HSP90在乳腺癌细胞增殖中的作用与机制研究

目的： 应用热休克蛋白90(HSP90)功能特异性抑制剂格尔德霉素(GA)研究HSP90在乳腺癌细胞增殖中的作用，进而探讨相关机制。方法: 培养人乳腺癌细胞株MDA-MB-435s，采用MTT法检测GA对MDA-MB-435s细胞的生长抑制作用，流式细胞术分析细胞周期，应用Western免疫印迹法检测细胞内Stat3磷酸化状态和突变型p53表达变化。结果: 不同浓度GA对乳腺癌MDA-MB-435s细胞有生长抑制作用，呈时效量效依赖关系，GA作用后细胞呈现G2/M期阻滞。400 nmol/L GA作用48h后，细胞内Stat3磷酸化水平明显低于对照组，同时突变型p53表达明显低于对照组。结论: 抑制HSP90功能可明显抑制乳腺癌MDA-MB-435s细胞的增殖，此与下调细胞内Stat3磷酸化水平和突变型p53表达有关。     

Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family.

Induction of heat shock-related stress proteins Pfhsp and Pfgrp, similar in sequence to hsp70 (heat shock protein) and grp78 (glucose-regulated protein), respectively, was studied in culture-derived parasite Plasmodium falciparum. Elevation in temperature from 26 degrees C to 37 degrees C and higher caused significant induction of Pfhsp with a moderate effect on the synthesis of Pfgrp also. Synthesis of Pfgrp, however, was not induced by partial glucose deprivation. On the contrary, lack of glucose in the medium resulted in cessation of protein synthesis in the parasites. Other known inducers of grp synthesis in mammalian cells, i.e., calcium ionophore A23187 and inhibitors of glycosylation (tunicamycin, 2-deoxy glucose) were also without any apparent effect on the synthesis of Pfgrp. Heat shock-induced responses were transient in nature: removal of stress caused repression of these responses. The effect of glucose deprivation was only partially reversible with better recovery if parasites were subjected to glucose starvation at 26 degrees C than at 37 degrees C. Northern blot analysis and in vitro translation of mRNA revealed a parallel increase in the levels of mRNA for Pfhsp upon heat shock. Immuno-gold electron microscopy with cultured parasites revealed nuclear location of Pfhsp and primarily cytoplasmic (probably endoplasmic reticulum) location of Pfgrp. These findings suggest that SDEL (carboxy terminal sequence of Pfgrp) might play a similar role in the cellular localization of Pfgrp as does the sequence KDEL in mammalian cells and HDEL in yeast.     

Potentiation of the antimalarial activity of atovaquone by doxycycline against Plasmodium falciparum in vitro

The effect of doxycycline, obtained from human volunteers administered doxycycline, on the minimum inhibitory concentration (MIC) of atovaquone was determined against the K1 and FC27 isolates of Plasmodium falciparum in vitro. Doxycycline concentrations ranging from 0.10-1.18 μg/ml added to atovaquone produced MIC ratios [atovaquone + doxycycline/atovaquone alone] ranging from 0.38 to 0.70. These results suggest that the antimalarial activity of atovaquone is potentiated by doxycycline. Additionally, these drugs may be rational partners for the treatment and prophylaxis of falciparum malaria. Received: 4 October 1996 / Accepted: 29 November 1996     

Metalloprotease activity in a small heat shock protein of the human malaria parasite Plasmodium vivax

The malaria parasite affects millions of people each year, lives and multiplies in two different hosts, and synthesizes a large number of proteases and heat shock proteins (HSPs) for its survival. We describe here the characterization of a metalloprotease activity which resides in the small HSP (PVHSP28) of the common but noncultivable human malaria parasite Plasmodium vivax. The protein is expressed by erythrocytic stages of the parasite. It is expressed as a approximately 55-kDa polypeptide which is then processed to the 28-kDa mature protein. The latter was found to be an active protease in gelatin zymography. This protease showed its optimal activity at 37 degrees C (pH 7.6). It also retained its proteolytic activity at higher temperatures of up to 55 degrees C. The enzyme belongs to the metalloprotease class, as its proteolytic activity was most effectively blocked by 1,10-phenanthroline and was restored to a maximal level by the addition of zinc metal ions. Inhibitors for the cysteine, serine, and aspartate classes of proteases were ineffective against this enzyme. A homology search indicates that PVHSP28 probably belongs to a new class of HSPs which possess the metalloprotease signature sequence.     

