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焦点是描述上皮钠通道(ENaC)的内质网相关降解(ERAD)的要求。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降解缺陷与导致高血压和低血压的利德尔综合征和假性低醛固酮增多症I型有关。由于ENaC在盐的动态平衡中的作用，其合成和运输在各个层面上都受到严格的控制。虽然ENaC在细胞表面和更多的外围细胞隔间的运输已被广泛研究，但目前对ENaC在内质网中的生物合成和质量控制知之甚少。内质网相关降解(ERAD)是进入分泌途径的蛋白质受到内质网质量控制系统的监控，当它们未能达到成熟构象时受到降解的过程。除了ENaC，许多其他与疾病相关的蛋白质也可以成为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)。我以前的工作表明，ENaC的降解需要一种独特的分子伴侣。例如，内质网管腔Hsp40是ENaC降解所必需的，但Hsp40作为辅助伴侣的Hsp70、Bip则不是。这项建议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器。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鉴定和表征ENaC效应器的人类同源物。将使用非洲爪哇卵母细胞过表达系统评估人类同系物在ENaC降解中的作用，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型生物，但我不熟悉使用电生理技术。幸运的是，汤姆·克利曼博士的实验室对这些技术非常有经验，并同意主持我的这部分培训。我非常有动力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控ENaC的贩运。我相信这一点，以及学习使用酵母遗传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外，这个奖项还将使我能够进一步发展我的教学、指导、写作、演示和管理技能，这些技能都是成为一名全面、独立的科学家的关键。我很幸运在我的联合赞助人Jeff Brodsky博士和Tom Kley man博士的指导下完成了这次培训，他们不仅是知名的研究人员，而且是熟练的教育工作者，我相信我将实现这个奖项中列出的目标。除了这项建议的技术方面，我还将充分利用这一职业奖项为我的专业发展提供的培训机会，参加期刊俱乐部、地方和全国会议，并定期与我的顾问委员会会面。我相信匹兹堡大学、生物科学系和肾脏电解质部门提供的培训环境将帮助我实现成为一名独立科学调查员的最终目标，在那里我将继续调查ENaC和其他与疾病相关的蛋白质的早期折叠、运输和降解事件。 公共卫生相关性：目前的应用建议调查上皮钠通道(ENaC)合成、折叠和降解过程中的早期事件。ENAC负责肾脏对钠的重新吸收，对调节血压至关重要。ENaC基因突变会导致包括利德尔综合征和1型假性醛固酮减少症在内的疾病；因此，了解早期的生物发生事件是至关重要的。

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. PUBLIC HEALTH RELEVANCE: The current application proposes to investigate early events during the synthesis, folding, and degradation of the Epithelial Sodium Channel (ENaC). ENaC is responsible for the reabsorbtion of sodium in the kidney and is critical for regulating blood pressure. Mutations in ENaC lead to diseases including Liddle's Syndrome and Pseudohypoaldosteronism Type 1; therefore, understanding the early biogenesis events is critical.