secretion from eggs into host tissues. These observations were confirmed by immunolocalization showing both HSPs are located in the Reynolds' layer within mature eggs, suggesting they are secreted by miracidia and accumulate between the envelope and the eggshell. Both HSPs are present in the musculature and parenchyma of adult males and in the vitelline cells of females; only Sjp90α is present on the tegument of adults. Sjp40 was able to enhance the expression of macrophages, dendritic cells, and eosinophilic cells in mouse liver non-parenchymal cells, whereas rSjp90α only stimulated the expression of dendritic cells. T helper 1 (Th1), Th2, and Th17 responses were increased upon rSjp40 stimulation in vitro, but rSjp90 only stimulated an increased Th17 response. Sjp40 has an important role in reducing the expression of fibrogenic gene markers in hepatic stellate cells in vitro. Overall, these findings provide new information on HSPs in S. japonicum, improving our understanding of the pathological roles they play in their interaction with host immune cells.

IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measurements, EGFP expression was monitored after double-transformation of pET28a-EGFP and pET21a-(Hsp27/Hsp40/Hsp70) plasmids into E. coli using fluorimetry. Moreover, the expression level, bacterial growth curve, and plasmid and expression stability were compared to an IPTG- inducible system using EGFP. Statistical analysis revealed a significant difference in EGFP expression between autoinducible and IPTG-inducible systems. The expression level was higher in Hsp27 system than Hsp70/Hsp40 systems. However, the highest amount of expression was observed for the inducible system. IPTG-inducible and Hsp70 systems showed more lag-time in the bacterial growth curve than Hsp27/Hsp40 systems. A relatively stable EGFP expression was observed in SILEX systems after several freeze-thaw cycles within 90 days, while, IPTG-inducible system showed a decreasing trend compared to the newly transformed bacteria. Moreover, the inducible system showed more variation in the EGFP expression among different clones than clones obtained by SILEX systems. All designed SILEX systems successfully self-induced the expression of protein models. In conclusion, Hsp27 system could be considered as a suitable autoinducible system for protein expression due to less metabolic burden, lower variation in the expression level, suitable plasmid and expression stability, and a higher expression level.

Most heat shock proteins (Hsps) function as molecular chaperones that help organisms to cope with stress. Although the best empirical evidence is related to heat shock, there is evidence that Hsps and their encoding genes are involved in resistance to other ecologically relevant types of stresses such as those imposed by high population density. We quantified density-dependent gene expression of large (i.e. Hsp40, Hsc70 and Hsp90) and small (Hsp20.5, Hsp20.6 and Hsp20.7) heat shock genes in neural tissue of fifth-instar nymphs of the Australian plague locust, Chortoicetes terminifera, using reverse transcription-quantitative PCR. Locusts are of particular interest when studying the influence of stress induced by high population density since they show an extreme form of phenotypic plasticity changing from a cryptic solitarious phase to a swarming gregarious phase. Crowding led to a synchronous and sustained 2-3 fold increase in the expression of only two Hsp genes, Hsp20.5 and Hsp20.7, which do not BLAST with any known animal sequences and therefore are likely to be unique to members of the Orthoptera. This study opens a range of experiments to investigate the possibility of specific roles for these two small Hsps in the resistance to stressful conditions imposed by crowded environments and/or the expression of gregarious behavior as well as their evolutionary significance to locusts whose populations are regularly exposed to high density conditions in the field.

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance.

Amyloid and amyloid-like protein aggregations are hallmarks of multiple, varied neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. We previously reported that spinocerebellar ataxia type 14 (SCA14), a dominant-inherited neurodegenerative disease that affects cerebellar Purkinje cells, is characterized by the intracellular formation of neurotoxic amyloid-like aggregates of genetic variants of protein kinase Cγ (PKCγ). A number of protein chaperones, including heat shock protein 70 (Hsp70), promote the degradation and/or refolding of misfolded proteins and thereby prevent their aggregation. Here, we report that, in various SCA14-associated, aggregating PKCγ variants, endogenous Hsp70 is incorporated into aggregates and that expression of these PKCγ mutants up-regulates Hsp70 expression. We observed that PKCγ binds Hsp70 and that this interaction is enhanced in the SCA14-associated variants, mediated by the kinase domain that is involved in amyloid-like fibril formation as well as the C2 domain of PKCγ. Pharmacological up-regulation of Hsp70 by the Hsp90 inhibitors celastrol and herbimycin A attenuated the aggregation of mutant PKCγ in primary cultured Purkinje cells. Up-regulation of Hsp70 diminished net PKCγ aggregation by preventing aggregate formation, resulting in decreased levels of apoptotic cell death among primary cultured Purkinje cells expressing the PKCγ variant. Of note, herbimycin A also ameliorated abnormal dendritic development. Extending our in vitro observations, administration of celastrol to mice up-regulated cerebellar Hsp70. Our findings identify heat shock proteins as important endogenous regulators of pathophysiological PKCγ aggregation and point to Hsp90 inhibition as a potential therapeutic strategy in the treatment of SCA14.

