Defining molecular mechanisms in the SLC26 family of proteins

 描述(申请人提供)：在SLC26家族的成员Pendrin和Prestin中发现了许多与综合征性和非综合征性听力障碍有关的突变。在大多数情况下，尚不清楚这些突变是如何扰乱垂蛋白和Prestin蛋白的正常功能的。因此，迫切需要对人类患者中与疾病相关的突变进行表征，以了解潜在的病理学。在这些实验中收集的数据将进一步加深我们对正常侧翼蛋白和Prestin对其转运和运动功能的分子机制的理解。利用这些信息，我们将开发蛋白质的机制模型，预计这些模型将推广到所有其他SLC26家族成员，并可能被利用来开发针对未来在SLC26蛋白质家族中发现的致病突变的药物。AIMS 1和AIMS 2的设计目的是利用在细胞系中异源表达的重组蛋白来确定已报道的与疾病相关的Pendrin(Aim 1)和Prestin(Aim 2)错义突变对其阴离子转运和电压依赖运动功能的影响。目标3将根据前述目标中获得的结果，开发解释运输(吊环)和运动(前置)功能的机械模型。此外，我们还将进行电生理测量，以剖析Prestin运动活动的动力学。标准的分子生物学方法将被用来产生突变的蛋白质结构，其功能评估将使用细胞系(体外)进行。这项研究不会使用动物。综上所述，拟议的研究不仅将填补我们目前对疾病相关突变及其功能后果的了解之间的空白，而且还将有助于阐明SLC26蛋白家族的详细分子机制。

 DESCRIPTION (provided by applicant): Numerous mutations that are associated with both syndromic and non-syndromic hearing impairment have been found in pendrin and prestin, which are members of the SLC26 family. In most cases, it is not clear how these mutations disrupt the normal function of pendrin and prestin proteins. Characterization of such disease- associated mutations in human patients is, therefore, urgently needed to understand the underlying pathology. Data collected in these experiments will further our understanding of the molecular mechanisms of normal pendrin and prestin on their transport and motor functions. Using this information, we will develop mechanistic models of the proteins, which are expected to generalize to all other SLC26-family members, and could be exploited to develop remedies against disease-causing mutations found in the SLC26 family of proteins in the future. Aims 1 and 2 are designed to determine the effects of disease-associated missense mutations reported for pendrin (Aim 1) and prestin (Aim 2) on their anion transport and voltage-dependent motor functions using recombinant proteins heterologously expressed in cell lines. Aim 3 will develop mechanistic models that explain transport (pendrin) and motor (prestin) functions based on the results obtained in the preceding Aims. In addition, we will carry out electrophysiological measurements to dissect the kinetics of prestin's motor activity. Standard molecular biological methods will be used to generate mutated protein constructs whose functional assessment will be performed using cell lines (in vitro). Animals will not be used in this study. Taken together, the proposed research will not only fill the gap between our current knowledge of disease-associated mutations and their functional consequences, but also help elucidate detailed molecular mechanisms of SLC26 family of proteins.