Identification of gentamicin-binding proteins in the inner ear

描述(由申请人提供)：像庆大霉素这样的氨基糖苷类抗生素对于对抗危及生命的细菌感染是必不可少的。在美国，氨基糖苷类药物每年还会导致超过12万人永久性耳聋/平衡障碍和肾毒性，特别是对婴儿和早产儿。这项研究的长期目标是通过调节庆大霉素结合蛋白的功能来预防氨基糖苷类药物的耳毒性，以保护听觉功能。在这项建议中，我们试图确定庆大霉素结合蛋白，并确定它们在内耳中的分布。我们假设在内耳中存在庆大霉素结合蛋白，这些蛋白在特定类型的细胞中保留庆大霉素，在体外有助于细胞毒作用，在体内起致病作用。本项目的具体目标是：首先，鉴定肾脏近端小管和耳蜗细胞中的庆大霉素结合蛋白。我们将使用庆大霉素-琼脂糖下拉实验和SDS-聚丙烯酰胺凝胶电泳法从肾脏近端小管和耳蜗细胞系中分离出这些蛋白质，并使用质谱仪确定它们的身份(目标1)。第二，用免疫细胞化学和共聚焦显微镜检测庆大霉素结合蛋白，包括热休克蛋白73和钙网织蛋白在内耳中的分布。我们还将利用庆大霉素免疫细胞化学定位的荧光标记的庆大霉素或未标记的庆大霉素，确定这些蛋白是否与系统给药的庆大霉素共定位(目标2)。如果我们能识别出庆大霉素结合蛋白，将药物隔离在细胞内，然后引起细胞毒性，我们就可以开发新的策略来抑制它们之间的相互作用，并减少药物滞留。或者，如果庆大霉素结合抑制了这些蛋白的功能，我们可以使用这些蛋白的药理激活剂来克服庆大霉素诱导的抑制。未来的研究将确定庆大霉素是否抑制它们与生理结合分子/蛋白的相互作用，以及庆大霉素持续治疗期间庆大霉素结合蛋白的表达水平是否发生变化。识别庆大霉素结合蛋白及其细胞内庆大霉素干扰细胞死亡的机制将为临床医生筛选患者先前存在的疾病和增加氨基糖苷类毒性和耳聋风险的药物提供新的见解。鉴定在庆大霉素耳毒性中起主要作用的庆大霉素结合蛋白对于了解庆大霉素引起耳毒性和耳聋的机制是至关重要的。这项拟议的研究将使我们能够开发策略来防止庆大霉素和庆大霉素结合蛋白之间的相互作用，并通过药物激活这些蛋白来克服庆大霉素诱导的抑制。这些策略将导致耳毒性和耳聋预防。

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics like gentamicin are essential for battling life-threatening bacterial infections. Aminoglycosides also cause permanent deafness/balance disorders and nephrotoxicity in more than 120,000 individuals each year in the US, particularly in infants and premature babies. The long-term goal of this research is to prevent aminoglycoside-induced ototoxicity by modulating the functions of gentamicin-binding proteins crucial for ototoxicity, to preserve auditory function. In this proposal, we seek to identify gentamicin-binding proteins, and determine their distribution in the inner ear. We hypothesize that there are gentamicin-binding proteins in the inner ear, and that these proteins retain gentamicin in specific types of cells, contributing to cytotoxicity in vitro and pathogenesis in vivo. The specific aims of this project are: First, to identify gentamicin-binding proteins specific in kidney proximal tubule and cochlear cells. We will use a gentamicin-agarose pull-down assay and SDS-polyacrylamide gel electrophoresis to isolate such proteins from kidney proximal tubule and cochlear cell lines, and determine their identity using mass spectrometry (Aim 1). Second, to determine the distribution of gentamicin-binding proteins, including HSP73 and calreticulin, in the inner ear by immunocytochemistry and confocal microscopy. We will also determine if these proteins are co-localized with systemically-administered gentamicin, using fluorescently-tagged gentamicin or untagged gentamicin localized by gentamicin immunocytochemistry (Aim 2). If we can identify gentamicin-binding proteins that sequester the drug within cells that then induce cytotoxicity, we can develop new strategies to inhibit their interactions, and attenuate drug retention. Alternatively, if gentamicin-binding inhibits the functions of these proteins, we could use pharmacological activators of these proteins to overcome gentamicin-induced inhibition. Future studies will determine if gentamicin inhibits their interactions with physiological binding molecules/proteins, and if expression levels of gentamicin-binding proteins change during sustained treatment with gentamicin. Identification of gentamicin-binding proteins and their intracellular mechanisms that gentamicin interferes with to initiate cell death will provide new insight for clinicians to screen patients for pre-existing conditions and medications that elevate the risk of aminoglycoside toxicity and deafness. Identification of gentamicin-binding proteins that play a major role in gentamicin- induced ototoxicity is crucial in understanding how gentamicin induces ototoxicity and deafness. The proposed research will enable us to develop strategies to prevent interactions between gentamicin and gentamicin-binding proteins, and to overcome gentamicin-induced inhibition by pharmacologically activating the proteins. These strategies will lead to ototoxicity and deafness prevention.