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+交换器介导阳离子与质子的跨膜交换，以调节盐，pH和水稳态。我们发现了一个进化古老的内体内体nhe子组，其中包括酵母NHX1和哺乳动物NHE6、7和9。在酵母中，NHX1定位于晚期的内体，在其中调节腔内pH，以控制囊泡的运输和多囊体（MVB）的囊泡运输和交付，以进行多囊体（MVB）进行污水处理。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。该途径中的缺陷可能导致溶酶体储存障碍以及伴随的神经系统和肾功能障碍。内体NHE的抑制剂为抵消Dent和Fanconi病中内体酸化缺陷的缺陷提供了治疗潜力，并作为抗病毒剂。该建议的目的是扩展我们对酵母NHX1功能的理解，并将我们的发现推断到哺乳动物细胞。在AIM 1中，我们将使用酵母遗传学，运输的生化测定和电子显微镜的组合来定义溶酶体生物发生的精确pH依赖性步骤。同时，我们将检验以下假设：NHE6和/或NHE9在哺乳动物细胞培养模型中在MVB体中定位和功能。在AIM 2中，我们将评估一种新型的Amiloride类似物外孔肽的合成变体，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，该研究试图通过NHX1来获得阳离子/质子交换的作用的全球视野（AIM 3）。为此，我们将继续对pH调节（PHOME）的遗传基础的分析，并确定与NHX1相互作用（PENMOME）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的晶体结构来评估NHE的新兴同源模型，并使用结构生物信息学驱动的诱变与酵母中的表型筛选。这些研究将集中于定义NHE的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，提供了对NHE超家族运输机制的见解，并充当新型NHE NHE抑制剂设计的模板。总而言之，我们提出了一种多学科方法，该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。公共卫生的相关性该提案针对一个新发现但进化上古老的离子转运蛋白家族，该家族调节所有细胞的边界和隔室中盐，水和酸等效物的运动。我们计划使用酵母和培养的哺乳动物细胞中的平行方法来定义这些蛋白质的功能，并使用新型筛查策略鉴定新药。这些药物可能会在肾脏储存疾病（Dent's和Fanconi）中提供治疗益处，并针对HIV等信封病毒。

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）

Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension.

DOI：
10.1016/j.jmb.2009.12.055
发表时间：
2010-03-12
期刊：
Journal of molecular biology
影响因子：
5.6
作者：
Schushan M;Xiang M;Bogomiakov P;Padan E;Rao R;Ben-Tal N
通讯作者：
Ben-Tal N

A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma.

DOI：
10.1038/ncomms7289
发表时间：
2015-02-09
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Kondapalli, Kalyan C.;Llongueras, Jose P.;Capilla-Gonzalez, Vivian;Prasad, Hari;Hack, Anniesha;Smith, Christopher;Guerrero-Cazares, Hugo;Quinones-Hinojosa, Alfredo;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.

DOI：
10.1074/jbc.m111.252650
发表时间：
2011-09-30
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Chanroj S;Lu Y;Padmanaban S;Nanatani K;Uozumi N;Rao R;Sze H
通讯作者：
Sze H

Functional evaluation of autism-associated mutations in NHE9.

DOI：
10.1038/ncomms3510
发表时间：
2013
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Kondapalli, Kalyan C.;Hack, Anniesha;Schushan, Maya;Landau, Meytal;Ben-Tal, Nir;Rao, Rajini
通讯作者：
Rao, Rajini

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