In order to look for proteins induced by heat and cold stresses, we performed a protein extraction followed by SDS PAGE after exposure of S. thermophilus cells to the two temperature shocks. In addition to a few bands most likely corresponding to well known proteins accumulated during the early phase of the heat shock response, a band of apparent molecular mass of 75 kDa was induced by both heat and cold (fig. 2). The two bands were eluted from the HS and CS lanes and N-terminal sequenced. The first 13 amino acids (MNNNFNNMDDLFN) were the same for both and were identical to those of the ClpL pro- tein of S. thermophilus  and to a previously reported 75 kDa heat shock induced protein . Based on the N-ter- minal 10 amino acids of the identified protein, we synthe- sized a 30 b oligonucleotide that was used to perform Southern hybridization with the KpnI-digested SFi39 chromosome. A 1500 bp fragment was isolated, cloned and sequenced; one 300 aa ORF starting with the expected 13 amino acids was identified (fig. 3) and resulted 99% identical to the class III heat shock protein ClpL of S. ther- mophilus CNRZ1066, a member of the ClpA/B ATPase family .
Abstract: Type 2 diabetes mellitus (T2DM) is still a global health problem. Current T2DM treatments are limited to curing the symptoms and have not been able to restore insulin sensitivity in insulin-sensitive tissues that have become resistant. In the past decade, some studies have shown the signi ﬁ cant role of a chaperone family, heat shock protein 70 (HSP70), in insulin resistance pathogenesis that leads to T2DM. HSP70 is a cytoprotective molecular chaperone that functions in protein folding and degradation. In general, studies have shown that decreased concentration of HSP70 is able to induce in ﬂ ammation process through JNK activation, inhibit fatty acid oxidation by mitochondria through mitophagy decrease and mitochondrial biogenesis, as well as activate SREBP-1c, one of the lipogenic gene transcription factors in ER stress. The overall molecular pathways are potentially leading to insulin resistance and T2DM. Increased expression of HSP70 in brain tissues is able to improve insulin sensitivity and glycemic control speci ﬁ cally. HSP70 modulation-targeting strategies (including long-term physical exercise, hot tub therapy (HTT), and administration of alfalfa-derived HSP70 (aHSP70)) in subjects with insulin resistance are proven to have therapeutic and preventive potency that are promising in T2DM management.
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hsp expression is mainly regulated at the transcriptional level via activation of one or more heat shock transcription factors (HSF) that bind to DNA at a specific site of the hsp gene promoter region, called the heat shock element (HSE) (7, 8). In the cytoplasm, HSFs are constitutively present in a non–DNA-binding state. Their activation is induced by vari- ous stresses and leads to hyperphosphorylation entailing oligo- merization of HSF to a trimeric DNA binding form and subse- quent transport to the cell nucleus (9–11). Two functionally different HSFs are known and the signals leading to their acti- vation appear to be different (12, 13). HSF1 has been shown to respond to stress factors such as elevated temperature, cytokines, heavy metal ions, and shear stress (9). On the other hand, HSF2 has been implicated in hsp expression during hemin-induced differentiation of human erythroleukemia cells (14).
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The following three genes encoding proteins with domains sharing similarity to known HSFs are present in Neurospora (4): hsf1 and hsf2, which are discussed in this paper, and a third gene not included in our study, NCU02413, which encodes response regulator-2 (rrg-2). rrg-2 contains a truncated HSF DNA-binding domain and is involved in Neurospora’s response to oxidative stress (2). HSF1, HSF2a, and HSF2b all contain conserved HSF binding domains and coiled-coil regions that in other HSFs are required for trimerization (43). HSF binding domains recognize and bind to heat shock elements (HSE), consensus sequences found in the promoters of hsp genes (28). The basic HSE shows similarity across a wide range of organisms and is based around GAA repeats, the spacing and orientation of which vary and may influence which sites are recognized by different HSFs or differ- ently phosphorylated HSFs (20). For Neurospora, HSE are not yet well defined, but nGAAn .. nTTCn motifs (where “n” can be any nucleotide) are present in the promoters of hsp30 (38) and hsp70 (21). Additionally, a factor in Neurospora protein extracts binds specifically to labeled oligonucleotides containing the classic yeast HSF binding sequence nTTCnnGAAnnTTCn (32). Band shift assays with purified Neurospora HSF(s) should aid in the identi- fication of the discriminating consensus binding sites for HSF1 and HSF2.
