smFRET Investigation of Aminoglycoside-induced Ototoxicity

描述（由申请人提供）：氨基糖苷类抗生素是治疗严重感染的最常用处方之一，由于其对多重耐药细菌的有效性，氨基糖苷类抗生素继续受到欢迎。氨基糖苷优先结合到细菌核糖体的几乎普遍保守的解码位点，在那里mRNA密码子和tRNA反密码子发生适当的匹配。细菌和人类核糖体之间解码位点结构的关键区别决定了氨基糖苷的特异性。人类线粒体核糖体的点突变减少了特异性决定因子的数量。相应地，这种突变与氨基糖苷引起的人类副作用有关，通常导致永久性听力丧失。虽然在了解氨基糖苷如何识别细菌核糖体解码位点方面取得了重大进展：1)翻译的分子机制，2)氨基糖苷靶向的过程的具体步骤，以及3)核糖体该区域的改变如何赋予特异性同时保留核糖体功能仍然不清楚。本文采用单分子荧光共振能量转移（smFRET）成像方法，提出了翻译机制的定量生物物理研究，这将使我们对氨基糖苷敏感和耐药核糖体的翻译分子机制有前所未有的了解。这些努力最终将导致开发一个平台，可以潜在地用于开发新的策略，以减轻这些重要抗生素的不良副作用。为了实现这一目标，最近用来描述细菌翻译延伸周期完整动力学机制的方法将应用于含有人类野生型和耳聋突变体解码位点的细菌核糖体。这些努力将导致对这些结构差异如何改变翻译机制的深刻理解，并使氨基糖苷超敏反应的分子起源得以描绘。由于拟议的研究需要的材料比传统的生物物理方法少几个数量级，在此基础上，还将对分离的人类线粒体核糖体进行类似的研究。研究目标的成功完成将对突变在氨基糖苷诱导的耳毒性中的作用和线粒体翻译的机制产生重要的见解，并为开发更安全的氨基糖苷类抗生素提供一个急需的平台。

DESCRIPTION (provided by applicant): Aminoglycoside ntibiotics are one of the most commonly prescribed treatments for serious infections, and continue to remain popular due to their effectiveness against multi-drug resistant bacteria. Aminoglycosides preferentially bind to the near-universally conserved decoding site of the bacterial ribosome, where proper matching of the mRNA codon and tRNA anticodon occurs. Key distinctions in the decoding site architecture between bacterial and human ribosomes determine aminoglycoside specificity. Point mutations in the human mitochondrial ribosome reduce the number of specificity determinants. Correspondingly, such mutations are associated with aminoglycoside-induced side effects in humans, commonly resulting in permanent hearing loss. While significant progress has been made towards understanding how aminoglycosides recognize the bacterial ribosome decoding site: 1) the molecular mechanism of translation, 2) the specific steps of the process that are targeted by aminoglycosides and 3) how alterations in this region of the ribosome confer specificity while retaining ribosomal functions remain obscure. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET) imaging methods, quantitative biophysical investigations of the translation mechanism are proposed that will enable unprecedented insights into the molecular mechanism of translation in both aminoglycoside -sensitive and -resistant ribosomes. These efforts will ultimately lead to development of a platform that can potentially be used to develop new strategies to mitigate unwanted side effects of these important antibiotics. Towards this goal, methods that have been recently employed to describe a complete kinetic mechanism of the bacterial translation elongation cycle will be applied to bacterial ribosomes engineered to contain human wild type and deafness mutant decoding sites. Such efforts will lead to a significantly deeper understanding of how these structural distinctions alter the translation mechanism and enable the molecular origins of aminoglycoside hypersensitivity to be delineated. As the proposed investigations require orders of magnitude less material than traditional biophysical methods, analogous investigations will also be performed on isolated human mitochondrial ribosomes that build upon this foundation. The successful completion of the research AIMs will yield significant insights into the role of mutations in aminoglycoside-induced ototoxicity and the mechanism of mitochondrial translation, as well as a much-needed platform that can potentially be used to develop safer aminoglycoside antibiotics. PUBLIC HEALTH RELEVANCE: The proposed research aims to significantly advance our understanding of the origins of antibiotic specificity for the bacterial ribosome and provide insights into the molecular mechanism of aminoglycoside hypersensitivity associated with mutations in the human mitochondrial ribosome, which often result in permanent hearing loss. Through the implementation of state-of-the-art single-molecule imaging techniques, the present study will provide novel platforms by which to directly investigate the performance of mitochondrial translation that can ultimately be used to design tailored aminoglycoside antibiotics with fewer side effects.