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

酵母和人类内体 Na /H 交换器：作用和调节

基本信息

批准号：
8091274
负责人：
RAJINI RAO
金额：
$ 30.74万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8091274
关键词：
Acids Amiloride Animal Model Antiviral Agents Binding Sites Biochemical Biogenesis Bioinformatics Biological Assay Cations Cell Culture Techniques Cell membrane Cell model Cells Critical Pathways Defect Disease Drug Metabolic Detoxication Drug usage Electron Microscopy Endosomes Erythrocytes Escherichia coli Eukaryota Family Functional disorder Funding Genes Goals Growth HIV Health Homeostasis Homology Modeling Human Intracellular Membranes Ions K562 Cells Kidney Lead Link Lysosomes Mammalian Cell Mammals Measurement Mediating Membrane Transport Proteins Modeling Molecular Molecular Models Monitor Movement Multivesicular Body Mutagenesis Mutation NHE1 Neurologic Neurologic Dysfunctions Orthologous Gene Pathway interactions Pharmaceutical Preparations Phenotype Phylogenetic Analysis Plants Positioning Attribute Proteins Protons Recycling Regulation Role Saccharomyces cerevisiae Screening procedure Sodium Chloride Sorting - Cell Movement Structure Subgroup System Testing Therapeutic Vacuole Variant Vesicle Water Yeasts analog antiport antiporter base chemical genetics deletion library design functional genomics genetic analysis genome wide association study inhibitor/antagonist insight interdisciplinary approach late endosome member molecular modeling mutant novel pH Homeostasis phenome protein degradation protein function trafficking trans-Golgi Network virus envelope yeast genetics

项目摘要

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.

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。我们发现了内体 NHE 的一个进化古老的亚群，其中包括酵母 Nhx1 和哺乳动物 NHE6、7 和 9。在酵母中，Nhx1 定位于晚期内体，在其中调节腔内 pH 值以控制囊泡运输和多囊泡体 (MVB) 的递送。降解的液泡。在哺乳动物中，MVB 途径在 HIV 生物合成、药物解毒、红细胞成熟和蛋白质降解中发挥重要作用。该途径的缺陷可能导致溶酶体储存障碍以及伴随的神经和肾功能障碍。内体 NHE 抑制剂具有治疗潜力，可以抵消 Dent 病和 Fanconi 病中出现的内体酸化缺陷，并可作为抗病毒药物。该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。在目标 1 中，我们将结合使用酵母遗传学、运输生化分析和电子显微镜来定义溶酶体生物合成中精确的 pH 依赖性步骤。与此同时，我们将在哺乳动物细胞培养模型中测试 NHE6 和/或 NHE9 在 MVB 体中定位和发挥作用的假设。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。该提案的近期目标是完成正在进行的研究，寻求对 Nhx1 阳离子/质子交换作用的全球看法（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 Nhx1（表型组）相互作用的基因和细胞途径。在目标 4 中，我们将基于大肠杆菌 NhaA 的晶体结构，使用结构生物信息学驱动的诱变结合酵母表型筛选来评估 NHE 的新兴同源模型。这些研究将重点定义 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，并作为设计新型 NHE 的模板抑制剂。总之，我们提出了一种多学科方法，针对临床和生理上重要的膜转运蛋白家族的功能和机制。公共健康相关性该提案针对的是新发现但进化上古老的离子转运蛋白家族，该家族调节盐、水和酸等价物跨所有细胞边界和区室的运动。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物。这些药物可能对肾贮积病（Dent's 和 Fanconi）以及 HIV 等包膜病毒具有治疗作用。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast.

Rab-GTP 酶激活蛋白抑制钠/质子交换可调节酵母中的内体运输。

DOI：
发表时间：
2004-02-06
期刊：
影响因子：
0
作者：
Ali, Rashid;Brett, Christopher L;Mukherjee, Sanchita;Rao, Rajini
通讯作者：
Rao, Rajini

Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting.

植物特异性阳离子/H 交换剂 17 及其同系物是内膜 K 转运蛋白，在蛋白质分选中发挥作用。

DOI：
发表时间：
2011-09-30
期刊：
影响因子：
0
作者：
Chanroj, Salil;Lu, Yongxian;Padmanaban, Senthilkumar;Nanatani, Kei;Uozumi, Nobuyuki;Rao, Rajini;Sze, Heven
通讯作者：
Sze, Heven

Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation.

内体 Na (K)/H 交换器 Nhx1/Vps44 在多囊泡体形成的下游独立发挥作用。

DOI：
发表时间：
2011-12-23
期刊：
影响因子：
0
作者：
Kallay, Laura M;Brett, Christopher L;Tukaye, Deepali N;Wemmer, Megan A;Chyou, Anthony;Odorizzi, Greg;Rao, Rajini
通讯作者：
Rao, Rajini

An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease.

内部调查：内体 Na( )/H( ) 交换器如何与自闭症和神经系统疾病联系起来。