Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition

We previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo.

Structured summary

MINT-7384283: HSP40 (uniprotkb:P25685) binds (MI:0407) to HSP70 (uniprotkb:P34930) by surface plasmon resonance (MI:0107)MINT-7384430: RNaseA (uniprotkb:P61823) binds (MI:0407) to HSP70 (uniprotkb:P34930) by surface plasmon resonance (MI:0107)     

Growth Inhibition of Helicobacter pylori by Monoclonal Antibody to Heat-Shock Protein 60

The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.     

A 70 kDa microtubule-associated protein in NIL8 cells comigrates with the 70 kDa heat shock protein

When eukaryotic cells are exposed to environmental stress such as elevated temperature, the synthesis of heat shock proteins (HSP) is stimulated. We have raised a monoclonal antibody to a 70 kDa cytoskeleton-associated protein; this antibody also appears to recognize HSPs 68, 70 and 90, as well as an additional 40 kDa non-heat shock protein. We have used this monoclonal antibody to study the localization of the 70 kDa protein in the cytoskeletons of NIL8 hamster fibroblasts. By selective sequential solubilization of the components of NIL8 cells and analysis of the resulting cytoskeletal preparations by Western blot technique and indirect immunofluorescence, we have shown that the 70 kDa protein is associated with microtubules in mitotic and interphase cells and comigrates with HSP70 on 2-dimensional gel electrophoretigrams.     

Microtubular Organization in Tobacco Cells: Heat-Shock Protein 90 can Bind to Tubulin in Vitro

Abstract: The heat-shock protein 90 (HSP90) from tobacco VBIO cells specifically binds to nitrocellulose that had been coated with polymerized microtubules or tubulin dimers. HSP90 is expressed preferentially during cell division and becomes down-regulated during cell elongation. HSP90 cofractionates with tubulin dimers during affinity chromatography with sepharose coupled to the tubulin-binding drug ethyl N-phenylcarbamate (EPC). Binding of HSP90 to EPC-sepharose depends on the presence of tubulin. Antibodies against tubulin and HSP90 immunoadsorb HSP90 and tubulin, respectively. These results demonstrate that HSP90 behaves as a microtubule-binding protein in vitro.     

Comparison of allosteric signaling in DnaK and BiP using mutual information between simulated residue conformations

The heat shock protein 70 kDa (Hsp70) chaperone system serves as a critical component of protein quality control across a wide range of prokaryotic and eukaryotic organisms. Divergent evolution and specialization to particular organelles have produced numerous Hsp70 variants which share similarities in structure and general function, but differ substantially in regulatory aspects, including conformational dynamics and activity modulation by cochaperones. The human Hsp70 variant BiP (also known as GRP78 or HSPA5) is of therapeutic interest in the context of cancer, neurodegenerative diseases, and viral infection, including for treatment of the pandemic virus SARS-CoV-2. Due to the complex conformational rearrangements and high sequential variance within the Hsp70 protein family, it is in many cases poorly understood which amino acid mutations are responsible for biochemical differences between protein variants. In this study, we predicted residues associated with conformational regulation of human BiP and Escherichia coli DnaK. Based on protein structure networks obtained from molecular dynamics simulations, we analyzed the shared information between interaction timelines to highlight residue positions with strong conformational coupling to their environment. Our predictions, which focus on the binding processes of the chaperone's substrate and cochaperones, indicate residues filling potential signaling roles specific to either DnaK or BiP. By combining predictions of individual residues into conformationally coupled chains connecting ligand binding sites, we predict a BiP specific secondary signaling pathway associated with substrate binding. Our study sheds light on mechanistic differences in signaling and regulation between Hsp70 variants, which provide insights relevant to therapeutic applications of these proteins.     

人肝癌细胞BEL-7402中热休克蛋白70相伴甲胎蛋白的实验研究

为研究人肝癌细胞BEL-7402中热休克蛋白70(HSP70)与甲胎蛋白(AFP)的相互作用,采用免疫化学和免疫荧光检测HSP70和AFP在肝癌细胞中的表达和定位.HSP70与AFP的相互关系通过免疫共沉淀和蛋白印迹杂交进行分析.结果免疫化学显示人肝癌细胞BEL-7402中存在高水平的HSP70和AFP共表达,均定位于细胞浆.AFP存在于HSP70单抗的免疫沉淀中,而HSP70则存在于AFP单抗的免疫沉淀中.结果表明人肝癌细胞BEL-7402中HSP70与AFP相伴.两者之间的相互关系研究将成为探讨肝癌的发生和免疫治疗的新途径.     

