Association of HSP90 with the heme-regulated eukaryotic initiation factor 2α kinase—A collaboration for regulating protein synthesis

Among the various heat shock proteins (HSPs), members of the HSP70 and HSP90 families have drawn particular attention due to their heat shock-unrelated functions. HSP90, an ubiquitous and abundant member of the HSP90 family has been shown to be associated with a large array of protein factors. These proteins reside in the nucleus as well as in the cytoplasm and are involved in various physiological processes, such as, regulation of chromatin structure, cell cycle, cytoskelelal architecture, protein trafficking and protein synthesis. In this article, we focus our interest on the role of HSP90 in protein synthesis. Recent data obtained from a few laboratories strongly suggest that HSP90 interacts with the heme-regulated eukaryotic initiation factor 2α (elF-2α) kinase, also called the heme-regulated inhibitor, and causes its activation which leads to inhibition of protein synthesis. On the basis of data reported from various laboratories, including our own, we propose a possible model on the mechanism of HSP90-mediated activation of heme-regulated inhibitor and regulation of protein synthesis.     

Characterization of the 90 kDa heat shock protein (HSP90)-associated ATP/GTPase

The 90 kDa heat shock protein (HSP90) is an ATP-binding molecular chaperone with an associated ATPase activity having nucleoplasmin and HSP70-binding homology domains and containing Ca-binding EF-hands and a nuclear localization signal. Here we characterize the HSP90-associated ATPase and show that it is (i) a P-type ATPase inhibited by molybdate and vanadate, (ii) able to hydrolyze methylfluorescein phosphate with a 5–6-fold higher affinity, (iii) a 3-times better GTPase than ATPase in the presence of calcium and (iv) HSP27 and F-actin, but not HSP10 can “convert” the HSP90-associated ATPase activity to HSP90 autokinase activity. The HSP90-associated ATP/GTPase may participate in the regulation of complex formation of HSP90 with other proteins, such as F-actin, tubulin and heat shock proteins.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.     

Variations on a theme: Combined molecular chaperone and proteolysis functions in Clp/HSP100 proteins

Most stress-inducible polypeptides are members of broader protein families that function either as molecular chaperones or constituents of proteolytic pathways. These systems control many aspects of protein structure and function throughout the cell under all types of growth regimes. The Clp/HSP1 00 protein family is a recently characterized representative, with constitutive and stress-inducible members found in many different organisms and various intracellular locations. Besides being regulators of energy-dependent proteolysis, Clp proteins may also function as molecular chaperones. Constitutive Clp proteins are involved foremost in cellular protein maintenance and repair, in cooperation with other chaperone and proteolytic systems. At high temperatures, additional Clp proteins are induced in response to rising levels of inactive polypeptides, resulting from either biosynthetic errors, thermal denaturation and aggregation. Clp proteins presumably help to stabilize selected polypeptides during severe thermal stress and enable resolubilization of non-functional protein aggregates, as well as promoting the degradation of irreversibly damaged polypeptides. The union of chaperone and proteolytic regulatory functions in one molecule suggests that certain Clp proteins play a decisive role in determining the destiny of proteins, not only during normal growth but also under conditions of extreme stress. This review briefly covers recent findings on the diversity of Clp proteins and their potential importance within the cell.     

Comparative analysis of differential gene expression of HSP40 and HSP70 family isoforms during heat stress and HIV-1 infection in T-cells

Heat shock proteins (HSPs) are a family of cellular proteins involved in a variety of biological functions including chaperone activity. HSPs are classified based on their molecular weight and each family has several isoforms in eukaryotes. HSP40 is the most diverse family acting as a co-chaperone for the highly conserved HSP70 family. Some of the isoforms are reported to be induced during heat stress. Few studies have also highlighted the diverse role of some isoforms in different stress conditions including viral infections. But till date, no study has comprehensively examined the expression profile of different HSP40 and 70 isoforms in either heat stress or HIV-1 infection, a virus that is responsible for the pandemic of AIDS. In the present study, we have compared the mRNA expression profile of HSP40 and HSP70 isoforms during heat stress and HIV-1 infection in a T-cell line and also validated the HIV-1 stress results in peripheral blood mononuclear cells. In case of HSP70, we observed that three isoforms (HSPA1A, HSPA1B, and HSPA6) are highly upregulated during heat stress, but these isoforms were found to be downregulated during the peak of HIV-1 infection. While in case of HSP40, we found that only DNAJA4, DNAJB1, and DNAJB4 showed significant upregulation during heat stress, whereas in HIV-1 infection, majority of the isoforms were induced significantly. Stress-dependent differential expression observed here indicates that different HSP40 and HSP70 isoforms may have specific roles during HIV-1 infection and thus could be important for future studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-020-01185-y.     

