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Analysis of protective roles for renal injury by heat shock proteins treansfected with HSP genes

热休克蛋白转染HSP基因对肾损伤的保护作用分析

基本信息

批准号：
14571011
负责人：
KOMATSUDA Atsushi
金额：
$ 2.24万
依托单位：
Akita University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571011/
关键词：
heat shock proteins HSP73 gentamicin chaperon renal toxicity HSP60 HSP105 dehydration HSP72 HSP90 シャペロン遺伝子導入耐性浸透圧

项目摘要

We investigated HSP60 import system. From immunoblotting and immunohistochemistry, HSP60 was detected in both cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial assay. HSP60 mRNA was increased in the renal papilla of rats that had been restricted water for three and five days, but no changes in HSP60 mRNA in the renal cortex or medulla. On immunoblotting, HSP60 was detected in both cytoplasm and mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the renal papilla of rats with water restriction, but the protein was increased in the mitochondria of the rat renal papilla. We also analyzed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from th … More e affinity column by an excess of signal peptide or by 5 mM ATP. On immunoblotting, the 70-kDa protein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytosolic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP60.We have characterized the biochemical properties of the testis and brain specific 105-kDa protein, which is cross-reacted with an anti-bovine HSP90 antibody. The 105-kDa protein inhibited the aggregation of citrate synthase as a molecular chaperone in vitro. ATP/MgCl2 has slightly influenced the suppression of the citrate synthase aggregation by 105-kDa protein. The protein was able to bind to ATP-Sepharose like the other molecular chaperone HSP70. A partial amino-acid sequence (24 amino-acid residues) of the protein was determined and coincided with those of the mouse testis-and brain-specific APG-1 and osmotic stress protein 94 (OSP94). The 105-kDa protein was detected only in the renal medulla similar to OSP94 upon immunoblotting. The purified 105-kDa protein was cross-reacted with an antibody against APG-1. These results suggested that APG-1 and OPS94 are both identical to the 105-kDa protein.Gentamicin (GM) has been used widely as an antibiotic. However, the specific binding protein of the drug has not yet been understood sufficiently. We investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the elutant. By immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known as a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury. Less

我们研究了HSP 60的导入系统。免疫印迹和免疫组化结果显示，HSP 60在细胞质和线粒体中均有表达。纯化的细胞质HSP 60显示分子伴侣活性，并通过线粒体测定将蛋白质在体外导入线粒体。限水3d和5d大鼠肾乳头HSP 60 mRNA表达增加，而肾皮质和髓质HSP 60 mRNA表达无明显变化。免疫印迹结果显示，HSP 60在正常大鼠肾皮质、髓质和肾乳头的胞浆和线粒体中均有表达。水限制大鼠肾乳头内HSP 60表达明显减少，线粒体内HSP 60蛋白表达明显增加。我们还分析了结合的蛋白质的信号序列的HSP 60使用信号序列亲和柱层析。在SDS/PAGE上仅鉴定出一条相对分子质量约为70 kDa的蛋白质条带。蛋白质被洗脱， ...更多信息通过过量的信号肽或通过5 mM ATP通过亲和柱。在免疫印迹中，70 kDa的蛋白质与针对HSP 70的抗体发生交叉反应。这些结果表明，哺乳动物的HSP 60既位于细胞质中作为一个稳定的胞浆HSP 60，也在线粒体在正常条件下。细胞质HSP 60在严酷条件下可迅速进入线粒体。我们对睾丸和脑特异性105-kDa蛋白的生化特性进行了表征，该蛋白可与抗牛HSP 90抗体发生交叉反应。105 kDa的蛋白质抑制柠檬酸合成酶的聚集作为分子伴侣在体外。ATP/MgCl 2对105-kDa蛋白抑制柠檬酸合酶聚集有轻微影响。该蛋白能够像其他分子伴侣HSP 70一样与ATP-Sepharose结合。该蛋白的部分氨基酸序列（24个氨基酸残基）与小鼠睾丸和脑特异性APG-1和渗透应激蛋白94（OSP 94）的氨基酸序列一致。105-kDa蛋白质仅在肾髓质中检测到类似于OSP 94的免疫印迹。纯化的105-kDa蛋白与抗APG-1的抗体发生交叉反应。这些结果表明APG-1和OPS 94与105-kDa蛋白相同。然而，该药物的特异性结合蛋白尚未被充分理解。我们使用GM亲和柱和猪肾胞质溶胶研究GM特异性结合蛋白。洗涤柱后，通过加入10 mm GM仅从柱上洗脱73-kDa蛋白质。洗脱液中未发现其他蛋白质。通过免疫印迹，该蛋白与HSP 73相同。CD谱分析表明，GM与HSP 73结合后，其蛋白质构象发生了变化。虽然热休克蛋白73防止聚集未折叠的罗丹酸在体外，在GM的存在下，抑制了热休克蛋白73的伴侣活性。利用TPCK-胰蛋白酶对HSP 73的有限蛋白水解，GM结合位点是三分之一的蛋白质的COOH末端，称为肽结合结构域。在免疫组化过程中，HSP 73和GM共定位于GM诱导的急性肾小管损伤大鼠肾脏扩大的溶酶体中。我们的研究结果表明，在体外和/或在体内，热休克蛋白73和GM之间的特异性关联可能会降低伴侣活性的热休克蛋白73，这可能有一个相互作用与GM的毒性在肾脏GM诱导的急性肾小管损伤。少