Chaperones of the 40-kDa family (Table 1) also localize to the mt nucleoid in yeast and human cells (33, 38), and Mdj1 and all three E. coli DnaJ proteins were shown to directly bind DNA, this binding being disrupted by the addition of protein substrate (29, 33). Taken together, these data indicate that mtHsp40 is predom- inantly localized in the nucleoid of E. coli, yeast, and human cells, where it performs its primary function; it is only under conditions in which protein substrates become available, such as upon a heat shock, that mtHsp40 dissociates to perform its chaperone func- tion. So far, any possible DNA binding properties of their T. brucei mtHsp40 homologue, which localizes throughout the mt lumen, remain to be determined. In yeast and mammals, the mt nucleoids are more or less evenly dispersed throughout the organellar ma- trix, making the chaperones available when needed. However, the T. brucei kDNA network is invariably present in the anterior para- basal region of a single tubular mitochondrion, and hence a strict DNA binding of the chaperones would be an impediment to their availability throughout the organelle in case of need. Therefore, uniform distribution of mtHsp40, with only its fraction bound to the kDNA, seems to be a more suitable solution for the flagellate. To further dissect the relationships between the mtHsp70/ mtHsp40 machinery and mtDNA, we took advantage of unique kDNA features, such as its exceptional size, catenated structure,
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We also found 80% similarity with A. mellifera and A. aegypti. After sequencing the gene that encodes HSP60 in Rhopalosiphum padi (Hemiptera), Li et al. (2017) observed 73-81% homology of this species with other insects. Hai-Hong et al. (2014) also observed significant homology and identity greater than 80% between the sequences of Frankliniella occidentalis (Thysanoptera) and other insects, including A. mellifera and D. melanogaster. The HSP70 family provides primary protection during exposure to heat shock (Evgen'ev et al., 2014). This family includes HSP70 inducible proteins that are highly expressed during stress and HSC70 cognate proteins expressed in normal conditions and weakly or not expressed during stress (Luo et al., 2015; Wang et al., 2015; Li et al., 2017). Many studies have found high homology and identity over 90% in the sequences of HSP70 among different groups of insects (Luo et al., 2015; Li et al., 2017). Wang et al. (2015) showed that the sequences of Hsc70 and HSP70 in Xestia c-nigrum (Lepidoptera) were more closely related to the sequences of the corresponding genes of other insect species than the genes of another individual of X. c-nigrum. Our results confirm this high homology since the polypeptide fragments of HSP70 in M. interrupta showed 90-100% similarity with HSP70 from A. mellifera, B. terrestris, B. impatiens, A. florea, A. dorsata and M. rotundata. HSP70 has a well-conserved ATPase domain in the aminoterminal part and a less conserved domain that binds to the substrate in the carboxi-terminal part (Zhang et al., 2016; Li et al., 2017).
