Investigating the nature of prestin-associated proteins

电运动性是哺乳动物耳蜗外毛细胞（OHC）的独特特征。这种电运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prestin蛋白的基因，并确定prestin是OHC电动力的关键分子。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描述。因此，我们提出的研究的总体目标是确定pap并表征其功能。首先，我们建议使用一种新开发的膜酵母双杂交选择方法（Aim I）来鉴定pap。建立适合该方法的OHC-cDNA文库，提高文库筛选的灵敏度，减少假阳性克隆。然后，以prestin为“诱饵”，我们将使用酵母双杂交方法鉴定pap。由于酵母双杂交方法具有高度的敏感性，因此能够识别出其他方法无法识别的pap。其次，我们建议使用第二种方法来鉴定pap：结合质谱分析和prestin共免疫沉淀（Aim II）。该方法的主要优点是能够识别存在于体内的生理相关pretin - pap相互作用。最后，在使用两种互补方法或从其他来源确定PAPs之后，我们将确定这些PAPs是否存在于体内的OHCs中，并将描述PAPs改变prestin功能的机制（Aim III）。综上所述，这些实验将进一步加深我们对ohc为基础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。这一知识可能使为治疗目的操纵OHC功能成为可能。

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.