Investigating the nature of prestin-associated proteins

电动是哺乳动物耳蜗外毛细胞的一种独特特征。这种电动能力被认为是哺乳动物听力精致的频率选择性和敏感度的基础。最近，我们克隆了蛋白质prestin的基因，并确定prestin是OHC电动的关键分子。然而，其他蛋白质已经被证明可以改变prestin的活性，从而影响听力功能。目前，这些Prestin相关蛋白(PAPs)的确切身份尚不清楚，也没有在分子水平上描述Prestin与PAPs之间的相互作用。因此，我们提出的研究的总体目标是识别PAPs并表征其功能。首先，我们建议使用一种新开发的基于膜的酵母双杂交选择方法(Aim I)来鉴定PAPs。为提高文库筛选的敏感性，减少假阳性克隆，将建立适合于该方法的OHC-cDNA文库。然后，以prestin为“诱饵”，采用酵母双杂交的方法对PAPs进行鉴定。酵母双杂交法由于其高度灵敏的性质，能够鉴定出其他方法不能识别的PAPs。其次，我们建议使用第二种方法来鉴定PAPs：结合质谱分析和Prestin免疫共沉淀(AIM II)。这种方法的主要优点是它能够识别存在于体内的生理上相关的Prestin-PAP相互作用。最后，在使用两种互补的方法或从其他来源鉴定PAPs后，我们将确定这些PAPs是否存在于体内的OHC中，并将表征PAPs修改prestin功能的机制(目标III)。综上所述，这些实验将进一步加深我们对基于OHC的耳蜗扩增的分子基础的理解，并可能导致识别与PAP相关的耳聋基因。这一知识可能使操控OHC功能用于治疗目的成为可能。

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.