The long and winding road: protein trafficking mechanisms in the Plasmodium falciparum infected erythrocyte

Mature human erythrocytes infected with the human malarial parasite Plasmodium falciparum are extensively modified to provide a more comfortable "home" for their intracellular guests. This process is mediated by parasite-encoded factors that are exported into, and through the host erythrocyte. This intra- yet simultaneously extra-cellular protein trafficking and sorting system has, in the past decades received much attention, also due to its unusual nature. Recent reports have highlighted the importance of a short peptide sequence, referred to individually as Plasmodium export element (PEXEL), vacuolar translocation signal (VTS) or generally as host cell targeting signal (HCT) in the export of both soluble and membrane bound proteins, allowing the partial definition of the parasite's "exportome". Mechanistically however, the discovery of this sequence raises as many questions as it answers. In this article, we comment on current models of protein transport to the host cell, discuss the mechanistic problems highlighted by these signals, and suggest what might be the next important steps in studying the protein export mechanisms of an obligate intracellular parasite that chooses to inhabit a de-nucleated host cell.     

热休克蛋白27,60和90在胃癌中表达及其临床价值

目的：探讨HSP-27、-60和-90在胃癌中的表达及其临床意义。 方法：采用免疫组织化学检测66例胃癌组织中HSP-27、-60和-90的表达情况,并结合临床病理特征、肿瘤增殖能力和患者生存期分析三种热休克蛋白表达的临床意义。结果： HSP-27、-60和-90在胃癌组织中异常高表达。HSP-27表达与肿瘤大小（pT,P=0.026）、器官转移（pM, P）及病理分期（P=0.041）相关,而HSP-27染色强度与淋巴腺状态明显相关（pN, P）。HSP-60表达与患者性别相关（P=0.011）,而HSP-60染色强度与患者年龄（P=0.027）和肿瘤病理组织学分级（P=0.031）相关。HSP-90表达与本研究分析的临床病理参数无相关性;但是,HSP-90染色强度与肿瘤大小存在着显著关系（pT,P=0.020）。单因素分析表明HSP-90高表达与生存期更长显著相关（P=0.033）,多因素分析证实HSP-90高表达是胃癌独立预后因素（P=0.026）。结论： HSP-27、-60和-90与某些临床病理参数有关,这些参数对于胃腺癌患者的治疗至关重要。胃腺癌患者HSP-90高表达是独立预后指标。=0.042=0.046     

Characterization of Plasmodium vivax heat shock protein 70 and evaluation of its value for serodiagnosis of tertian malaria

We have characterized Plasmodium vivax heat shock protein 70 (PvHSP70) and evaluated serodiagnostic applicability of recombinant PvHSP70 (rPvHSP70). In enzyme-linked immunosorbent assays and immunoblot analyses, rPvHSP70 showed high sensitivity (88.8%; 203/228 cases). P. falciparum-infected sera revealed positive reactions (78.8%). The predominant immunoglobulin G (IgG) subclasses were segregated with IgG1 and IgG3.     

Plasmodium falciparum line-dependent association of in vitro growth-inhibitory activity and risk of malaria

Plasmodium falciparum's ability to invade erythrocytes is essential for its survival within the human host. Immune mechanisms that impair this ability are therefore expected to contribute to immunity against the parasite. Plasma of humans who are naturally exposed to malaria has been shown to have growth-inhibitory activity (GIA) in vitro. However, the importance of GIA in relation to protection from malaria has been unclear. In a case-control study nested within a longitudinally followed population in Tanzania, plasma samples collected at baseline from 171 individuals (55 cases and 116 age-matched controls) were assayed for GIA using three P. falciparum lines (3D7, K1, and W2mef) chosen based on their erythrocyte invasion phenotypes. Distribution of GIA differed between the lines, with most samples inhibiting the growth of 3D7 and K1 and enhancing the growth of W2mef. GIA to 3D7 was associated with a reduced risk of malaria within 40 weeks of follow-up (odds ratio, 0.45; 95% confidence interval [CI], 0.21 to 0.96; P = 0.04), whereas GIA to K1 and W2mef was not. These results show that GIA, as well as its association with protection from malaria, is dependent on the P. falciparum line and can be explained by differences in erythrocyte invasion phenotypes between parasite lines. Our study contributes knowledge on the biological importance of growth inhibition and the potential influence of P. falciparum erythrocyte invasion phenotypic differences on its relationship to protective immunity against malaria.     

Molecular docking simulation analysis of the interaction of dietary flavonols with heat shock protein 90

Hsp90 is a major protein involved in the stabilization of various proteins in cancer cells. The present investigation focused on the molecular docking simulation studies of flavanols as inhibitors of Hsp90 at the high affinity adenosine triphosphate (ATP) binding site and analyzed absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity). The molecular docking analysis revealed that the flavanols showed competitive inhibition with ATP molecule at the active site and enhanced pharmacological parameters.     