Seasonal patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants

Although considerable effort has been directed at identifying and understanding the function and regulation of stress-induced proteins in herbaceous plants, reports concerning woody plants are limited. Studies with herbaceous crops have revealed similarities in the types of proteins that accumulate in response to a wide array of abiotic stresses and hormonal cues such as the accumulation of abscisic acid. Many of the identified proteins appear to be related to dehydrins (the D-11 subgroup of late-embryogenesis-abundant proteins). The objective of the present study was to determine if seasonal induction of dehydrins is a common feature in woody plants and to see if seasonal patterns existed for other stress-induced proteins. Bark tissues from eight species of woody plants were collected monthly for a period of 1.5 years. The species included: peach (Prunus persica) cv. Loring; apple (Malus domestica) cv. Golden Delicious; thornless blackberry (Rubus sp.) cv. Chester; hybrid poplar (Populus nigra); weeping willow (Salix babylonica); flowering dogwood (Cornus florida); sassafras (Sassafras albidum); and black locust (Robinia pseudo-acacia). Immunoblots of bark proteins were probed with a polyclonal antibody recognizing a conserved region of dehydrin proteins, and monoclonal antibodies directed against members of the HS70 family of heat-shock proteins. Some proteins, immunologically related to dehydrins, appeared to be constitutive; however, distinct seasonal patterns associated with winter acclimation were also observed in all species. The molecular masses of these proteins varied widely, although similarities were observed in related species (willow and poplar). Identification of proteins using the monoclonal antibodies (HSP70, HSC70, BiP) was more definitive because of their inherent specificity, but seasonal patterns were more variable among the eight species examined. This study represents only a precursory examination of several proteins reported to be stress related in herbaceous plants, but the results indicate that these proteins are also common to woody plants and that further research to characterize their regulation and function in relation to stress adaptation and the perennial life cycle of woody plants is warranted.     

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.     

热休克反应中小鼠肾HSP70和乳酸脱氢酶同工酶表达变化的研究

用免疫组织化学和聚丙烯酰胺凝胶同工酶电泳方法研究了小鼠肾在热休克(46℃,30分钟)恢复期(4h和12h)HSP70的表达和乳酸脱氢酶(LDH)同工酶的变化。结果表明:(1)HSP70主要定位于肾小管上皮细胞胞质中,细胞核内未见表达;(2)HSP70免疫阳性反应在肾髓质较肾皮质强,肾小管较肾小球强;(3)热休克诱导小鼠肾LDH同工酶活性增强。提示:LDH同工酶可能对细胞热耐受性的建立有重要作用。     

Sorghum bicolor SbHSP110 has an elongated shape and is able of protecting against aggregation and replacing human HSPH1/HSP110 in refolding and disaggregation assays

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.     

热休克反应中小鼠心脏HSP70表达的免疫组织化学研究

用免疫组织化学方法研究了小鼠心脏在不同温度（40、41、44、46℃）热休克处理后,各恢复期（2、4、8、12、24小时）HSP70的表达。结果表明,（1）44、46℃热处理能诱导心肌细胞合成HSP70,以46℃为多（P＜0．01）,且于恢复期4－8小时为合成高峰（P＜0．01）。（2）阳性免疫反应定位于心肌细胞质中,核呈阴性反应。提示了心脏有较强的耐热能力。     

T cell recognition of naturally presented epitopes of self-heat shock protein 70

Self-reactive T cells have shown to have a potential role as regulators of the immune system preventing or even suppressing autoimmunity. One of the most abundant proteins that can be eluted from human HLA molecules is heat shock protein 70 (HSP70). The aims of the current study are to identify HSP70 epitopes based on published HLA elution studies and to investigate whether T cells from healthy individuals may respond to such self-epitopes. A literature search and subsequent in silico binding prediction based on theoretical MHC binding motifs resulted in the identification of seven HSP70 epitopes. PBMCs of healthy controls proliferated after incubation with two of the seven peptides (H167 and H290). Furthermore H161, H290, and H443 induced CD69 expression or production of cytokines IFNγ or TNFα in healthy controls. The identification of these naturally presented epitopes and the response they elicit in the normal immune system make them potential candidates to study during inflammatory conditions as well as in autoimmune diseases.     

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (相似文献   

Restriction fragment length polymorphisms at the heat shock protein HSP70 gene(s) in pigs*

Pigs from a population consisting of eight US breeds or strains and three Chinese breeds were examined by restriction fragment length polymorphism (RFLP) analysis of the heat shock protein HSP70 gene(s). Limited polymorphisms with PstI and PvuII restriction enzymes were observed, but there were no polymorphisms with BomIII and BglI.     

Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin

Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa. Heat stress at 42.5 °C appeared to be the optimum temperature for HSP formation in cells grown at 30 °C.The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h.The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli. To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity. There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942. Moreover, the purified HSP64 cross-reacted to anti-E. coli GroEL antibody. To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.     

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。