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

• 批准号: 10382327
• 负责人: Teresa M Buck
• 金额: $ 33.66万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 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/10382327
• 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; Evaluation; Event; Fluid Balance; 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; Surface; System; Testing; Thyroid Gland; Time; Transmembrane Domain; Ubiquitination; Water; Work; Xenopus oocyte; Yeast Model System; Yeasts; base; blood pressure control; 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

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.

项目摘要 该建议的重点是研究保守分子链酮的机制， GRP170/LHS1，调节上皮钠通道ENAC的降解，组装和贩运。 ENAC负责跨肾脏和肺上皮的盐吸收，并控制两个血液 压力和离子和液体稳态。 ENAC的功能丧失突变会导致疾病，并且 ENAC活性还与其他与上皮故障相关的疾病有关。 ENAC是一个 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域， 一个大的细胞外环，以及短的胞质N-和C末端。合成后不久，ENAC受到 内质网相关降解（ERAD），靶向错误折叠的蛋白质和孤儿 胞质26S蛋白酶体破坏多个复合物的亚基。毫不奇怪，恩纳克 亚基是针对ERAD的，但即使这三个 ENAC亚基存在。为了逃避Erad，如何发生足够的亚基组装是神秘的。 但是，该调查人员团队的数据发现了LHS1伴侣的新角色（GRP170 IN 哺乳动物细胞）在ENAC生物发生过程中。具体而言，LHS1准备了α亚基的降解，但有 对β或γ亚基降解没有影响，但是当表达所有三个ENAC亚基时， 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，但促进了组装的运输 异三个通道。三个模型系统将用于进一步了解这些事件：1）已建立的， 遗传上容易的酵母系统将用于定义区分区分所需的结构元素 孤立的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介导的氨基酸基序 渠道议会和贩运； 3）有条件的GRP170敲除鼠标，该鼠标缺乏肾脏的GRP170 小管将用于确定GRP170伴侣的ENAC调节如何影响肾脏生理。 总体而言，该提案将使用多系统方法来定义单个分子伴侣如何调节 ENAC和 - 首次表明伴侣也可以选择一个孤立的亚基以降解 作为寡聚蛋白的促进组装。同时了解GRP170的作用机理将 提供有关ENAC功能和相关疾病状态的新见解。更一般地，这项工作将有所帮助 解读了ER中多光蛋白的膜组装如何导致稳定和贩运，这 对于肾脏中许多其他离子转运蛋白的功能至关重要。在此描述的实验 提案将由多学科的调查员团队以及与当地专家的合作准备 在ENAC生理学，成像技术，鼠病模型和ERAD中。