smFRET Investigation of Aminoglycoside-induced Ototoxicity

描述（由申请人提供）：氨基糖苷类抗生素是严重感染最常用的处方治疗方法之一，由于其对多重耐药细菌的有效性，因此仍然很受欢迎。氨基糖苷类优先结合到细菌核糖体的几乎普遍保守的解码位点，在那里发生mRNA密码子和tRNA反密码子的正确匹配。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类的特异性。人类线粒体核糖体中的点突变减少了特异性决定簇的数量。相应地，这些突变与人类中氨基糖苷类诱导的副作用有关，通常导致永久性听力损失。虽然在了解氨基糖苷类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）氨基糖苷类靶向的过程的具体步骤和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。在这里，使用单分子荧光共振能量转移（smFRET）成像方法，定量生物物理研究的翻译机制，提出了将使前所未有的见解的分子机制的翻译在两个氨基糖苷类敏感和耐药核糖体。这些努力最终将导致开发一个平台，该平台可能用于开发新的策略，以减轻这些重要抗生素的不良副作用。为了实现这一目标，最近已经采用的方法来描述一个完整的动力学机制的细菌翻译延长周期将被应用到细菌核糖体工程改造，含有人类野生型和耳聋突变体的解码位点。这些努力将导致这些结构差异如何改变翻译机制，并使氨基糖苷类超敏反应的分子起源被描绘一个显着更深入的了解。由于拟议的调查需要的数量级比传统的生物物理方法少的材料，类似的调查也将在此基础上建立的分离的人类线粒体核糖体上进行。研究AIM的成功完成将对氨基糖苷类抗生素诱导的耳毒性和线粒体翻译机制中突变的作用产生重要的见解，以及一个急需的平台，可用于开发更安全的氨基糖苷类抗生素。

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.