Interactions of the antimalarial drug methylene blue with methemoglobin and heme targets in Plasmodium falciparum: a physico-biochemical study

AIMS: Resistance of Plasmodium falciparum to drugs has led to renewed interest of redox-active methylene blue (MB) for which no resistance has been reported so far. Moreover, MB displays unique interactions with glutathione reductase (GR). However, the mechanisms of action/interaction with potential targets of MB are yet to be elucidated. Our physico-biochemical study on MB and relevant hematin-containing targets was performed under quasi-physiological conditions. RESULTS: The water deprotonation of the Fe(III)protoporphyrin dimer, the major building block of β-hematin, was studied. At pH 6, the predominant dimer possesses water coordinated to both metals. Below pH 6, spontaneous precipitation of β-hematin occurred reminiscent of hemozoin biomineralization at pH 5.0-5.5 in the food vacuole of the malarial parasite. MB also forms dimers (K(Dim)=6800 M(-1)) and firmly binds to hematin in a 2:1 hematin:MB sandwich complex (K(D)=3.16 μM). MB bioactivation catalyzed by GR induces efficient methemoglobin(Fe(III)) [metHb(Fe(III))] reduction to hemoglobin(Fe(II)). The reduction rate, mediated by leucomethylene blue (LMB), was determined (k(metHb)(red)=991 M(-1)·s(-1)) in an assay coupled to the GR/reduced form of nicotinamide adenine dinucleotide phosphate system. INNOVATION AND CONCLUSION: Our work provides new insights into the understanding of (i) how MB interacts with hematin-containing targets, (ii) other relevant MB properties in corroboration with the distribution of the three major LMB species as a function of pH, and (iii) how this redox-active cycler induces efficient catalytic reduction of metHb(Fe(III)) to hemoglobin(Fe(II)) mediated by oxidoreductases. These physico-biochemical parameters of MB open promising perspectives for the interpretation of the pharmacology and pathophysiology of malaria and possibly new routes for antimalarial drug development.     

Tubulin和HSP90在氧化应激预适应中的作用

目的：探讨热休克蛋白90和细胞骨架蛋白tubulin在氧化应激预适应中的作用。方法:应用不同浓度H2O2作用于HepG2细胞,建立HSP90不同表达量的模型后,MTT比色法检测细胞活力,Western blotting 定量检测各模型中HSP90、tubulin量,激光扫描共聚焦显微镜测定该两种蛋白在对照、预适应、氧化应激、预适应后氧化应激下的分布和共定位。 结果:MTT比色法结果提示预适应可提高应激状态下的细胞生存活力;Western blotting结果显示氧化应激预适应可提高细胞内HSP90 和 tubulin含量,减轻再次应激所致的HSP90 和 tubulin总量下降;激光共聚焦显示该两种蛋白有相似的细胞分布。结论:Tubulin可能协同HSP90在氧化应激预适应中起保护作用。     

Protective effect of human heat shock protein 60 suggested by its association with decreased seropositivity to pathogens

The presence of heat shock protein 60 (Hsp60) in human plasma has been linked with cardiovascular disease (CVD). In this study, the examination of the relationship between Hsp60 in plasma and seropositivity for three microbial agents, which are thought to be risk factors for CVD, surprisingly revealed a negative association between Hsp60 and seropositivity, suggesting a protective effect of this circulating stress protein.     

In vitro antimalarial activity of traditionally used Western Ghats plants from India and their interactions with chloroquine against chloroquine-resistant Plasmodium falciparum

Malaria is a major global public health problem, and the alarming spread of drug resistance and limited number of effective drugs now available underline how important it is to discover new antimalarial compounds. An ethnopharmacological investigation was undertaken on Western Ghats plants traditionally used to treat malaria in India; 50 plants were very carefully selected from a total of 372 plants, and 200 extracts were prepared and tested for in vitro antiplasmodial activity alone and in combination with chloroquine (CQ) against CQ-resistant Plasmodium falciparum (strain MRC-Pf-43). In in vitro antiplasmodial activity, when plant extract alone is used, 29 extracts (or 14.5%) showed significant high in vitro antiplasmodial activity with IC₅₀ values ranging from 3.96 to 4.85 μg/ml, 53 extracts (or 26.5%) showed significant good in vitro antiplasmodial activity with IC₅₀ values ranging from 5.02 to 9.87 μg/ml, and 28 extracts (or 14%) showed significant moderate in vitro antiplasmodial activity with IC₅₀ values ranging from 10.87 to 14 μg/ml, respectively. In combination with CQ, 103 extracts (or 51.5%) showed significant synergistic in vitro antiplasmodial activities with synergistic factor values ranging from 1.03 to 1.92, and these activities were up to a fold higher with CQ, suggesting synergistic interactions of the two drugs. Our investigation has confirmed that above 62.1% of the plant extracts showed moderate to high in vitro antiplasmodial activity when used alone, and in combination with CQ, 55.7% of the extracts showed borderline to good synergistic activity.     