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Eukaryotic cells respond to heat stress by activating the transcription factor HSF1. In addition to its role in stress response, HSF1 also functions in protein homeostasis, aging, innate immunity, and cancer. Despite prominent HSF1 involvement in processes pertinent to human health and disease, there are still gaps in our understanding of HSF1. For example, controversy exists regarding the localization of HSF1, the identity of HSF1 regulators, and the function and conservation of heat-induced HSF1 stress granules. Many of the physiological roles for HSF1 have been defined using the model organism Caenorhabditis elegans, yet little is known about how the molecular and biological properties of HSF-1 in C. elegans compare to HSF1 in other organisms, including humans. To address these questions, we generated animals expressing physiological levels of a GFP-tagged C. elegans HSF-1 protein. We studied the localization of HSF-1::GFP in vivo and observed its behavior upon heat shock in C. elegans. Furthermore, we conducted a genome-wide, RNAi- based screen for regulators of an HSF-1-dependent, heat shock-inducible transcriptional reporter. We found that in live C. elegans, HSF-1 localizes predominantly to the nucleus before and after heat shock. Following heat shock, HSF-1 redistributes into subnuclear puncta that share many characteristics with human nuclear stress granules, including rapid formation, reversibility, and colocalization with markers of active
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Background: Deciphering of the information content of eukaryotic promoters has remained confined to universal landmarks and conserved sequence elements such as enhancers and transcription factor binding motifs, which are considered sufficient for gene activation and regulation. Gene-specific sequences, interspersed between the canonical transacting factor binding sites or adjoining them within a promoter, are generally taken to be devoid of any regulatory information and have therefore been largely ignored. An unanswered question therefore is, do gene-specific sequences within a eukaryotic promoter have a role in gene activation? Here, we present an exhaustive experimental analysis of a gene-specific sequence adjoining the heat shock element (HSE) in the proximal promoter of the small heat shock protein gene, α B-crystallin (cryab). These sequences are highly conserved between the rodents and the humans. Results: Using human retinal pigment epithelial cells in culture as the host, we have identified a 10-bp gene-specific promoter sequence (GPS), which, unlike an enhancer, controls expression from the promoter of this gene, only when in appropriate position and orientation. Notably, the data suggests that GPS in comparison with the HSE works in a context-independent fashion. Additionally, when moved upstream, about a nucleosome length of DNA ( − 154 bp) from the transcription start site (TSS), the activity of the promoter is markedly inhibited, suggesting its involvement in local promoter access. Importantly, we demonstrate that deletion of the GPS results in complete loss of cryab promoter activity in transgenic mice.
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The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive transcription factors, such as Msn2, Msn4, and Gis1, were unable to rescue the temperature-sensitive growth phenotype of the hsf1 mutant. Another class of suppressors encoded cell wall stress sensors, Wsc1, Wsc2, and Mid2, and the GDP/GTP exchange factor Rom2 that interacts with these cell wall sensors. Activation of a protein kinase, Pkc1, which is induced by these cell wall sensor proteins upon heat shock, but not activation of the Pkc1-regulated mitogen-activated protein kinase cascade, was necessary for the hsf1 suppression. Like Wsc-Pkc1 pathway mutants, hsf1 cells exhibited an osmotic remedial cell lysis phenotype at elevated temperatures. Several of the other suppressors were found to encode proteins functioning in cell wall organization. These results suggest that Hsf1 in concert with Pkc1 regulates cell wall remodeling in response to heat shock.
It has been known for some time that heat shock proteins bind peptide (Refs 4, 147) and that heat shock proteins purified from cells chaperone a large number of peptides derived from the cells from which they are isolated – the so-called ‘antigenic repertoire’ of that cell (Ref. 148). Early studies showed that fractionated tumour cell lysates have the capacity to reduce tumour cell growth in mice (Ref. 149). Since then, it has been well established that immunisation of mice with Hsp70, Hsp90 and gp96 isolated from murine tumour cells induces anti-tumour immunity and tumour-specific cytolytic T cells, and that the immunity results from tumour- derived peptides associated with the heat shock protein rather than from the heat shock proteins themselves (Ref. 150). More recently, it has been reported that calreticulin, Hsp110 and grp170 can also be used in heat shock protein-based cancer immunotherapy (Refs 151, 152). The finding that the immunological properties of heat shock proteins and the capacity of Hsp70 and gp96 to induce tumour protection as shown in rodent models are also observed in amphibians (Xenopus) (Ref. 153) indicates the evolutionary conserved nature of these functions, and strongly supports the successful translation of these strategies into the clinical environment. In that regard, preliminary clinical trials have demonstrated the induction of cancer-specific CD8 + T-cell responses in 6/12
