Identification and Characterization of Factors Involved in ENaC Biogenesis

描述(申请人提供)：进入分泌途径的蛋白质由ER质量控制系统监测，如果它们不能正确折叠，则会受到内质网相关降解(ERAD)的影响。ERAD途径的缺陷与囊性纤维化、亨廷顿病和肾源性尿崩症等疾病有关。由于ERAD在大量和几种类型的疾病中发挥着重要作用，了解这一过程无疑对治疗学的发展至关重要。然而，该领域的最新进展揭示了这一过程的巨大复杂性和特异性，表明充分阐明每种疾病相关底物的ERAD途径将是至关重要的。这项研究的主要重点是描述ERAD对上皮钠通道(ENaC)处理的要求。ENAC负责跨越肺和肾脏等上皮细胞的盐重吸收，ENaC降解缺陷与利德尔综合征和假性醛固酮减少症1型有关。此外，ENaC功能也与囊性纤维化的发病有关。虽然存在大量关于ER ENaC后降解和贩运的文献，但针对ERAD很大比例的蛋白质的早期、ER相关的“决定”基本上没有研究。在这项研究中，我建议：(1)通过分析具有已知ERAD效应器突变的酵母菌株的降解来定义ENaC ERAD途径；(2)通过转录图谱确定参与早期ENaC生物发生和降解的新因素。这里提出的实验将在一个新开发的酵母表达系统中进行，在这个系统中，高效的ENaC ERAD已经被重组，并且脊椎动物细胞中ENaC质量控制的信息可以从该系统中转移。

DESCRIPTION (provided by applicant): Proteins entering the secretory pathway are monitored by the ER quality control system and subject to Endoplasmic Reticulum Associated Degradation (ERAD) in the event they fail to properly fold. Defects in the ERAD pathway are associated with diseases such as cystic fibrosis, Huntington's disease and nephrogenic diabetes insipidus. Because ERAD plays an important role in a large number and several types of diseases, understanding this process will undoubtedly be critical for the development of therapeutics. However, recent progress in the field has brought to light the tremendous complexity and specificity of this process, suggesting that it will be critical to fully elucidate the ERAD pathway for each disease related substrate. The major focus of the study proposed here is to characterize the ERAD requirements for the disposal of the Epithelial Sodium Channel (ENaC). ENaC is responsible for salt reabsorption across epithelia including lung and kidney, and defects in ENaC degradation are associated with Liddle's syndrome and pseudohypoaldosteronism type 1. In addition, ENaC function has also been connected to the pathogenesis of cystic fibrosis. While a significant literature exists regarding post-ER ENaC degradation and trafficking, the early, ER associated "decisions" that target a large proportion of the protein for ERAD have gone largely unstudied. In this study I propose to: (1) Define the ENaC ERAD pathway by assaying degradation in yeast strains with mutations in known ERAD effectors; (2) Identify novel factors involved in early ENaC biogenesis and degradation by transcriptional profiling. The experiments proposed here will be carried out in a newly developed yeast expression system in which efficient ENaC ERAD has been reconstituted, and from which information on ENaC quality control in vertebrate cells is transferable.