Calreticulin Binds to Gentamicin and Reduces Drug-Induced Ototoxicity
Calreticulin Binds to Gentamicin and Reduces Drug-Induced Ototoxicity
复制标题
DOI：
10.1093/toxsci/kfr196
复制
发表时间：
2011-12-01
影响因子：
3.8
通讯作者：
Steyger, Peter S.
中科院分区：
文献类型：
--
作者：
Karasawa, Takatoshi;Wang, Qi;Steyger, Peter S.
氨基糖苷类药物如庆大霉素是临床实践中最常用的抗生素之一，对于治疗危及生命的结核病和革兰氏阴性细菌感染至关重要。然而，氨基糖苷类也具有肾毒性和耳毒性。虽然已经提出了许多机制，但氨基糖苷类药物如何诱导听觉感觉上皮细胞死亡和随后的耳聋仍不清楚。氨基糖苷类与各种细胞内分子结合，如RNA和磷酸肌醇。我们假设，氨基糖苷类，基于其组织特异性的敏感性，也结合细胞内的蛋白质，在药物诱导的耳毒性中发挥作用。通过将氨基糖苷庆大霉素与琼脂糖珠结合并进行庆大霉素-琼脂糖下拉测定，我们从小鼠Corti器官HEI-OC 1的永生化细胞中分离出庆大霉素结合蛋白（GBP）。质谱鉴定钙网蛋白（CRT）为GBP。免疫荧光显示，CRT表达集中在耳蜗边缘细胞和毛细胞静纤毛，药物摄取和细胞毒性的主要位置。在用庆大霉素处理的HEI-OC 1细胞中，使用小干扰RNA（siRNA）减少CRT表达降低了细胞内药物水平。CRT缺陷的小鼠胚胎成纤维细胞（MEF）细胞以及CRT siRNA转染的野生型MEF在庆大霉素处理后也具有降低的细胞活力。使用CRT的缺失突变体的下拉测定确定CRT的羧基C-结构域与庆大霉素结合。与转染全长CRT或其他缺失突变体的细胞相比，转染CRT C结构域缺失突变体构建体的HeLa细胞对庆大霉素诱导的细胞毒性更敏感。因此，我们得出结论，CRT结合庆大霉素是对庆大霉素诱导的细胞毒性的保护。
Aminoglycosides like gentamicin are among the most commonly used antibiotics in clinical practice and are essential for treating life-threatening tuberculosis and Gram-negative bacterial infections. However, aminoglycosides are also nephrotoxic and ototoxic. Although a number of mechanisms have been proposed, it is still unclear how aminoglycosides induce cell death in auditory sensory epithelia and subsequent deafness. Aminoglycosides bind to various intracellular molecules, such as RNA and phosphoinositides. We hypothesized that aminoglycosides, based on their tissue-specific susceptibility, also bind to intracellular proteins that play a role in drug-induced ototoxicity. By conjugating an aminoglycoside, gentamicin, to agarose beads and conducting a gentamicin-agarose pull-down assay, we have isolated gentamicin-binding proteins (GBPs) from immortalized cells of mouse organ of Corti, HEI-OC1. Mass spectrometry identified calreticulin (CRT) as a GBP. Immunofluorescence revealed that CRT expression is concentrated in strial marginal cells and hair cell stereocilia, primary locations of drug uptake and cytotoxicity in the cochlea. In HEI-OC1 cells treated with gentamicin, reduction of CRT expression using small interfering RNA (siRNA) reduced intracellular drug levels. CRT-deficient mouse embryonic fibroblast (MEF) cells as well as CRT siRNA-transfected wild-type MEFs also had reduced cell viability after gentamicin treatment. A pull-down assay using deletion mutants of CRT determined that the carboxyl C-domain of CRT binds to gentamicin. HeLa cells transfected with CRT C-domain deletion mutant construct were more susceptible to gentamicin-induced cytotoxicity compared with cells transfected with full-length CRT or other deletion mutants. Therefore, we conclude that CRT binding to gentamicin is protective against gentamicin-induced cytotoxicity.