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Hsp70 has been cloned from rainbow trout (Oncorhynchus mykiss) (Kothary et al., 1984; Airaksinen et al., 1998), medaka (Oryzias latipes) (Arai et al., 1995), zebrafish (Lele et al., 1997; Santacruz et al., 1997), tilapia (Oreochromis mossambicus) (Molina et al., 2000), carp (Cyprinus carpio) (Yin et al., 1999) and pufferfish (Fugu rubripes) (Lim and Brenner, 1999) and heat stress- related increases in mRNA levels have been investigated. The fish hsp70 genes are highly conserved at the amino acid level (Molina et al., 2000; Deane and Woo, 2006). Keller et al. (2008) explored that heat stress-induced Hsp70 expression was altered by activation of ERK (Extracellular signal regulated kinase) in the zebrafish Pac2 fibroblast cell line as occurs in mammalian cells. Heat stress induced both Hsp70 mRNA expression and phosphorylation of both ERK1 and ERK2 (ERK1/2) in Pac2 cells. ERK inhibitors, PD98059 and U0126 we reported to block both heat stress-induced and platelet-derived growth factor (PDGF)-induced ERK1/2 phosphorylation, and also diminished heat-induced Hsp70 expression. Pac2 cell viability was not affected by either the ERK inhibitors or heat stress. This knowledge demonstrates that induction of Hsp70 as a response to heat stress is dependent on ERK activation in Pac2 cells. The available knowledge suggests that the heat shock response in zebrafish utilizes a similar signaling pathway to that of mammals (Elicker and Hutson, 2007; Keller et al., 2008).
E2 and Heat Shock Protein Expression E2 treatment, both in vivo and in vitro, leads to increased expression of HSF-1, HSP 72, and HSP 90 in female cardiac myocytes and uterine tissue (83,94–97). In addition, E2 stimulates the phospho- rylation of HSP 27 and αβ-crystallin via p38 MAP kinase, essential for the protec- tive properties of these proteins (28). Fur- ther evidence of E2 increasing expression of HSPs are the observations that intact females have higher levels of HSP 72 than males, both basally and following ischemia/reperfusion, in cardiac and renal tissue (95,98). Reduction of female rat cardiac HSP 72 expression to that of males occurred 9 weeks after ovariec- tomy, suggesting the increase in HSP 72 is indirectly due to estrogen. Basal levels of HSP 27 and 90 were found to be de- creased in female heart tissue compared with males, suggesting that not all HSPs are upregulated by E2 (99). Interestingly, the inbred Fischer rats did not show the same male/female difference in cardiac levels of HSP 72, while inbred Norway Brown rats did (AA Knowlton and J Stal- lone, unpublished data).
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Despite the recent advances in proteomic research, and improved 2-DE systems, the analysis of the Halobacterium NRC-1 proteome is a considerable challenge . Because of its extreme acidity, and relatively high hydrophobicity, proteins tend to precipitate at their isoelectric point dur- ing IEF analysis [10,11]. Moreover, presence of salt in the sample and excess DTT at acidic pH leads to streaking and skewed results. These problems have previously been approached using low molecular weight cut-off columns, ultrazoom immobilized pH gradient strips, and combina- tions of IPGphor/Multiphor systems [7,12]. However, streaking at the high molecular weight region of the 2-D gels still poses considerable challenge in obtaining repro- ducible results, which are essential for comparative study and global protein analysis in extreme halophiles. Heat shock response is an important homeostatic mecha- nism that enables cells to survive a variety of environmen- tal stresses . Some heat shock proteins are
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There is a dramatic up-regulation of Hsp expression in cells upon induction of the heat shock response (HSR). The activation of this pathway is a primary defense mechanism that protects cells from stress conditions that promote protein misfolding, aggregation and cell death. It has previously been shown that components of the HSR may be neuroprotective due to their ability to interact with the earliest misfolded proteins that trigger pathogenic aggregation. Indeed, numerous studies have shown that Hsps can prevent the aggregation of various disease-associated proteins in vitro, for example, mutant superoxide dismutase 1 (SOD1) in ALS [11–15]. Heat shock proteins can also interact with pathogenic pro- teins in vivo and have been found co-localized with pla- ques and inclusions in transgenic mouse models of NDs and patient post-mortem tissues [16–19]. For example, Hsc70 was co-localized with inclusion bodies in spinal cord sections of SOD1 G93A , SOD1 G85R , and SOD1 G37R transgenic mice, and human sporadic ALS cases . The co-localization of Hsps with inclusions suggests that Hsps are diverted into inclusions and therefore unavail- able to perform normal “housekeeping” functions.
