Role of GRP170 in ENaC Biogenesis and Renal Physiology

GRP170 在 ENaC 生物发生和肾脏生理学中的作用

基本信息

批准号：
9886238
负责人：
Teresa M Buck
金额：
$ 33.75万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9886238
关键词：
26S proteasome Ablation Address Adult Affect Amino Acid Motifs Amino Acids Anabolism Area Bartter Disease Binding Biochemical Biogenesis Biological Assay Biological Models Biology Blood Pressure Cell surface Cells Cellular Assay Collaborations Complement Complex Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Development Disease Disease model Elements Endocytosis Endoplasmic Reticulum Epithelial Epithelium Evaluation Event Goals Holoenzymes Homeostasis Homologous Gene Hypertension Hypokalemia Hyponatremia Hypotension Imaging technology In Vitro Inbred F344 Rats Individual Ion Channel Ions KCNJ1 gene Kidney Knockout Mice Lead Liddle syndrome Liquid substance Lung Mammalian Cell Mediating Membrane Membrane Proteins Molecular Molecular Chaperones Monitor Mus Orphan Phenotype Physiology Play Process Proteins Pseudohypoaldosteronism Quality Control Regulation Renal tubule structure Research Personnel Role Sodium Sodium Chloride Structure Surface System Testing Thyroid Gland Time Transmembrane Domain Ubiquitination Water Work Xenopus oocyte Yeast Model System Yeasts base blood pressure regulation epithelial Na+ channel experimental study extracellular gain of function mutation genetic analysis glucose-regulated protein 170 hyperkalemia insight link protein loss of function mutation membrane assembly misfolded protein monomer mouse model multidisciplinary novel overexpression protein folding trafficking vasopressin resistant diabetes insipidus

项目摘要

Project Summary The focus of this proposal is to investigate the mechanism by which the conserved molecular chaperone, GRP170/Lhs1, regulates the degradation, assembly, and trafficking of the epithelial sodium channel, ENaC. ENaC is responsible for salt reabsorption across epithelia of the kidney and lung, and controls both blood pressure and ion and fluid homeostasis. Gain- and loss-of-function mutations in ENaC lead to disease, and ENaC activity is also associated with other diseases associated with epithelial malfunction. ENaC is a heterotrimeric channel composed of an α, β, and γ subunit. Each subunit contains two transmembrane domains, a large extracellular loop, and short cytosolic N- and C-termini. Soon after synthesis, ENaC is subject to Endoplasmic Reticulum Associated Degradation (ERAD), which targets misfolded proteins and orphaned subunits of multimeric complexes for destruction by the cytosolic 26S proteasome. Not surprisingly, ENaC subunits individually are targeted for ERAD, but a significant percent of ENaC is degraded even when all three ENaC subunits are present. How sufficient subunit assembly occurs in order to escape ERAD is mysterious. However, data from this team of investigators uncovered a new role for the Lhs1 chaperone (GRP170 in mammalian cells) during ENaC biogenesis. Specifically, Lhs1 facilitated the degradation of the α subunit but had no effect on β or γ subunit degradation, yet when all three ENaC subunits were expressed, intersubunit interactions between the transmembrane domains blocked Lhs1-dependent ERAD. Consistent with these data, GRP170 also targeted the α subunit for ERAD in mammalian cells but promoted trafficking of the assembled heterotrimeric channel. Three model systems will be used to further understand these events: 1) An established, genetically facile yeast system will be used to define the structural elements required to differentiate between an orphaned ENaC subunit and the assembled heterotrimeric channel; 2) A Fischer rat thyroid (FRT) cell system will be used to confirm results from the yeast system and define amino acid motifs required for GRP170-mediated channel assembly and trafficking; 3) A conditional GRP170 knock out mouse, which lacks GRP170 in kidney tubules, will be used to determine how ENaC regulation by the GRP170 chaperone affects renal physiology. Overall, this proposal will use a multi-system approach to define how a single molecular chaperone regulates ENaC and—for the first time—indicate how chaperones can select an orphaned subunit for degradation as well as facilitate assembly of an oligomeric protein. Together, understanding the mechanism of action of GRP170 will provide novel insights into ENaC function and associated disease states. More generally, this work will help decipher how membrane assembly of a multimeric protein in the ER results in stabilization and trafficking, which is vital for the function of numerous other ion transporters in the kidney. The experiments described in this proposal will be facilitated by a multi-disciplinary team of investigators and by collaborations with local experts in ENaC physiology, imaging technologies, murine disease models and ERAD.

项目概要该提案的重点是研究保守分子伴侣的机制， GRP170/Lhs1 调节上皮钠通道 ENaC 的降解、组装和运输。 ENaC 负责肾和肺上皮细胞的盐重吸收，并控制血液 ENaC 中的压力、离子和液体稳态的功能获得和丧失突变会导致疾病，以及 ENaC 活性还与其他与上皮功能障碍相关的疾病有关。由 α、β 和 γ 亚基组成的异三聚体通道，每个亚基包含两个跨膜结构域， ENaC 具有大的细胞外环和短的胞质 N 端和 C 端。内质网相关降解 (ERAD)，针对错误折叠和孤儿蛋白毫不奇怪，ENaC 可以被胞质 26S 蛋白酶体破坏。 ERAD 的目标是单独的亚基，但即使所有三个亚基都被降解，很大一部分 ENaC 也会被降解。 ENaC 亚基的存在如何进行足够的亚基组装以逃避 ERAD 是一个谜。然而，该研究小组的数据发现了 Lhs1 伴侣（GRP170）的新作用。具体来说，Lhs1 促进了 α 亚基的降解，但对 β 或 γ 亚基降解没有影响，但当所有三个 ENaC 亚基都表达时，亚基间跨膜结构域之间的相互作用阻断了 Lhs1 依赖性 ERAD，与这些数据一致， GRP170 还针对哺乳动物细胞中 ERAD 的 α 亚基，但促进了组装后的转运将使用三个模型系统来进一步了解这些事件：1）已建立的、遗传简便的酵母系统将用于定义区分不同酵母所需的结构元件孤儿 ENaC 亚基和组装的异三聚体通道 2) Fischer 大鼠甲状腺 (FRT) 细胞系统；将用于确认酵母系统的结果并定义 GRP170 介导所需的氨基酸基序通道组装和运输；3) 条件性 GRP170 敲除小鼠，其肾脏中缺乏 GRP170 肾小管，将用于确定 GRP170 伴侣对 ENaC 的调节如何影响肾生理学。总体而言，该提案将使用多系统方法来定义单分子伴侣如何调节 ENaC 首次表明分子伴侣如何选择孤儿亚基进行降解促进寡聚蛋白的组装，了解 GRP170 的作用机制将有助于促进寡聚蛋白的组装。为 ENaC 功能和相关疾病状态提供新的见解更一般地说，这项工作将有所帮助。破译内质网中多聚蛋白的膜组装如何导致稳定和运输，这对于肾脏中许多其他离子转运蛋白的功能至关重要。多学科研究小组以及与当地专家的合作将促进提案的提出 ENaC 生理学、成像技术、小鼠疾病模型和 ERAD 领域的研究。