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和水的稳态。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定位于内核体晚期，在那里调节腔内pH值，以控制囊泡运输和将多泡体（MVB）运送到液泡进行降解。在哺乳动物中，MVB通路在HIV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贮积障碍和伴随的神经和肾脏功能障碍。内体NHE抑制剂提供了一种治疗潜力，可以弥补在邓氏病和范可尼病中看到的内体酸化缺陷，并作为抗病毒药物。本提案的目标是扩展我们对酵母Nhx1功能的理解，并将我们的发现外推到哺乳动物细胞中。在Aim 1中，我们将结合酵母遗传学，生物化学分析和电子显微镜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验证NHE6和/或NHE9在MVB体中定位和功能的假设。在Aim 2中，我们将评估exoporide（一种新型阿米洛利类似物）的合成变体，以寻找细胞内NHE的选择性抑制剂。本提案的直接目标是完成正在进行的研究，以寻求获得Nhx1阳离子/质子交换作用的全局视图（目的3）。为此，我们将继续分析pH调节（pHome）的遗传基础，并确定与Nhx1 （phenome）相互作用的基因和细胞途径。在Aim 4中，我们将基于大肠杆菌NhaA的晶体结构，利用结构生物信息学驱动的诱变与酵母表型筛选相结合，评估一种新兴的NHE同源模型。这些研究将侧重于确定NHE细胞内亚型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，并为设计新型NHE抑制剂提供模板。总之，我们提出了一种多学科的方法，以临床和生理上重要的膜转运蛋白家族的功能和机制为目标。这一建议的目标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、水和酸的等价物在所有细胞的边界和隔室中的运动。我们计划在酵母和培养的哺乳动物细胞中使用平行方法确定这些蛋白质的功能，并使用新的筛选策略确定新药。这些药物可能对肾储存疾病（邓氏病和范可尼病）和抗包膜病毒（如HIV）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.