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

描述(申请人提供)：NHE超家族的Na+/H+交换器调节阳离子与质子的跨膜交换，以调节盐、pH和水的动态平衡。我们已经发现了一个进化上古老的内体NHE亚群，包括酵母Nhx1和哺乳动物NHE6、7和9。在酵母中，Nhx1定位于晚期内体，在那里它调节管腔pH来控制囊泡的运输和将多囊泡体(MVB)运送到液泡进行降解。在哺乳动物中，MVB途径在HIV的生物发生、药物解毒、红细胞成熟和蛋白质降解中起重要作用。这一途径的缺陷可能导致溶酶体储存障碍，并伴随神经和肾脏功能障碍。内体NHE的抑制剂提供了一种治疗潜力，可以弥补登特氏病和Fanconi病中出现的内体酸化缺陷，并可用作抗病毒药物。这项建议的目的是扩大我们对酵母Nhx1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、运输生化分析和电子显微镜相结合的方法来定义溶酶体生物发生中精确的pH依赖步骤。同时，我们将在哺乳动物细胞培养模型中测试NHE6和/或NHE9在MVB小体中定位和功能的假设。在目标2中，我们将评估一种新的阿米洛利类似物--外洛普利的合成变种，以寻找一种选择性的细胞内NHE抑制剂。这项提议的一个直接目标是完成正在进行的研究，寻求对Nhx1阳离子/质子交换的作用有一个全球看法(目标3)。为此，我们将继续分析pH调节的遗传基础(PHOME)，并确定与Nhx1(PHOME)相互作用的基因和细胞通路。在目标4中，我们将评估一个新的NHE同源模型，基于E.ColiNhaA的晶体结构，使用结构-生物信息学驱动的突变结合酵母中的表型筛选。这些研究将集中于确定NHE细胞内和质膜亚型之间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。综上所述，我们提出了一个多学科的方法，目标是一个临床和生理上重要的膜转运蛋白家族的功能和机制。公共卫生相关性这项建议针对的是一个新发现的但进化上古老的离子转运蛋白家族，它调节盐、水和酸当量跨越所有细胞的边界和隔间的移动。我们计划在酵母和培养的哺乳动物细胞中使用并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。这些药物可能对肾脏储存性疾病(登特氏病和范可尼病)以及对HIV等包膜病毒有治疗作用。

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.