Characterization of the ER associated Biogenesis and Degradation of ENaC

ER 相关的 ENaC 生物发生和降解的表征

基本信息

批准号：
8234160
负责人：
Teresa M Buck
金额：
$ 9.94万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-03-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8234160
关键词：
Advisory Committees Anabolism Animal Model Autophagocytosis Award Biochemical Genetics Biogenesis Biological Assay Biological Models Biological Sciences Blood Pressure Calnexin Cell surface Cells Complement Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Development Disease Educational process of instructing Electrodes Electrolytes Electrophysiology (science)Endoplasmic Reticulum Degradation Pathway Environment Epithelial Epithelium Event Genes Goals Guanine Nucleotide Exchange Factors Health Homeostasis Homologous Gene Human Hypertension Hypotension Individual Journals Kidney Laboratories Lead Learning Lectin Liquid substance Lung Membrane Mentors Microarray Analysis Modeling Molecular Molecular Chaperones Molecular Conformation Monitor Mutation Nephrogenic Diabetes Insipidus Nucleotides Oocytes Organism Parkinson Disease Pathway interactions Peripheral Play Process Proteins Pseudohypoaldosteronism Publishing Quality Control Reading Regulation Research Personnel Role Saccharomyces cerevisiae Scientist Series Sodium Sodium Channel Sodium Chloride Specificity Stress Surface Syndrome System Techniques Testing Training Universities Work Writing Xenopus oocyte Yeast Model System Yeasts base blood pressure regulation career epithelial Na+ channel experience methionylmethionine novel overexpression skills structure-specific endonuclease I trafficking voltage clamp yeast genetics

项目摘要

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.

描述（由申请人提供）：此处提出的研究的主要焦点是表征上皮钠通道（ENaC）的 ER 相关降解（ERAD）的要求。 ENaC 负责肾脏和肺上皮细胞的盐重吸收，在控制血压和气道液量方面发挥着关键作用。 ENaC 降解缺陷与利德尔综合征和 I 型假性醛固酮增多症相关，分别导致高血压和低血压。由于 ENaC 在盐稳态中的作用，其合成和运输在各个层面都受到严格监管。虽然细胞表面和更外周细胞区室的 ENaC 运输已被广泛研究，但目前对 ER 中 ENaC 的生物合成和质量控制知之甚少。 ER 相关降解 (ERAD) 是进入分泌途径的蛋白质由 ER 质量控制系统监控的过程，当蛋白质未能达到成熟构象时，蛋白质就会被降解。除了ENaC之外，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（肾性尿崩症）和Pael-R（帕金森病）。我之前的工作表明 ENaC 的降解需要独特的分子伴侣的补充。例如，ENaC 降解需要 ER 腔 Hsp40，但 Hsp70、BiP（Hsp40 作为共伴侣）则不需要。该提案的目标是使用两个模型系统来识别和表征 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 的作用，以及与质量控制相关的凝集素在 ENaC 降解中的作用。其次，我将使用在酵母中获得的数据来识别和表征 ENaC 效应子的人类同源物。将使用爪蟾卵母细胞过表达系统评估人类同源物在 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式生物，但我不熟悉使用电生理技术。幸运的是，Tom Kleyman 博士的实验室对这些技术非常有经验，并同意主办我的这部分培训。我非常有动力掌握两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 的运输。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之外，该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，这些技能对于成为一名全面发展的独立科学家至关重要。我很幸运能够在我的共同发起人 Jeff Brodsky 博士和 Tom Kleyman 博士的指导下完成这次培训，他们不仅是经验丰富的研究人员，而且是熟练的教育家，我相信我将获得该奖项概述的目标。除了该提案的技术方面之外，我还将充分利用该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会面。我相信匹兹堡大学生物科学系和肾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研究者的最终目标，在那里我将继续研究早期折叠， ENaC 和其他疾病相关蛋白的运输和降解事件。公共健康相关性：当前申请建议研究上皮钠通道 (ENaC) 合成、折叠和降解过程中的早期事件。 ENaC 负责肾脏中钠的重吸收，对于调节血压至关重要。 ENaC 突变会导致利德尔氏综合征和 1 型假性醛固酮减少症等疾病；因此，了解早期生物发生事件至关重要。