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

• 批准号: 8423344
• 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
• 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/8423344
• 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

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.

描述（由申请人提供）：此处提出的研究的主要重点是表征上皮钠通道（ENAC）的ER相关降解（ERAD）的要求。 ENAC负责在肾脏和肺上皮上吸收盐，并且在控制血压和气道液体积中起着至关重要的作用。 ENAC降解中的缺陷与Liddle综合征和假氧化醛型分别导致高血压和低血压有关。由于ENAC在盐稳态中的作用，综合和贩运在各个层面都受到严格的调节。虽然已经对细胞表面的ENAC运输和更多的外围细胞室进行了广泛的研究，但目前，关于ER中的ENAC生物合成和质量控制鲜为人知。 ER相关的降解（ERAD）是ER质量控制系统监测进入分泌途径的蛋白质的过程，并在无法达到成熟构象时会降解。除ENAC外，许多其他相关的蛋白质也可以成为ERAD底物，包括CFTR（囊性纤维化），AQP2（AQP2）（肾病性糖尿病）和Pael-R（帕金森氏病）。我以前的工作表明，ENAC的降解需要独特的分子伴侣补体。例如，ER Lumenal HSP40是ENAC降解所需的，但是HSP70，HSP40的HSP70，HSP40不作为共伴侣。该提案的目的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子。首先，我将使用酵母模型系统来表征在表达酵母的ENAC转录分析中被上调的基因。我还将分析核苷酸交换因子SIL1和LHS1的作用，以及质量控制与ENAC降解中相关的凝集素的作用。其次，我将使用我在酵母中获得的数据来识别和表征ENAC效应子的人类同源物。人类同源物在ENAC降解中的作用将使用爪蟾卵母细胞过表达系统进行评估，以获得功能性的电生理读数（钠电流）以进行ENAC表面表达。虽然我已经精通酵母作为模型生物，但我不熟悉使用电生理技术。幸运的是，汤姆·克莱曼（Tom Kleyman）博士的实验室对这些技术非常有经验，并同意主持我的培训部分。我非常有动力掌握两电极电压夹电生理学，这将使我能够使用功能读数来监视ENAC运输。我相信这一点，以及学习使用酵母遗传方法将补充我当前的技术技能，并为我提供成为成功的独立科学家的技术能力。除了获得新的技术技能外，该奖项还将使我能够进一步发展自己的教学，指导，写作，呈现和管理技能，这对于成为一名全面的独立科学家至关重要。我很幸运能够在我的共同提案家杰夫·布罗德斯基（Jeff Brodsky）博士和汤姆·克莱曼（Tom Kleyman）博士的指导下完成这项培训，他们不仅是成熟的调查员，而且是熟练的教育工作者，而且我有信心我会实现该奖项中概述的目标。除了本提案的技术方面，我还将通过参加期刊俱乐部，本地和国家会议，并定期与我的咨询委员会会面，从而充分利用该职业奖的培训机会。我相信，匹兹堡大学，生物科学系和肾功能电解质部提供的培训环境将有助于我实现成为一名独立的科学研究者的最终目标，我将继续研究ENAC和其他疾病相关蛋白质的早期折叠，贩运，贩运和退化事件。