热休克蛋白HSP70和gp96在抗病毒感染中的作用

热休克蛋白(HSP)是一组在进化上高度保守、具有重要生理功能的蛋白质家族,是生物在应激条件下产生的一种非特异性防御产物,在调节免疫应答和抗病毒反应中起重要作用。现简要介绍HSP70、gp96(HSP96,GRP94)这两种HSP与病毒感染的关系及在抗病毒感染中的作用。     

Biochemical and immunofluorescence analysis of the constitutively expressed HSP27 stress protein in monkey CV-1 cells

The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particu late fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.     

Photoinhibition of photosynthesis without net loss of photosystem II components inPopulus deltoides

The photoinhibition of photosynthesis was investigated on intact attached leaves and isolated thylakoid membranes of Populus deltoides.Our studies demonstrate that in intact leaves photoinhibition takes place under high irradiance which is more pronounced at higher temperatures. No net loss of Dl and other proteins associated with photosystem II (PSII) were observed even after 64 % photoinhibition suggesting that the degradation of polypeptides associated with PSII is not the only key step responsible for photoinhibition as observed by other workers. Electron transport studies in isolated thylakoid membranes suggested water oxidation complex as one of the damaged site during high light exposure. The possible mechanisms of photoinhibition without net loss of D1 protein are discussed.     

Characterisation and subcellular localisation of the GroEL-like and DnaK-like proteins isolated from Fusobacterium nucleatum ATCC 10953

Fusobacterium nucleatum is associated with periodontitis in humans, and is a central member of the dental biofilm. Heat shock proteins (HSPs) of many different bacteria have been considered to play important roles during inflammations and infections. We have identified and characterised the HSP60 and HSP70, the Escherichia coli GroEL and DnaK homologues, respectively, in F. nucleatum ATCC 10953. The N-terminal 22 amino acid residues of HSP60 exhibited up to 63.6% identity with members of the HSP60 heat shock protein family of some selected bacterial species, while the N-terminal of 25 residues of HSP70 revealed up to 80% identity with members of the HSP70 family. The subcellular localisation of HSP60 and HSP70 was analysed by immunoblotting of bacterial cell fractions and immunoelectron microscopy of whole cells. HSP60 and HSP70 were localised in the cytosol, associated with membranes and extracellular fractions. These results are consistent with localisation for HSPs found in other micro-organisms, which further lead to the suggestion of a potential role in the pathogenesis of infectious diseases.     

The BglG group of antiterminators: a growing family of bacterial regulators

The product of the bglG gene of Escherichia coli was among the first bacterial antiterminators to be identified and characterized. Since the elucidation ten years ago of its role in the regulation of the bgl operon of E. coli,a large number of homologies have been discovered in both Gram-positive and Gram-negative bacteria. Often the homologues of BglG in other organisms are also involved in regulating β-glucoside utilization. Surprisingly, in many cases, they mediate antitermination to regulate a variety of other catabolic functions. Because of the high degree of conservation of the cis-acting regulatory elements, antiterminators from one organism can function in another. Generally the antiterminator protein itself is negatively regulated by phosphorylation by a component of the phosphotransferase system. This family of proteins thus represents a highly evolved regulatory system that is conserved across evolutionarily distant genuses.     

Immunoprophylactic studies on cell wall associated proteins ofMycobacterium tuberculosis H37Ra

The cell wall protein peptidoglycan complex (CW-PPC) of Mycobacterium tuberculosis H₃₇Ra was isolated through sequential extraction of lipids, carbohydrates and soluble proteins. CW-PPC emulsified in FIA was found to induce significant protection in mice against challenge with LD₅₀ dose ofM. tuberculosis H₃₇Rv. To identify the immunoprotective components of CW-PPC, the proteins in avid association with peptidogican were dissociated by chemical treatment with trifluoromethanesulthonic acid (CF₃CO₃H): anisole (2:1). Immunoreactivity of total (CW-Pr) as well as its component proteins i.e., 71, 60 and 45 kDa proteins of cell wall was studied in animals immunized with CW-Pr-FIA. The 71 kDa protein was found to be most immunoreactive giving higher T-cell sensitization and humoral responses. Further, immunization of mice with 71 kDa-FIA demonstrated enhanced T- and B- cell responses. Mice immunized with 71 kDa-FIA gave significantly higher protection (P ≤ 0.05) against intravenous challenge with LD₅₀ dose ofM. tuberculosis H₃₇Rv, than BCG immunized animals. The results indicate the potential of 71 kDa cell wall protein as a suitable candidate for Cthe subunit vaccine.     

