Role of GRP170 in ENaC Biogenesis and Renal Physiology

项目摘要 这项建议的重点是研究保守的分子伴侣分子的机制， Grp170/Lhs1调节上皮钠通道ENaC的降解、组装和运输。 ENAC负责跨越肾和肺上皮细胞的盐重吸收，并控制这两种血液。 压力、离子和流体的动态平衡。ENaC的功能获得和丧失突变会导致疾病，以及 ENAC活性也与其他与上皮功能障碍相关的疾病有关。ENAC是一家 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， 胞外环较大，胞质N-和C-末端较短。合成后不久，ENaC将受到 针对错误折叠的蛋白质和孤儿的内质网相关降解(ERAD) 胞浆26S蛋白酶体破坏的多聚体复合体的亚基。毫不奇怪，ENaC 单独的亚基是ERAD的目标，但ENaC的相当大比例即使在三个亚基都被降解的情况下也是如此 ENAC亚基存在。如何发生足够的亚基组装才能逃脱ERAD是一个谜。 然而，这组调查人员的数据揭示了Lhs1伴侣的新角色(Grp170 in 哺乳动物细胞)在ENaC生物发生过程中。具体来说，LHS1促进了α亚基的降解，但 对β或γ亚基的降解没有影响，但当三个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依赖的ERAD。与这些数据一致， Grp170还针对哺乳动物细胞中ERAD的α亚单位，但促进了组装的 异三聚体通道。将使用三个模型系统来进一步理解这些事件：1)已建立的， 遗传易用性酵母系统将被用来定义区分 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 通道组装和贩运；3)肾脏缺乏Grp170的有条件Grp170基因敲除小鼠 小管，将用于确定Grp170伴侣对ENaC的调节如何影响肾脏生理。 总体而言，这项提案将使用多系统方法来定义单个分子伴侣如何调节 ENAC，并首次指出伴侣如何也可以选择一个孤立的亚单位进行降解 因为有助于低聚蛋白的组装。总之，了解Grp170的作用机制将 提供有关ENaC功能和相关疾病状态的新见解。更广泛地说，这项工作将有所帮助 破译内质网中多聚体蛋白的膜组装如何导致稳定和运输， 对肾脏中许多其他离子转运体的功能至关重要。本文件中描述的实验 提案将由一个由多学科调查人员组成的团队以及通过与当地专家的合作来推动 在ENaC生理学、成像技术、小鼠疾病模型和ERAD方面。

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.