Transfection of human HSP27 in rodent cells: Absence of compensatory regulation between small heat shock proteins

1. 1. We examined rodent cells transfected with an expression plasmid encoding a human small heat shock protein for possible compensatory expression of endogenous heat shock genes. For these investigations, human hsp27 was transfected into CHO cells which express endogenous HSP25.

2. 2. Both endogenous HSP25 and transfected HSP27 were expressed and multiple phosphorylated isoforms were detected upon exposure to thermal stress.

3. 3. Levels of endogenous HSP70 and HSP25 did not appear to be altered by expression of the heterologous heat shock protein.

4. 4. These results suggest that compensatory interactions are not exhibited in the expression of the heat shock genes examined, and that independent regulation may exist not only between the large and small heat shock proteins, but also between individual small heat shock proteins as well.

     

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.     

C-terminal modulators of heat shock protein of 90 kDa (HSP90): State of development and modes of action

Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as “C-terminal inhibitors” have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites.     

A Fluorescence‐Based Sensor Assay that Monitors General Protein Aggregation in Human Cells

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ‐peptide. Exposure to proteome destabilizers lead to re‐localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide‐genetic screens to identify genes and pathways involved in protein misfolding and aggregation.     

HDJC9, a novel human type C DnaJ/HSP40 member interacts with and cochaperones HSP70 through the J domain

HSP40s are a subfamily of heat shock proteins (HSPs) and play important roles in regulation of cell proliferation, survival and apoptosis by serving as chaperones for HSP70s. Up to date hundreds of HSP40 proteins derived from various species ranging from Escherichia coli to homo sapiens have been identified. Here we report the cloning and characterization of a novel human type C DnaJ homologue, HDJC9, containing a typical N-terminal J domain. HDJC9 is upregulated at both mRNA and protein levels upon various stress and mitogenic stimulations. HDJC9 is mainly localized in cell nuclei under normal culture conditions while it is transported into cytoplasm and plasma membrane upon heat shock stress through a non-classical and lipid-dependent pathway. HDJC9 can interact with HSP70s and activate the ATPase activity of HSP70s, both of which are dependent on the J domain. Our data suggest that HDJC9 is a novel cochaperone for HSP70s.     

Activation mechanism of HSP16.5 from Methanococcus jannaschii

The small heat shock proteins are the ubiquitous proteins found in a wide range of organisms and function as molecular chaperones by binding to the folding intermediates of their substrates. Although the crystal structure of HSP16.5, a small heat shock protein from Methanococcus jannaschii, revealed that it is a hollow sphere composed of 24 identical subunits, its activation mechanism remains unclear. We found out that HSP16.5 is active only at high temperatures and forms a stable complex with substrate in a stoichiometric manner. We also observed that the conformational change of HSP16.5 is correlated with the increasing hydrophobic site and its activation as a molecular chaperone. However, it is revealed that the conformational change is not accompanied with the change of the secondary structure of a subunit, but correlated with the increasing diameter of HSP16.5. Therefore, it is proposed that the activation mechanism of HSP16.5 involves temperature induced conformational change with size increment of the complex resulting in the exposure of hydrophobic substrate-binding site.     

Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects,patients with coronary heart disease and inflammatory bowel disease

Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.     

TNF-a在乳鼠心肌细胞中诱导HSP70蛋白表达的研究

热休克蛋白70 (HSP70) 在细胞修复、存活和维持细胞正常功能方面有着重要作用。作为分子伴侣,它起着心肌保护的作用。已经对重症心脏病人的心肌组织进行了蛋白组学研究,得到了HSP70在心衰病人心肌组织中较正常人心肌组织表达升高的结论,并且在血液中得到了进一步的验证。在进一步的离体细胞实验中用不同剂量的肿瘤坏死因子-alpha (TNF-α) 刺激乳鼠心肌细胞,以观察不同时间点HSP70的动态表达情况。培养乳鼠心肌细胞,分别对细胞进行热休克（42 ℃）、TNF-α和缺血缺氧处理,在不同的时间点收获细胞,以观察HSP70的动态表达情况。用免疫化学、ELISA以及Western blotting的方法对HSP70蛋白进行分析。结果表明,在正常对照细胞中基本没有阳性信号出现,而在经缺血缺氧、热休克（42 ℃）以及TNF-α处理的细胞中有明显的阳性表达。以上研究首次在乳鼠心肌细胞中证明TNF-α诱导的HSP70表达具有时间和浓度依赖性。通过运用TNF-α对HSP70蛋白表达影响的研究,初步推断HSP70的表达模式,为体内诱导产生HSP70从而发挥心肌保护作用的研究提供一定的理论基础。     