几种热激蛋白在细胞凋亡信号通路中的调控作用

热激蛋白(heat shock proteins, HSPs)作为进化保守的蛋白家族 之一,普遍存在于各种生物体中,并在生物体内发挥着重要的生理功能.大 量的实验证据表明,热激蛋白与细胞凋亡密切相关,参与细胞凋亡信号通 路的多个环节. 近年来有关该领域的研究已获得了重要的突破与进展.一方 面,热激蛋白主要起着抑制细胞凋亡、促进细胞存活的作用;另一方面, 某些热激蛋白又能够作为凋亡蛋白的分子伴侣,促进细胞凋亡,比如HSP70 能够激活DNase来促使细胞凋亡,线粒体内HSP60能够促进caspase依赖的细 胞凋亡途径.本文在阐明细胞凋亡信号通路的基础上,综述了近年来几种不 同热激蛋白家族（HSP90、 HSP70 、HSP60和小分子HSPs）在细胞凋亡调控 中作用的研究进展,重点阐述了几种主要热激蛋白与细胞凋亡信号通路上 相关因子的相互作用,并绘制了热激蛋白在细胞凋亡信号通路中的调控图 ,为进一步完善细胞凋亡调控网络研究提供一定的参考.     

Comprehensive approach to genes involved in cell wall modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant cell wall is of supermolecular architecture, and is composed of various types of heterogeneous polymers. A few thousand enzymes and structural proteins are directly involved in the construction processes, and in the functional aspects of the dynamic architecture in Arabidopsis thaliana. Most of these proteins are encoded by multigene families, and most members within each family share significant similarities in structural features, but often exhibit differing expression profiles and physiological functions. Thus, for the molecular dissection of cell wall dynamics, it is necessary to distinguish individual members within a family of proteins. As a first step towards characterizing the processes involved in cell wall dynamics, we have manufactured a gene-specific 70-mer oligo microarray that consists of 765 genes classified into 30 putative families of proteins that are implicated in the cell wall dynamics of Arabidopsis. By using this array system, we identified several sets of genes that exhibit organ preferential expression profiles. We also identified gene sets that are expressed differentially at certain specific growth stages of the Arabidopsis inflorescence stem. Our results indicate that there is a division of roles among family members within each of the putative cell wall-related gene families.     

Modulation of HSP70 GlcNAc-directed lectin activity by glucose availability and utilization

It is well-accepted that protein quality control (occurring either after protein synthesis or after cell damage) is mainly ensured by HSP, but the mechanism by which HSP decides whether the protein will be degraded or not is poorly understood. Within this framework, it has been hypothesized that O-GlcNAc, a cytosolic and nuclear-specific glycosylation whose functions remain unclear, could take a part in the protection of proteins against degradation by modifying both the proteins themselves and the proteasome. Because the synthesis of O-GlcNAc is tightly correlated to glucose metabolism and Hsp70 was endowed with GlcNAc-binding property, we studied the relationship between GlcNAc-binding activity of both Hsp70 and Hsc70 (the nucleocytoplasmic forms of HSP70 family) and glucose availability and utilization. We thus demonstrated that low glucose concentration, inhibition of glucose utilization with 2DG, or inhibition of glucose transport with CytB led to an increase of Hsp70 and Hsc70 lectin activities. Interestingly, the response of Hsp70 and Hsc70 lectin activities toward variations of glucose concentration appeared different: Hsp70 lost its lectin activity when glucose concentration was >5 mM (i.e., physiological glucose concentration) in contrast to Hsc70 that exhibited a maximal lectin activity for glucose concentration approximately 5 mM and at high glucose concentrations. This work also demonstrates that HSP70 does not regulate its GlcNAc-binding properties through its own O-GlcNAc glycosylation.     

The co-chaperone SGT of <Emphasis Type="Italic">Leishmania donovani</Emphasis> is essential for the parasite's viability

Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative co-chaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.