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3. Induction and regulation of heat shock protein ex- pression: Regulation of transcription of heat shock pro- tein genes is mediated by the interaction of heat shock factor transcription factors with heat shock elements in the heat shock protein gene promoter regions. In verte- brates, four HSFs have been identified, of which HSF1 and HSF2 are ubiquitously expressed and conserved. 4. Heat shock proteins as intercellular signaling mole- cules: The usual view of eukaryotic heat shock proteins is that they are intracellular molecules that are released from necrotic, but not apoptotic cells, and that their re- lease into the extracellular environment indicates non- physiological tissue damage and therefore induces a range of proinflammatory responses 10 .
The HSP70 also provides protection when induced by methods other than heat shock, such as treatment of human breast cancer T47-D cells with estrogen (Kiang et al. 1997), rat vascular smooth muscle cells with nitric oxide generating agents (Xu et al. 1997), and rat lung cells with arsenate or glutamine (Wischmeyer et al., 1997). Cells transfected with the HSP70 gene are protected from many harmful agents (Li et al. 1991; Kiang et al. 1998; Samali and Cotter 1996; Uney et al. 1993). It is reported that induction of HSP70 enhances the hydrogen peroxide cytotoxicity in Drosophila (Love et al. 1986) and the TCR/CD3- and Fas/APO-1/CD95-mediated apoptotic cell death in Jurkat cells (Liossis et al. 1997). Inhibition of HSP70 expression diminishes cell survival (Riabowol et al. 1988). These results further support the view that HSP70 is important for the cell survival. Evidence presented above suggests that harnessing endogenous protective systems such as HSPs can possibly provide therapeutic benefit. Harmless manipulation that increases the synthesis of cytoprotective HSP70 may prove to be of clinical use in organ transplantation and functional recovery of organs that require reperfusion as a result of lost blood supply.
. Kioishi M, Hosokava N, Sato M et al. Quercetin, an inhibitor of heat shock protein synthesis, inhibits the acquisition of thermotolerance in a human colon carcinoma cell line. Jpn J Cancer Res 1992; 83: 1216-22. . Aalinkeel P, Bindu Kumar B, Raynolds
Abstract: The present study used a genetic engineering method to express a truncated heat shock protein 110 (tHSP110) isoform in Escherichia coli and verified its ability to bind to and present macromolecular antigens. Poly- merase chain reaction (PCR) was used to obtain the truncated HSP110 gene, which was expressed in E. coli. The tHSP110 protein was non-covalently coupled to the intracellular domain (ICD) of human epidermal growth factor receptor 2 (HER2/Neu) in vitro to construct the antigen peptide complex tHSP110-ICD, which was identified by a co-immunoprecipitation assay. BALB/c mice were immunized 14-day interval for three times with the HSP110, tHSP110, HSP110-ICD, tHSP110-ICD, HSP110-P 851-859 (a complex formed by full-length HSP110 with a cytotoxic T lymphocyte (CTL) epitope peptide of the Her2/neu ICD) and tHSP110-P 851-859 complexes. Fourteen days after the last immunization, D 2 F 2 cells were inoculated into BALB/c mice. The in vivo tumor volume of each group was measured every three days after cell inoculation to evaluate the immunization efficacy of the vaccine in each group. The level of the IFN-γ secreted by activated lymphocytes, the specific CTLs activity was detected. Immunohistochemical stain- ing of bcl-2 and bax were measured on the tumor tissues of each group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the expressed tHSP110 protein was 66 kDa in size. The non-covalent coupling of tHSP110 with ICD and peptide were confirmed by a co-immunoprecipitation assay. The in vivo tumor experiment results indicated no differences in the tumor volumes of the tHSP110-ICD and HSP110-ICD groups. In contrast, the tumor volume of the tHSP110-ICD group was significantly different compared with the tumor volume of the tHSP110-P 851-859 group. After the mice immunized with tHSP110-ICD, tHSP110-P 851-859 complexes, the complexes have potential immunogenicity, and can induce specific CTLs activity and apoptosis in BALB/c mice. As a tumor vac- cine to inhibit in vivo tumor growth, the tHSP110 has the same ability to bind macromolecular antigens and activate tumor immune responses as full-length HSP110.
