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.

电活动性是哺乳动物耳蜗外毛细胞（OHC）的一个独特特性。这种电活动性被认为是哺乳动物听觉的精密频率选择性和灵敏度的基础。最近，我们克隆了蛋白质普雷斯廷的基因，并已确定普雷斯廷是负责OHC电活动的关键分子。然而，其他蛋白质已经被证明可以改变普雷斯廷的活性，从而影响听力功能。目前，这些prestin相关蛋白（PAP）的确切身份是未知的，普雷斯廷和PAP之间的相互作用还没有在分子水平上描述。因此，我们提出的研究的总体目标是确定PAP和表征其功能。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（目的I）来识别PAP。建立适合该方法的OHC-cDNA文库，以提高灵敏度，减少文库筛选的假阳性克隆。然后，以普雷斯廷为“诱饵”，利用酵母双杂交方法鉴定PAP。由于其高度敏感的性质，酵母双杂交方法能够鉴定其他方法无法识别的PAP。其次，我们建议使用第二种方法识别PAP：结合质谱分析与普雷斯廷免疫共沉淀（Aim II）。该方法的主要优点是其能够识别存在于体内的生理相关Prestin-PAP相互作用。最后，后PAP已确定使用两种互补的方法或从其他来源，我们将确定这些PAP是否驻留在OHC在体内，并将表征PAP修改普雷斯廷的功能的机制（目的III）。综上所述，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，并可能导致PAP相关耳聋基因的鉴定。这种知识可能使操纵OHC功能用于治疗目的成为可能。