二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

腾冲嗜热厌氧菌热休克蛋白60及其基因表达的热激动态变化分析

热休克蛋白60(HSP60)是细菌体内一种非常重要的分子伴侣,其可以协助蛋白质或肽链的正确折叠和构型,防止变性和降解。基于本实验室的早期观察,腾冲嗜热厌氧菌的HSP60是一个典型的温度相关蛋白质,在80℃的表达水平最高。为了进一步了解嗜热菌应急的分子机制,继续进行了在热激后HSP60基因表达的动态研究。将最适温度(75℃)下培养的腾冲嗜热厌氧菌迅速地转移至80℃继续培养,然后在不同的时间点上分别取样,并通过双向电泳、Western blot和Real_time PCR等方法,分析了HSP60在mRNA和蛋白质水平上的表达量的改变。试验结果表明,在80℃热处理4h内的短期应急过程中,HSP60蛋白水平一直呈上升趋势,而它的mRNA水平则表现为先升高后下降的一个非对称性的峰形变化。HSP60的mRNA和蛋白质的对温度的应答快慢程度是不同的。HSP60的mRNA水平的显著变化在1h内便可观察到,而蛋白质水平的显著改变要延迟3h左右。此外,HSP60的mRNA和蛋白质对温度的应答量变大小也是不同的。     

HSP90及其抑制剂对肿瘤免疫反应的影响

HSP90作为一种热休克蛋白参与调控蛋白质的正确折叠、装配和水解等多种生理过程,其在肿瘤组织中异常表达与活化,与恶性肿瘤的发生发展密切相关,是肿瘤药物研发的重要靶标,目前已有多个HSP90抑制剂进入临床研究。近年来研究发现,HSP90在调控机体固有性免疫和适应性免疫反应中也发挥着重要的作用,包括抗原呈递、T细胞、NK细胞活化和DC(树突状细胞)的成熟,以及肿瘤微环境的免疫抑制等。抑制HSP90导致免疫抑制和免疫激活双重反应,因此,HSP90在机体免疫中作用复杂,有待人们进一步研究。本文主要综述了HSP90及其抑制剂与肿瘤免疫之间的联系,为今后相关研究人员的工作提供参考。     

Light-inducible geneHSP70B encodes a chloroplast-localized heat shock protein inChlamydomonas reinhardtii

The nuclear heat shock geneHSP70B ofChlamydomonas reinhardtii is inducible by heat stress and light. Induction by either environmental cue resulted in a transient elevation in HSP70B protein. Here we describe the organization and nucleotide sequence of theHSP70B gene. The deduced protein exhibits a distinctly higher homology to prokaryotic HSP70s than to those of eukaryotes, including the cytosolic HSP70A ofChlamydomonas reinhardtii. The HSP70B protein, as previously demonstrated by in vitro translation, is synthesized with a cleavable presequence. Using an HSP70B-specific antibody, this heat shock protein was localized to the chloroplast by cell fractionation experiments. A stromal location was suggested by the presence of a conserved sequence motif used for cleavage of presequences by a signal peptidase of the stroma. Amino acid alignments of HSP70 proteins from various organisms and different cellular compartments allowed the identification of sequence motifs, which are diagnostic for HSP70s of chloroplasts and cyanobacteria.     

HSP90, HSP70, and GAPDH directly interact with the cytoplasmic domain of macrophage scavenger receptors.

The macrophage scavenger receptor (MSR) is a trimeric membrane protein which binds to modified low-density lipoprotein (LDL) and has been indicated in the development of atherosclerosis. It has recently been demonstrated that the N-terminal cytoplasmic domain of MSR has an important role in the efficient internalization and cell-surface expression of the receptor. This study shows that the N-terminal cytoplasmic domain in bovine was constructed using a peptide architecture technique in which the peptide chain was bundled at their C-terminus to yield a trimeric form and that this did not form an ordered structure. Furthermore, the binding proteins to the cytoplasmic domain of MSR were determined for the first time using a peptide affinity column. Sequence analyses of the specific binding proteins in bovine revealed that heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), leucine aminopeptidase (LAP), adenocylhomocysteinase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were included. GST-pull-down assay and immunoprecipitation analyses on HSP90, HSP70, and GAPDH showed that all these proteins could bind to the cytoplasmic domain of MSR in vitro and in vivo. These proteins interact with the cytoplasmic domain directly and may have an effect on the functions of MSR such as internalization, cell-surface expression, and signal transduction.     

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。     

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.     

Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells.

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.