Investigating the nature of prestin-associated proteins

研究 prestin 相关蛋白的性质

• 批准号: 6923414
• 负责人: Jing Zheng
• 金额: $ 29.64万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923414
• 关键词: SDS polyacrylamide gel electrophoresis; cochlea; complementary DNA; deafness; ear hair cell; genetic library; hearing; laboratory mouse; mass spectrometry; polymerase chain reaction; protein protein interaction; protein structure function; sound perception; western blottings; yeast two hybrid system

Electromotility is a unique characteristic of the outer hair cells (OHC) of the mammalian cochlea. This electromotility is thought to underlie the exquisite frequency selectivity and sensitivity of mammalian hearing. Recently, we cloned the gene for the protein prestin, and have determined that prestin is the key molecule responsible for OHC electromotility. However, other proteins have been shown to modify prestin's activity, thereby affecting hearing function. Currently, the precise identities of these prestin-associated proteins (PAPs) are unknown, and the interactions between prestin and PAPs have not been described at the molecular level. Thus, the overall aim of our proposed research is to identify PAPs and to characterize their function. First, we propose to identify PAPs using a newly developed membrane-based yeast two-hybrid selective method (Aim I). An OHC-cDNA library suitable for that method will be built to increase the sensitivity and to decrease the false positive clones of library screening. Then, using prestin as "bait," we will use the yeast two-hybrid method to identify PAPs. Because of its highly sensitive nature, the yeast two-hybrid method is able to identify PAPs that can not be recognized by other methods. Second, we propose to identify PAPs using a second method: combined mass spectrometry analysis with prestin coimmunoprecipitation (Aim II). The major advantage of this method is its ability to identify physiologically relevant Prestin-PAP interactions that exist in vivo. Finally, after PAPs have been identified using either the two complementary methods or from other sources, we will determine whether those PAPs reside in OHCs in vivo, and will characterize the mechanisms by which PAPs modify prestin's function (Aim III). Taken together, these experiments will further our understanding of the molecular basis of OHC-based cochlear amplification, and potentially lead to identification of PAP-related deafness genes. This knowledge may make it possible to manipulate OHC function for therapeutic purposes.

电动性是哺乳动物耳蜗外毛细胞 (OHC) 的独特特征。这种电动性被认为是哺乳动物听觉精致的频率选择性和敏感性的基础。最近，我们克隆了prestin蛋白的基因，并确定prestin是负责OHC电动性的关键分子。然而，其他蛋白质已被证明可以改变 prestin 的活性，从而影响听力功能。目前，这些 prestin 相关蛋白 (PAP) 的确切身份尚不清楚，并且 prestin 和 PAP 之间的相互作用尚未在分子水平上得到描述。因此，我们提出的研究的总体目标是识别 PAP 并表征其功能。首先，我们建议使用新开发的基于膜的酵母双杂交选择性方法（目标 I）来鉴定 PAP。将建立适合该方法的OHC-cDNA文库，以提高灵敏度并减少文库筛选的假阳性克隆。然后，以prestin为“诱饵”，我们将使用酵母双杂交方法来识别PAP。由于其高度灵敏的特性，酵母二杂交方法能够识别其他方法无法识别的 PAP。其次，我们建议使用第二种方法来识别 PAP：结合质谱分析与 prestin 免疫共沉淀 (Aim II)。该方法的主要优点是能够识别体内存在的生理相关的 Prestin-PAP 相互作用。最后，在使用两种互补方法或从其他来源鉴定出 PAP 后，我们将确定这些 PAP 是否存在于体内 OHC 中，并将表征 PAP 改变 prestin 功能的机制（目标 III）。总而言之，这些实验将进一步加深我们对基于 OHC 的耳蜗放大的分子基础的理解，并有可能导致 PAP 相关耳聋基因的鉴定。这些知识可能使操纵 OHC 功能达到治疗目的成为可能。