Convergence of heat shock protein 90 with ubiquitin in filamentous alpha-synuclein inclusions of alpha-synucleinopathies

Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are alpha-synucleinopathies characterized by filamentous alpha-synuclein (alpha-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with alpha-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with alpha-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble alpha-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like alpha-syn pathologies. Cell culture studies also revealed that alpha-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of alpha-syn inclusions in PD and related alpha-synucleinopathies.     

热休克蛋白90和凋亡抑制蛋白Livin在病理性瘢痕中的表达

背景：近年来研究表明热休克蛋白90和凋亡抑制蛋白Livin在细胞的凋亡过程中扮演着非常重要的角色,而两者在病理性瘢痕形成过程中的作用研究至今鲜有报道。 目的：从基因和蛋白水平分别检测热休克蛋白90和凋亡抑制蛋白Livin在病理性瘢痕中的表达情况及两者之间的关系。 方法：分别采用RT-PCR方法和免疫组织化学方法检测热休克蛋白90和Livin在24例瘢痕疙瘩、26例增生性瘢痕、22例非病理性瘢痕和20例正常皮肤组织中的表达水平。 结果与结论：瘢痕疙瘩,增生性瘢痕中热休克蛋白90和Livin的阳性表达水平高于非病理性瘢痕及正常皮肤组织(P < 0.05)。病理性瘢痕中热休克蛋白90和Livin表达呈正相关(rs=0.436, P < 0.05)。RT-PCR检测结果与免疫组织化学结果基本一致。结果提示热休克蛋白90和Livin在病理性瘢痕的形成过程中可能起着重要作用,且二者之间可能具有协同作用。      

The ring-infected erythrocyte surface antigen protein of Plasmodium falciparum is phosphorylated upon association with the host cell membrane

The ring-infected erythrocyte surface antigen (RESA) is a 155-kDa malarial polypeptide which is released from merozoites and becomes associated with the erythrocyte membrane at the time of invasion. Inside-out vesicles (IOVs) prepared from Plasmodium falciparum-infected erythrocytes contain RESA, presumably bound to the membrane skeleton, as it is largely insoluble in Triton X-100. When these IOVs were incubated with [gamma-32P]ATP, a 155-kDa polypeptide was labeled in IOVs from infected, but not from uninfected erythrocytes. Immunoprecipitation using specific rabbit antisera confirmed that RESA is indeed a phosphoprotein. Phosphoamino acid analysis revealed phosphoserine and a small amount of phosphothreonine, but no phosphotyrosine. Labeling of intact parasitized erythrocytes with inorganic [32P]phosphate for several hours in culture resulted in RESA in Triton-insoluble extracts being phosphorylated. Labeling of synchronized parasites showed that RESA was phosphorylated only when it became associated with the erythrocyte membrane, and although RESA was abundant in mature parasites, it was not phosphorylated. RESA, released into the culture supernatants during the growth of P. falciparum, bound to IOVs prepared from normal uninfected erythrocytes, and subsequent labeling with [gamma-32P]ATP resulted in the phosphorylation of RESA. The evidence suggests that RESA is phosphorylated by an erythrocyte membrane kinase and probably not by a parasite-encoded enzyme.     

热休克蛋白70、90在子宫内膜癌中的表达

目的:探讨子宫内膜癌组织中热休克蛋白(HSP)70、90的表达及意义。方法:用免疫组化Envision法及图象分析仪,检测 30例正常子宫内膜、30例增生过长子宫内膜和53例子宫内膜癌中HSP70、HSP90的表达。结果:子宫内膜癌中HSP70表达的灰度值为(209.06±5.36),明显高于正常内膜[(145.21±4.09),P<0.01]和增生过长内膜[(148.59±4.23),P<0.01];子宫内膜癌中HSP90表达的灰度值为(166.98±5.71),明显低于正常子宫内膜[(208.57±31.14),P<0.05]和增生过长子宫内膜[(249.73±4.94),P<0.01]。子宫内膜癌中HSP70的表达随肿瘤病理分级的增加而增强(P<0.01),非内膜样癌(229.90±3.77)较内膜样癌表达强[(198.37±3.15),P<0.01];子宫内膜癌中HSP90表达随肿瘤病理分级的升高而表达减弱,非内膜样癌(140.21±3.22)较内膜样癌表达减弱[(176.59±2.79),P<0.01]。子宫内膜癌中HSP70、90的表达与肌层浸润深度、临床分期及淋巴结转移未见显著相关性(P>0.05)。结论:HSP70、90可能与子宫内膜癌的发生及预后有关。