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chaperones” in the development, progression, metas- tasis and drug resistance of cancers [6, 7]. Therefore, HSPs have recently been proposed as potential thera- peutic targets for tumor therapy . HSPH1 (also named HSP105), a member of the HSP70 superfamily, is a component of the β-catenin degradation complex. Previous studies have demonstrated that overexpres- sion of HSPH1/HSP105 in various cancers is associ- ated with increased levels of nuclear β-catenin protein and upregulation of Wnt target genes . HSPD1 is a molecular chaperone primarily localised in the mito- chondrial matrix. It has been described as a poten- tial prognostic and diagnostic biomarker for cancer. Recent studies have demonstrated that HSPD1 not only regulates the stability of survivin protein, but also regulates the mRNA expression of survivin . SER- PINH1, also called heat shock protein 47 (HSP47), is a collagen specific molecular chaperone. Several studies have confirmed that SERPINH1 participates in numer- ous steps of collagen synthesis, blocking the aggrega- tion of procollagen and inducing the hydroxylation of proline and lysine residues. Abnormal expression lev- els of SERPINH1 are frequently found in a variety of cancers, including cervical, lung and gastric cancers [11–13]. Heat shock protein A4 (HSPA4), a member of the HSP110 family, is widely expressed in a variety of organs and can be induced under different condi- tions, including carcinogenic stress [14–16]. Recent studies have indicated that knockdown of HSPA4 can significantly reduce the migration, invasion, and trans- formation activities of tumor cells . Heat shock protein 90α (Hsp90α) is the major cytosolic chaper- one in eukaryotes. It is involved in cell protection and intracellular signaling transduction, controls intra- cellular homeostasis and assemblies of endoplasmic reticulum-secreted peptides, and regulates the translo- cation of proteins across the membranes of organelles after translation. Upregulated expression of Hsp90α is observed in a variety of cancer tissues, including liver, breast, and pancreatic cancers [18–20]. However, there is limited understanding of the underlying mechanisms and the unique roles of these genes in HNSC.
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For each seasonal phenotype, each species of bird was further divided into an acute treatment group and a 5 day chronic treatment group. Within acute and chronic treatments, birds were further randomly assigned to a control, heat shock treatment (33°C) or recovery from heat shock treatment group. Control acute treatment birds were held at 22°C for 9 h after collection, acutely heat-shocked birds were placed in a Percival chamber with an IntellusUltra control system (CTH-7272) and held at 33°C for 9 h, and acute recovery birds were also heat shocked for 9 h, but were then allowed to recover from heat shock overnight (9 – 10 h) at 22°C. Control chronic treatment birds were held at 22°C for 5 days after collection, heat-shocked chronic treatment birds were heat shocked to 33°C for 6 h each day for 5 days, and recovery chronic treatment birds were also heat shocked, but then allowed to recover after the day 5 heat shock for 9 – 10 h at 22°C (Fig. 1). However, we were not aiming to test maximal increases in temperature, which, it could be argued, these animals may never see in the wild or in the near future. Therefore, we used 33°C as an ecologically relevant temperature for the central New York area, and one that has been common in recent years across all