73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin
73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin
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DOI：
10.1074/jbc.m312217200
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发表时间：
2004-04-23
影响因子：
4.8
通讯作者：
Itoh, H
中科院分区：
文献类型：
--
作者：
Miyazaki, T;Sagawa, R;Itoh, H
虽然庆大霉素（GM）已被广泛用作抗生素，但其特异性结合蛋白尚未被充分了解。在这里，我们表明GM特异性地与73-kDa分子伴侣HSP73结合，并在体外降低其伴侣活性。在本研究中，我们使用转基因亲和柱和猪肾细胞质来研究转基因特异性结合蛋白。洗涤柱后，加入10 mM GM将73-kDa蛋白从柱中洗脱，洗脱液中未发现其他蛋白。经免疫印迹，该蛋白与HSP73相同。CD谱分析表明，GM与HSP73的结合导致了该蛋白的构象变化。尽管HSP73在体外阻止未展开罗丹斯的聚集，但在GM存在下，HSP73的伴侣活性被抑制。利用tpk -胰蛋白酶对HSP73进行有限的蛋白水解，GM结合位点是已知肽结合结构域三分之一的蛋白的cooh末端。免疫组化结果显示，HSP73和GM在GM诱导的急性肾小管损伤大鼠肾脏溶酶体中共定位。我们的研究结果表明，HSP73和GM之间的特异性关联可能会降低HSP73在体外和/或体内的伴侣活性，这可能与GM对肾脏急性肾小管损伤的毒性相互作用。
Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, 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 eluant. Upon 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 to be 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.