Investigating the role of GRP170 in ENaC biogenesis

 描述(由申请人提供)：本研究的主要重点是确定内质网管腔伴侣Grp170在上皮钠通道(ENaC)的质量控制和运输中的作用。Enac是由α，β和γ亚基组成的异源三聚体蛋白。ENAC负责跨越肾和肺上皮细胞的盐重吸收，并在控制血压和呼吸道液体量方面发挥关键作用。ENaC降解缺陷与利德尔综合征和假性醛固酮减少症I型有关，后者分别导致高血压和低血压。ENAC由ER质量控制机制进行监测，并针对ER相关降级(ERAD)。内质网相关降解(ERAD)是进入分泌途径的蛋白质在未能达到成熟构象时发生降解的过程。事实上，即使是很大比例的WT ENaC也是针对ERAD机器的。许多其他疾病相关蛋白也可以成为ERAD的底物，包括CFTR(囊性纤维化)、AQP2(肾源性尿崩症)和Pael-R(帕金森氏病)。这项关于Grp170在ENaC质量控制和贩运中的作用的提案的重点是出于以下考虑。首先，Grp170含量丰富，是高分子量Hsp70样蛋白家族的成员。第二，Grp170具有两种独特的活性：Grp170作为内质网管膜Hsp70的核苷酸交换因子(NEF)，以及在体外作为“保持酶”与分泌途径中错误折叠的蛋白质结合。第三，Grp170针对ERAD的αENaC亚基，但对β和γENaC亚基没有影响。第四，与其对单个亚基的影响不同，初步数据表明，Grp170促进了非洲爪哇卵母细胞中ENaC通道(αβγ)的组装和表面表达。Fisher大鼠甲状腺(FRT)细胞系统将被用来研究Grp170对单体和组装的ENaC通道的不同作用。FRT细胞在培养中形成极化单层，允许通过阿米洛利敏感的Na+短路电流测量ENaC亚单位的稳定性、表面表达、蛋白分解处理和ENaC功能。这些产出将被量化，以应对Grp170基因的敲除或过度表达。为了进一步研究Grp170对ENaC的作用机制，还将分析一系列结构域缺失的Grp170构建体。除了调节ENaC和血压外，Grp170的功能还与其他相关过程有关，包括胰岛素分泌、血管内皮生长因子(VEGF)的分泌、肿瘤存活和程序性细胞死亡。此外，Grp170在缺血和动脉粥样硬化过程中起到细胞保护作用。总之，了解Grp170的作用机制将为ENaC功能和相关疾病状态提供新的见解。

 DESCRIPTION (provided by applicant): The major focus of the study proposed here is to characterize the role of the ER lumenal chaperone, GRP170, on the quality control and trafficking of the Epithelial Sodium Channel (ENaC). ENaC is a heterotrimeric protein composed of α, β, and γ subunits. ENaC is responsible for salt reabsorption across the epithelia of the kidney and lung, and plays a critical role in controlling blood pressure and airway fluid volume. Defects in ENaC degradation are associated with Liddle's Syndrome and pseudohypoaldosteronism type I that result in hyper- and hypotension, respectively. ENaC is monitored by the ER quality control machinery and targeted for ER associated degradation (ERAD). ER associated degradation (ERAD) is the process whereby proteins entering the secretory pathway are subject to degradation when they fail to attain a mature conformation. In fact, even a large percentage of WT ENaC is targeted for the ERAD machinery. Many other disease relevant proteins can also become ERAD substrates, including CFTR (cystic fibrosis), AQP2 (nephrogenic diabetes insipidus), and Pael-R (Parkinson's disease). The focus of this proposal on the role of GRP170 in ENaC quality control and trafficking arises from the following considerations. First, GRP170 is abundant and is a member of the high molecular weight Hsp70-like family of proteins. Second, GRP170 possesses two unique activities: GRP170 acts as a nucleotide exchange factor (NEF) for the ER lumenal Hsp70, and it acts as a "holdase" in vitro and binds to misfolded proteins within the secretory pathway. Third, GRP170 targets the α ENaC subunit for ERAD, but has no effect on the β and γENaC subunits. Fourth, in contrast to its effect on individual subunits, preliminary data suggest that GRP170 promotes the assembly and surface expression of the ENaC channel (αβγ) in Xenopus oocytes. A Fisher Rat Thyroid (FRT) cell system will be used to investigate the differential effect of GRP170 on the monomeric versus assembled ENaC channel. FRT cells form polarized monolayers in culture, allowing for measurement of ENaC subunit stability, surface expression, proteolytic processing, and ENaC function, as measured by amiloride-sensitive Na+ short circuit current. These outputs will be quantified in response to knockdown or overexpression of GRP170. To further characterize the mechanism of GRP170 action on ENaC a series of GRP170 constructs with domain deletions will also be assayed. In addition to its role in regulating ENaC and therefore blood pressure, GRP170 function has been tied to other relevant processes, including insulin secretion, secretion of vascular endothelial growth factor (VEGF), tumor survival, and programmed cell death. Moreover, GRP170 plays a cytoprotective role during ischemia and atherosclerosis. Together, understanding GRP170 mechanism of action will provide novel insights into ENaC function and associated disease states.