Identification and Characterization of Factors Involved in ENaC Biogenesis

描述（由申请方提供）：通过ER质量控制系统监测进入分泌途径的蛋白质，如果蛋白质未能正确折叠，则进行内质网相关降解（ERAD）。ERAD途径的缺陷与诸如囊性纤维化、亨廷顿病和肾源性尿崩症等疾病相关。由于ERAD在大量和几种类型的疾病中起着重要作用，因此了解这一过程无疑对治疗方法的发展至关重要。然而，该领域的最新进展揭示了该过程的巨大复杂性和特异性，这表明完全阐明每种疾病相关底物的ERAD途径至关重要。本文提出的研究的主要重点是表征上皮钠通道（ENaC）处置的ERAD要求。ENaC负责跨上皮细胞（包括肺和肾）的盐重吸收，并且ENaC降解的缺陷与Liddle综合征和1型假性醛固酮减少症相关。此外，ENaC功能也与囊性纤维化的发病机制有关。虽然存在关于ER后ENaC降解和运输的重要文献，但早期ER相关的“决定”（靶向ERAD的大部分蛋白质）在很大程度上未被研究。在这项研究中，我建议：（1）确定ENaC ERAD途径，通过测定已知ERAD效应子突变的酵母菌株的降解;（2）通过转录谱确定参与早期ENaC生物合成和降解的新因子。这里提出的实验将在一个新开发的酵母表达系统中进行，其中有效的ENaC ERAD已被重建，并从其中的信息ENaC在脊椎动物细胞中的质量控制是可转移的。

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.