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

我们对HSP60导入系统进行了研究。免疫印迹和免疫组织化学结果显示，HSP60在细胞质和线粒体中均有表达。纯化的胞质HSP60具有伴侣活性，通过线粒体检测，该蛋白被导入体外线粒体。限水3天和5天的大鼠肾乳头组织中HSP60基因表达增加，而肾皮质和髓质中HSP60基因表达无明显变化。免疫印迹法检测到HSP60在正常大鼠肾皮质、髓质和乳头的胞浆和线粒体中的表达几乎相同。限水大鼠肾乳头HSP60蛋白表达明显减少，线粒体HSP60蛋白表达增加。我们还利用信号序列-亲和柱层析分析了该蛋白与HSP60信号序列的结合。在SDS/PAGE上只鉴定出一条相对分子质量为70 kDa的蛋白条带。该蛋白是从…中洗脱出来的。通过过量的信号肽或5 mM的三磷酸腺苷获得更多的E亲和柱。在免疫印迹中，70 kDa的蛋白与抗HSP70的抗体发生交叉反应。这些结果表明，哺乳动物的HSP60既以稳定的胞浆HSP60的形式存在于细胞质中，在正常情况下也存在于线粒体中。在严酷的条件下，细胞质HSP60能迅速将细胞质HSP60导入线粒体。我们鉴定了睾丸和脑特异性105 kDa蛋白的生化性质，该蛋白与抗牛HSP90抗体发生交叉反应。该105 kDa蛋白在体外作为分子伴侣抑制柠檬酸合成酶的聚集。ATP/MgCl2对105-kDa蛋白抑制柠檬酸合成酶聚集有轻微影响。与其他分子伴侣热休克蛋白70一样，该蛋白能够与ATP-琼脂糖凝胶结合。测定了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和渗透压应激蛋白94(OSP94)的氨基酸序列一致。免疫印迹显示，该105 kDa蛋白仅在肾髓质中检测到，与OSP94相似。纯化的105 kDa蛋白与抗APG-1抗体发生交叉反应。这些结果表明，APG-1和OPS94都与105 kDa的蛋白相同。庆大霉素(GM)已被广泛用作抗生素。然而，药物的特定结合蛋白还没有得到充分的了解。我们使用GM亲和柱和猪肾胞浆研究了GM特异性结合蛋白。洗脱柱后，加入10 mM GM，仅73 kDa蛋白从柱上洗脱。在突变体中没有发现其他蛋白。经免疫印迹分析，该蛋白与HSP73蛋白完全相同。CD光谱分析表明，GM与HSP73的结合导致了蛋白质构象的变化。虽然HSP73在体外可以抑制未折叠的罗丹明的聚集，但在GM的存在下，HSP73的伴侣活性被抑制。利用TPCK-胰酶对HSP73的有限蛋白分解，GM结合部位是三分之一蛋白质的COOH末端，称为多肽结合域。在免疫组织化学中，HSP73和GM共定位于GM诱导的急性肾小管损伤大鼠肾脏内增大的溶酶体内。我们的结果提示，在体外和/或体内，HSP73与GM之间的特异性关联可能会降低HSP73的伴侣活性，这可能与GM致急性肾小管损伤的GM毒性作用有关。较少

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