Investigating the PROTO Targets in Regulating Drug-Induced Hair Cell Death

描述（由申请人提供）：氨基糖苷类抗生素（AGA）广泛用于治疗各种难治性细菌感染。AGA最重要的副作用和治疗局限性是内耳损伤（耳毒性），其可产生不可逆的听力损失和肾损伤（肾毒性），其可导致肾衰竭。预防AGA诱导的耳毒性和肾毒性一直是生物医学研究的主要治疗目标。Julian Simon博士实验室的一个主要目标是确定耳保护性PROTO 1类似物（PROTO）的生物学靶点，以确定它们在预防AGA诱导的内耳毛细胞死亡中的作用机制。为了实现这一目标，该实验室最近通过光亲和下拉结合质谱法在斑马鱼中确定了几个候选PROTO目标。在该奖学金提案中，设计了三个具体目标来检验以下假设，即这些质谱鉴定的PROTO候选靶标之一在控制毛细胞响应AGA的死亡和存活中起关键作用：1）使用蛋白质印迹和竞争下拉法验证PROTO与最有希望的质谱鉴定的候选靶标之间的相互作用; 2）使用表面等离子体共振（SPR）生物传感器动力学分析PROTO与其靶标之间的相互作用; 3）使用哺乳动物细胞系和条件敲除小鼠研究PROTO靶标对AGA诱导的毛细胞毒性的影响。生物PROTO靶标的鉴定有望促进对调节毛细胞死亡和存活的细胞途径的理解，并促进预防听力和平衡障碍的新治疗策略的开发。该奖学金提案将提供听力保护研究和临床前药物开发方面的宝贵实践经验和全面知识，为作为听觉和生物医学研究领域的调查员/科学家的独立研究生涯奠定坚实的基础。

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics (AGAs) are widely used in the treatment of a variety of refractory bacterial infections. The most important side effects and therapeutic limitations of AGAs are inner ear damage (ototoxicity) which can produce irreversible hearing loss and kidney damage (nephrotoxicity) which can lead to kidney failure. Prevention of AGA-induced ototoxicity and nephrotoxicity has been a major therapeutic objective in biomedical research. One major goal at the laboratory of Dr. Julian Simon is to identify the biological target of the otoprotective PROTO1 analogs (PROTO) to determine their mechanism of action in preventing AGA-induced inner ear hair cell death. To achieve this goal, the laboratory recently identified several candidate PROTO targets in zebrafish through photoaffinity pulldowns coupled with mass spectrometry. In this fellowship proposal, the three specific aims are designed to test the hypothesis that one of these mass spectrometry- identified PROTO candidate targets plays a critical role in controlling hair cell death and surviva in response to AGAs: 1) Validation of the interactions between PROTO and the most promising mass spectrometry-identified candidate targets using western blotting and competition pulldowns; 2) kinetic analysis of interactions between PROTO and its targets using a surface plasmon resonance (SPR) biosensor; 3) investigation of the effects of the PROTO targets on AGA-induced hair cell toxicity using mammalian cell lines and conditional knockout mice. Identification of the biological PROTO target is expected to advance the understanding of the cellular pathways that regulate hair cell death and survival and promote the development of new therapeutic strategies to prevent hearing and balance disorders. The fellowship proposal will provide an invaluable hands-on experience and comprehensive knowledge in hearing protection research and preclinical drug development paving a solid foundation towards an independent research career as an investigator/scientist in the field of auditory and biomedical research.