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.     

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.     

过量表达内质网小分子热激蛋白增强番茄的衣霉素抗性

真核细胞内质网腔内未折叠蛋白的过度积累会引起内质网胁迫（ER胁迫）,继而激活未折叠蛋白应答（UPR）信号途径,诱导内质网定位的分子伴侣的大量表达（如BiP和calnexin等）。本工作将CaMV35S启动子驱动的内质网小分子热激蛋白基因（ER-sHSP）导入番茄,发现ER-sHSP的过量表达提高了转基因番茄整株对衣霉素的抗性。衣霉素处理使未转基因番茄中BiP和calnexin基因的表达迅速升高,转基因番茄中这两个基因的表达也有增加,但表达强度明显低于未转基因番茄。说明ER-sHSP能够减轻ER胁迫,并可能参与UPR信号转导途径。     

Tolerating diabetes: an alternative therapeutic approach for diabetic neuropathy

It is becoming apparent that a number of pathogenic mechanisms contribute to diabetic neuropathy, so that therapeutic interventions that target one particular mechanism may have limited success. A recently published preclinical study has adopted an alternative approach by using a novel small molecule to induce heat-shock protein 70. This confers upon neurons, and perhaps other cells of the nervous system, the ability to better tolerate the diverse stresses associated with diabetes rather than intervening in their production.     

热激蛋白90抑制剂

分子伴侣热激蛋白90(heat-shock protein 90,Hsp90)在生物体内具有重要的生理功能,它在许多肿瘤细胞中表达增加。临床研究发现Hsp90抑制剂单一用药或者联合用药都具有较好的抗肿瘤效果,因此目前Hsp90被认为是癌症治疗一个非常有潜力的靶标。本文总结了Hsp90的结构功能、Hsp90抑制剂的作用机理以及Hsp90抑制剂的临床应用前景,希望为设计和开发新的Hsp90抑制剂提供一定的参考。     

蛋白激酶CK2与PrP蛋白的体外相互作用研究

为了探讨蛋白激酶CK2与PrP是否存在分子间相互作用,利用RT-PCR方法从人源细胞系SH-SY5Y cDNA中分别扩增出蛋白激酶CK2a和CK2β基因,并在E coli中表达了融合蛋白HIS-CK2α和GST-HISCK2β.利用pU-doWn及免疫共沉淀实验检测PrP与CK2的分子间相互作用.结果显示,重组PrP可与CK2α出现特异性结合,但不与CK2β发生反应.天然状态下脑组织中的CK2与PrPc也发生相互作用.PrP与CK2α亚基相互作用的区域位于PrP的C-端90～231位氨基酸.本研究从分子水平上提供了人重组和天然CK2和PrP蛋白相互作用的实验依据,为深入探讨CK2与朊蛋白作用的生物学意义和在朊病毒病发病过程中的作用提供了科学线索.     

Identification of Mixed Lineage Leukemia 1(MLL1) Protein as a Coactivator of Heat Shock Factor 1(HSF1) Protein in Response to Heat Shock Protein 90 (HSP90) Inhibition

Heat shock protein 90 (HSP90) inhibition inhibits cancer cell proliferation through depleting client oncoproteins and shutting down multiple oncogenic pathways. Therefore, it is an attractive strategy for targeting human cancers. Several HSP90 inhibitors, including AUY922 and STA9090, show promising effects in clinical trials. However, the efficacy of HSP90 inhibitors may be limited by heat shock factor 1 (HSF1)-mediated feedback mechanisms. Here, we identify, through an siRNA screen, that the histone H3 lysine 4 methyltransferase MLL1 functions as a coactivator of HSF1 in response to HSP90 inhibition. MLL1 is recruited to the promoters of HSF1 target genes and regulates their expression in response to HSP90 inhibition. In addition, a striking combination effect is observed when MLL1 depletion is combined with HSP90 inhibition in various human cancer cell lines and tumor models. Thus, targeting MLL1 may block a HSF1-mediated feedback mechanism induced by HSP90 inhibition and provide a new avenue to enhance HSP90 inhibitor activity in human cancers.     

Detection and Characterization of Antibodies to Bacterial Heat-Shock Protein 60 in Sera of Patients with Primary Biliary Cirrhosis

The enzyme-linked immunosorbent assay (ELISA) with bacterial heat-shock protein 60 (HSP60) purified from Yersinia enterocolitica (Ye) revealed that the antibodies directed against YeHSP60 existed in sera of patients with primary biliary cirrhosis (PBC). To characterize the epitope specificity of the antibodies in patients, the epitope mapping of HSP60 by means of the antibodies was performed. The results have suggested that the epitope recognized with anti-HSP60 antibodies in PBC relates to the amino acid sequence of YeHSP60 molecule as follows: DLGQA-KRVVINKDTTIIIDGVGDEAAIQGRLAQIRQQIEEATSDYDKEK.     

