Characterization of the ER associated Biogenesis and Degradation of ENaC

描述（由申请人提供）：这里提出的研究的主要重点是表征上皮钠通道（ENaC）内质网相关降解（ERAD）的要求。ENaC负责肾和肺上皮的盐重吸收，并在控制血压和气道液体量方面起关键作用。ENaC降解缺陷与Liddle综合征和I型假性醛固酮减少症有关，分别导致高血压和低血压。由于ENaC在盐稳态中的作用，它的合成和运输在每一个水平上都受到严格的管制。虽然ENaC在细胞表面和更多外周细胞间室的转运已被广泛研究，但目前对内质网中ENaC的生物合成和质量控制知之甚少。内质网相关降解（ERAD）是指进入分泌途径的蛋白质受到内质网质量控制系统的监测，当它们未能达到成熟构象时就会被降解的过程。除ENaC外，许多其他疾病相关蛋白也可以成为ERAD底物，包括CFTR（囊性纤维化）、AQP2（肾源性尿囊症）和Pael-R（帕金森病）。我之前的工作表明，ENaC的降解需要一种独特的分子伴侣补充。例如，内质网管腔hsp40是ENaC降解所必需的，但hsp40作为共同伴侣的Hsp70、BiP则不是。本提案的目标是使用两个模型系统识别和表征ENaC降解和生物发生的其他效应物。首先，我将使用酵母模型系统来表征在表达ENaC的酵母的转录分析中上调的基因。我还将分析核苷酸交换因子Sil1和Lhs1的作用，以及质量控制相关凝集素在ENaC降解中的作用。其次，我将使用我在酵母中获得的数据来识别和表征ENaC效应物的人类同源物。人类同源物在ENaC降解中的作用将通过非洲爪蟾卵母细胞过表达系统进行评估，以获得ENaC表面表达的功能性电生理读数（钠电流）。虽然我已经熟练地使用酵母作为模式生物，但我不熟悉使用电生理技术。幸运的是，汤姆·克莱曼博士的实验室对这些技术很有经验并且同意主持我的这部分培训。我非常渴望掌握双电极电压钳电生理学，这将使我能够使用功能读数来监测ENaC的贩运。我相信这一点，以及学习使用酵母基因方法将补充我目前的技术技能，并为我提供成为一名成功的独立科学家的技术能力。除了获得新的技术技能，这个奖项将使我进一步发展我的教学、指导、写作、演讲和管理技能，这些都是成为一个全面的、独立的科学家的关键。我很幸运能够在我的共同赞助者Jeff Brodsky博士和Tom Kleyman博士的指导下完成这次培训，他们不仅是知名的研究者，而且是熟练的教育者，我相信我将达到这个奖项所规定的目标。除了这项提案的技术方面，我还将充分利用这个职业奖为我的专业发展提供的培训机会，参加期刊俱乐部、地方和国家会议，并定期与我的咨询委员会开会。我相信匹兹堡大学生物科学系和肾电解质学部提供的培养环境将帮助我实现成为一名独立科学研究者的最终目标，在那里我将继续研究ENaC和其他疾病相关蛋白的早期折叠、转运和降解事件。

DESCRIPTION (provided by applicant): The major focus of the study proposed here is to characterize the requirements for the ER associated degradation (ERAD) of the Epithelial Sodium Channel (ENaC). 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. Because of ENaC's role in salt homeostasis, the synthesis and trafficking are tightly regulated at every level. While ENaC trafficking at the cell surface and more peripheral cellular compartments has been extensively studied, there is currently little known about ENaC biosynthesis and quality control in the ER. ER associated degradation (ERAD) is the process whereby proteins entering the secretory pathway are monitored by the ER quality control system and subject to degradation when they fail to attain a mature conformation. In addition to ENaC, many other disease relevant proteins can also become ERAD substrates, including CFTR (cystic fibrosis), AQP2 (nephrogenic diabetes insipidus), and Pael-R (Parkinson's disease). My previous work has shown that the degradation of ENaC requires a unique complement of molecular chaperones. For example, the ER lumenal Hsp40s are required for ENaC degradation, but the Hsp70, BiP for which Hsp40s serve as co-chaperones are not. The goal of this proposal is to identify and characterize additional effectors of ENaC degradation and biogenesis using two model systems. First, I will use the yeast model system to characterize genes that were upregulated in a transcriptional analysis of ENaC expressing yeast. I will also assay the role of the nucleotide exchange factors Sil1 and Lhs1, as well as the quality control associated lectins in ENaC degradation. Second, I will use the data I obtain in yeast to identify and characterize the human homologues of the ENaC effectors. The role of the human homologues in ENaC degradation will be assessed using a Xenopus oocyte overexpression system to obtain a functional, electrophysiological readout (sodium current) for ENaC surface expression. While I have become proficient in using yeast as a model organism, I am unfamiliar with using electrophysiological techniques. Fortunately, the laboratory of Dr. Tom Kleyman is very experienced with these techniques and has agreed to host this portion of my training. I am extremely motivated to master two- electrode voltage clamp electrophysiology, which will allow me to monitor ENaC trafficking using a functional readout. I believe this, as well as learning to use yeast genetic approaches will complement my current technical skills and provide me with the technical ability to become a successful independent scientist. In addition to acquiring new technical skills this award will enable me to further develop my teaching, mentoring, writing, presenting, and management skills, which are all critical to becoming a well-rounded, independent scientist. I am fortunate to be completing this training under the direction of my co-sponsors, Dr. Jeff Brodsky and Dr. Tom Kleyman, who are both not only well-established investigators, but skilled educators, and I am confident that I will attain the goals outlined in this award. In addition to the technical aspects of this proposal I will take full advantage of the training opportunities this career award will provide for my professional development by participating in journal clubs, local and national meetings, and meeting with my advisory committee on a regular basis. I am confident that the training environment of the University of Pittsburgh, the Department of Biological Sciences, and the Renal-Electrolyte Division provides will help me attain my ultimate goal of becoming an independent scientific investigator, where I will continue to investigate the early folding, trafficking and degradation events of ENaC and other disease relevant proteins.