Endosomal Na+/H+ Exchangers From Yeast and Human: Role and Regulation

描述（由申请人提供）：NHE超家族的Na+/H+交换剂介导阳离子与质子的跨膜交换，以调节盐、pH和水稳态。我们已经发现了一个进化上古老的亚组内体NHE，包括酵母Nhx 1和哺乳动物NHE 6，7和9。在酵母中，Nhx 1定位于晚期内体，在那里它调节管腔pH以控制囊泡运输和将多泡体（MVB）递送到液泡进行降解。在哺乳动物中，MVB途径在HIV生物发生、药物解毒、红细胞成熟和蛋白质降解中很重要。该途径的缺陷可能导致溶酶体贮积症以及伴随的神经和肾功能障碍。内体NHE抑制剂提供了治疗潜力，以抵消在登特病和范可尼病中观察到的内体酸化缺陷，并作为抗病毒剂。这项提案的目标是扩展我们对酵母Nhx 1功能的理解，并将我们的发现外推到哺乳动物细胞。在目标1中，我们将使用酵母遗传学，生化分析的贩运，和电子显微镜的组合，以确定精确的pH值依赖的步骤，在溶酶体的生物合成。同时，我们将在哺乳动物细胞培养模型中检验NHE 6和/或NHE 9在MVB体中定位和功能的假设。在目标2中，我们将评估exoporide（一种新型阿米洛利类似物）的合成变体，以找到细胞内NHE的选择性抑制剂。该提案的一个直接目标是完成正在进行的研究，这些研究旨在得出Nhx 1阳离子/质子交换作用的全球观点（目标3）。为此，我们将继续分析pH调节（pHome）的遗传基础，并确定与Nhx 1（表型组）相互作用的基因和细胞通路。在目标4中，我们将评估一个新兴的同源性模型的NHE，基于晶体结构的E。coliNhaA中，使用结构-生物信息学驱动的诱变结合酵母中的表型筛选。这些研究将侧重于定义NHE的细胞内和质膜亚型之间的离子选择性和抑制剂敏感性差异的分子基础，深入了解NHE超家族的转运机制，并作为设计新型NHE抑制剂的模板。总之，我们提出了一个多学科的方法，目标的功能和机制的临床和生理上重要的膜转运蛋白家族。公共卫生相关性该提案针对一个新发现的但在进化上古老的离子转运蛋白家族，该家族调节盐、水和酸等价物穿过所有细胞的边界和隔室的运动。我们计划在酵母和培养的哺乳动物细胞中使用平行方法来定义这些蛋白质的功能，并使用新的筛选策略来识别新药。这些药物可能对肾储存疾病（Dent's和Fanconi）和包膜病毒（如HIV）提供治疗益处。

DESCRIPTION (provided by applicant): Na+/H+ exchangers of the NHE superfamily mediate the transmembrane exchange of cations with protons to regulate salt, pH and water homeostasis. We have uncovered an evolutionarily ancient subgroup of endosomal NHE that includes yeast Nhx1 and mammalian NHE6, 7 and 9. In yeast, Nhx1 localizes to the late endosome where it regulates luminal pH to control vesicle trafficking and delivery of the multivesicular body (MVB) to the vacuole for degradation. In mammals, the MVB pathway is important in HIV biogenesis, drug detoxification, erythrocyte maturation, and protein degradation. Defects in this pathway are likely to lead to lysosomal storage disorders and concomitant neurological and kidney dysfunction. Inhibitors of endosomal NHE offer a therapeutic potential to offset defects in endosome acidification seen in Dent's and Fanconi disease, and as antiviral agents. The goal of this proposal is to extend our understanding of yeast Nhx1 function and extrapolate our findings to mammalian cells. In Aim 1, we will use a combination of yeast genetics, biochemical assays of trafficking, and electron microscopy to define the precise pH-dependent step in lysosomal biogenesis. In parallel, we will test the hypothesis that NHE6 and/or NHE9 localize and function in MVB bodies in a mammalian cell culture model. In Aim 2, we will evaluate synthetic variants of exoporide, a novel amiloride analog, to find a selective inhibitor of intracellular NHE. An immediate goal of this proposal is to complete ongoing studies that seek to derive a global view of the role of cation/proton exchange by Nhx1 (Aim 3). To this end, we will continue our analysis of the genetic basis for pH regulation (pHome) and identify genes and cellular pathways that interact with Nhx1 (phenome). In Aim 4, we will assess an emerging homology model of NHE, based on the crystal structure of E. coli NhaA, using structure- bioinformatics driven mutagenesis in conjunction with phenotype screening in yeast. These studies will focus on defining the molecular basis for differences in ion selectivity and inhibitor sensitivity between the intracellular and plasma membrane subtypes of NHE, provide insight into the mechanism of transport by the NHE superfamily, and serve as a template for the design of novel NHE inhibitors. In summary, we propose a multidisciplinary approach that targets the function and mechanism of a clinically and physiologically important family of membrane transport proteins. PUBLIC HEALTH RELEVANCE This proposal targets a newly discovered but evolutionarily ancient family of ion transporters that regulate the movement of salt, water and acid equivalents across the boundaries and compartments of all cells. We plan to define the function of these proteins using parallel approaches in yeast and cultured mammalian cells, and identify new drugs using a novel screening strategy. These drugs may offer therapeutic benefits in kidney storage diseases (Dent's and Fanconi), and against envelope viruses such as HIV.