细胞外钙调素对烟草悬浮培养细胞蛋白质磷酸化作用的影响

用液体闪烁计数法研究了细胞外钙调素对烟草悬浮培养细胞质蛋白质磷酸化的作用。结果表明烟草细胞细胞质蛋白质磷酸化活性在细胞培养过程中逐渐增加,达到最高峰后又开始下降。在细胞质蛋白质磷酸化强度高峰时,加入抗CaM血清后,细胞质蛋白质磷酸化活性受到了部分抑制。加抗CaM血清后再补加CaM能够部分解除抗CaM血清对细胞质部分与细胞核部分蛋白质磷酸化的抑制作用。外加纯化钙调素可以引起烟草悬浮培养细胞细胞质蛋白质磷酸化的活性增强,并且这种增强作用具有时间（高峰为70min）与剂量（最适为CaM10-7mmol/L）依赖性。CaM引起的细胞质蛋白质磷酸化变化与红光所引起的细胞质蛋白质磷酸化变化在时间进程上是不相同的。     

Extended conformational states dominate the Hsp90 chaperone dynamics

The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a β-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.     

盐适应和非盐适应烟草愈伤组织蛋白质组分变化

通过在培养基中逐代增加氯化钠浓度方法获得的适应0.5％到1.0％NaCl的盐适应细胞系,与非盐适应细胞系的蛋白质电泳图谱相比,其蛋白质SDS-PAGE图谱中出现了26kD蛋白质,但量不多;此外,40.3和16.5kD蛋白质量增加,其中16.5kD蛋白质增加显著;而33.8和18kD蛋白质则减少。上述16.5,26和40.3kD蛋白质的诱导产生或含量的增加和细胞在盐环境中表现出的适应性相伴随发生,表明这3种蛋白质可能与抗盐性有关。     

Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.     

热休克蛋白90对血小板源衍生因子诱导的大鼠主动脉平滑肌细胞增殖的影响

目的:研究热休克蛋白90(HSP90)对血小板衍生因子(Platelet derived growth factor,PDGF)诱导的大鼠主动脉平滑肌细胞增殖的影响。方法:采用胶原酶消化法原代培养大鼠胸主动脉平滑肌细胞,应用脂质体细胞转染siRNA的方法抑制HSP90的表达,定量PCR和western blot的方法检测抑制效率。利用PDGF-bb诱导刺激平滑肌细胞增殖,CCK8法检测细胞增殖能力的变化,流式细胞术检测细胞生长周期的改变。结果:平滑肌细胞中转染HSP90的siRNA后,HSP90的mRNA和蛋白水平明显降低,分别为对照组的65.3%和57.6%(P0.05);PDGF-bb刺激明显促进平滑肌细胞生长,而降低HSP90水平显著影响PDGF-bb诱导的细胞增殖(P0.05);流式细胞术检测发现降低HSP90水平引起平滑肌细胞生长停滞,分布在细胞周期G1期的细胞比例明显增多(P0.05)。结论:HSP90通过调控平滑肌细胞的生长周期参与调节细胞增殖过程。     

The role of Hsp90 in cell response to hyperthermia

(1) Preincubation of SKOV3 human ovarian carcinoma cells with a non-toxic dose of Geldanamycin resulted in exacerbation of hyperthermia-induced cytotoxicity and re-distribution of dying cells toward necrosis. (2) Exposure of primary human ovarian carcinoma cells to mild hyperthermia (42 °C for 2 h) led, after a recovery period of 16 h, to several-fold increase in the levels of Hsp90 and ErbB2, to a moderate decrease in the levels of phospho-Erk1/2, whereas the level of β-catenin appeared to be unchanged. (3) The inhibitors of Hsp90 (Geldanamycin and Novobiocin) significantly affected the cell signaling of heat-pretreated cultures. (4) The results suggest that Hsp90 plays a pivotal role in cell response to hyperthermia. (5) A combination of Hsp90 inhibitors with hyperthermia may considerably increase the efficacy of thermotherapy.     

Protective Role of Genetic Variants in HSP90 Genes-Complex in COPD Secondary to Biomass-Burning Smoke Exposure and Non-Severe COPD Forms in Tobacco Smoking Subjects

Background: Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory disease characterized by airflow obstruction, commonly present in smokers and subjects exposed to noxious particles product of biomass-burning smoke (BBS). Several association studies have identified single-nucleotide polymorphisms (SNP) in coding genes related to the heat shock proteins family-genes that codify the heat shock proteins (Hsp). Hsp accomplishes critical roles in regulating immune response, antigen-processing, eliminating protein aggregates and co-activating receptors. The presence of SNPs in these genes can lead to alterations in immune responses. We aimed to evaluate the association of SNPs in the HSP90 gene complex and COPD. Methods: We enrolled 1549 participants, divided into two comparison groups; 919 tobacco-smoking subjects (cases COPD-TS n = 294 and, controls SWOC n = 625) and 630 chronic exposed to BBS (cases COPD-BBS n = 186 and controls BBES n = 444). We genotyped 2 SNPs: the rs13296 in HSP90AB1 and rs2070908 in HSP90B1. Results: Through the dominant model (GC + CC), the rs2070908 is associated with decreased risk (p < 0.01, OR = 0.6) to suffer COPD among chronic exposed BBS subjects. We found an association between rs13296 GG genotype and lower risk (p = 0.01, OR = 0.22) to suffer severe COPD-TS forms in the severity analysis. Conclusions: single-nucleotide variants in the HSP90AB1 and HSP90B1 genes are associated with decreased COPD risk in subjects exposed to BBS and the most severe forms of COPD in tobacco-smoking subjects.     

SNX-2112, a Novel Hsp90 Inhibitor,Induces G2/M Cell Cycle Arrest and Apoptosis in MCF-7 Cells

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.