Heat shock proteins (HSPs), a family of conserved proteins that are produced by cells in response to stresses, are known as molecular chaperones with a range of housekeeping and cellular protective functions. The 40 kD heat shock protein (HSP40) is a co-chaperone for HSP70 in the regulation of ATP hydrolysis. Unlike its well-documented cofactor HSP70, little is currently known regarding the biological functions of HSP40 in crustacean species such as penaeid shrimp. In the present study, the cDNA encoding HSP40 (Lv-HSP40) was identified from the Pacific white shrimp Litopenaeus vannamei, a highly significant commercial culture species. The structural organization indicates that Lv-HSP40 belongs to the type-I HSP40s. The muscle, gill, and hepatopancreas are the main sites of Lv-HSP40 transcript expression. Within these tissues, Lv-HSP40 mRNA were predominantly exhibited in the myocytes, epithelial cells and hepatopancreatic cells, respectively. Under acute thermal stress in the culture environment, Lv-HSP40 transcript levels are significantly induced in these three tissues, while low pH stress only upregulates Lv-HSP40 mRNA in the hepatopancreas and gill. During ontogenesis, Lv-HSP40 transcript levels are high at early embryonic stages and drop sharply at late embryonic and early larval stages. The ovary is another major organ of Lv-HSP40 mRNA expression in female shrimp, and Lv-HSP40 transcripts were mainly presented in the follicle cells but only weekly detected in the oocytes. Ovarian Lv-HSP40 mRNA levels increase continuously during gonadal development. Silencing of the Lv-HSP40 gene by RNA interference may effectively delay ovarian maturation after unilateral eyestalk ablation. The roles of Lv-HSP40 in ovarian development are speculated to be independent of its cofactor HSP70, and the vitellogenesis factor vitellogenin (Vg) and vitellogenin receptor (VgR). Our study, as a whole, provides new insights into the roles of HSP40 in multiple physiological processes in L. vannamei: (1) HSP40 is a responding factor during stressful conditions; and (2) HSP40 participates in embryonic and ovarian development.

The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a Hsp20 (StHsp20) and a Hsp40 (StHsp40) in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, Scrippsiella trochoidea. We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR. The results revealed StHsp20 and StHsp40 exhibited mRNA accumulation patterns that were highly similar in response to heat stress but completely different toward cold stress, which implies that the mechanisms underlying thermal and cold acclimation in dinoflagellates are regulated by different sets of genes. The StHsp20 was probably related to the heat tolerance of the species, and StHsp40 was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of StHsp40 was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied StHsp40 might be engaged in resting cyst formation of S. trochoidea. Our findings enriched the knowledge about possible cross-talk of different Hsp members in dinoflagellates and provided clues to further explore the molecular underpinnings underlying resting cyst production and broad temperature tolerance of this group of HABs contributors.

Breast cancer is the most commonly occurring cancer among women. MicroRNAs as noncoding small RNA molecules play pivotal roles in cancer-related biological processes. Increased levels of microRNA-29a in the serum of breast cancer patients have been reported. Since heat shock proteins (HSPs) play important roles in cell events, the quantitative fluctuations in their cellular levels could be deemed as key indicators of how the exerted treatment alters cell behavior. In this regard, using an antisense small RNA, we attempted to investigate the effects of miR-29a knockdown on the expression of HSPs genes in the MCF-7 breast cancer cell line. MCF-7 cells were cultured in high-glucose Dulbecco's modified Eagle's medium with 10% FBS. Studied cells were subdivided into five groups: treated with scramble, anti-miR-29a, anti-miR-29a + Taxol, Taxol, and control. Taxol was added 24 h post-anti-miR transfection and RNA extraction, and cDNA synthesis was done 48 h later. The changes in expression of HSP27, HSP40, HSP60, HSP70, and HSP90 were evaluated by real-time PCR. Our results revealed that inhibitors of microRNA-29a promote apoptosis through upregulation of HSP60 level and downregulation of HSP27, HSP40, HSP70, and HSP90 levels and could be contemplated as a compelling alternative for Taxol employment with similar effects and/or to sensitize cancer cells to chemotherapy with fewer side effects.

Heat shock proteins (HSPs) have molecular chaperone functions in protein biogenesis as well as cytoprotective functions against deleterious environmental stresses, and they work mainly inside of the cells. HSPs are usually induced in living cells that have been exposed to mild stresses or have recovered from severe stresses. Here, we show the enhanced synthesis of HSPs in gradually and necrotically dying cells that were treated with a high concentration of acrylamide (10 mM). This treatment caused irreversible cell death. The synthesis of HSPs, which was enhanced before cell death, was mediated by the activation of heat shock transcription factor 1 (HSF1); that is, the treatment led to the phosphorylation of HSF1, formation of characteristic HSF1 granules in the nucleus, and acquisition of DNA binding ability of HSF1. The induction of HSPs by acrylamide treatment was dependent on the consensus sequence of heat shock element (HSE) as demonstrated by a reporter assay. Also, several HSPs (Hsp90, Hsc70, Hsp70, Hsp60, Hsp47, Hsp40, and Hsp27) were detected outside of the cells after the treatment with acrylamide, indicating that these HSPs are released from necrotically dead cells. These results suggest that when cells are slowly and irreversibly dying, they augment the expression of HSPs and release them outside of the cells as a danger signal or dying messages.

Heat shock protein 90 (HSP90) plays a critical role in the survival of cancer cells including muscle invasive bladder cancer (MIBC). The addiction of tumor cells to HSP90 has promoted the development of numerous HSP90 inhibitors and their use in clinical trials. This study evaluated the role of inhibiting HSP90 using STA9090 (STA) alone or in combination with the HSP70 inhibitor VER155008 (VER) in several human MIBC cell lines. While both STA and VER inhibited MIBC cell growth and migration and promoted apoptosis, combination therapy was more effective. Therefore, the signaling pathways involved in MIBC were systematically interrogated following STA and/or VER treatments. STA and not VER reduced the expression of proteins in the p53/Rb, PI3K and SWI/SWF pathways. Interestingly, STA was not as effective as VER or combination therapy in degrading proteins involved in the histone modification pathway such as KDM6A (demethylase) and EP300 (acetyltransferase) as predicted by The Cancer Genome Atlas (TCGA) data. This data suggests that dual HSP90 and HSP70 inhibition can simultaneously disrupt the key signaling pathways in MIBC.

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms.