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:
    8423344
  • 负责人:
  • 金额:
    $ 9.94万
  • 依托单位:
  • 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

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.
描述(由申请方提供):此处拟定研究的主要重点是表征上皮钠通道(ENaC)ER相关降解(ERAD)的要求。ENaC负责跨肾脏和肺的上皮细胞的盐重吸收,并且在控制血压和气道流体体积中起关键作用。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水平上都受到严格的调控。虽然ENaC在细胞表面和更多外周细胞区室的运输已被广泛研究,但目前对ER中的ENaC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质量控制系统监测并在未能达到成熟构象时发生降解的过程。除了ENaC,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),AQP 2(肾源性尿崩症)和Pael-R(帕金森病)。我以前的工作表明,ENaC的降解需要一个独特的分子伴侣补充。例如,ER内腔Hsp 40是ENaC降解所需的,但Hsp 70、BiP(其中Hsp 40充当共分子伴侣)不是。该提案的目标是使用两个模型系统来识别和表征ENaC降解和生物发生的额外效应物。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。我也将分析的作用,核苷酸交换因子Sil 1和Lhs 1,以及质量控制相关的凝集素在ENaC降解。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。将使用非洲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学读数(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,并同意主持我的这部分培训。我非常积极地掌握双电极电压钳电生理学,这将使我能够监测ENaC贩运使用功能读出。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,这个奖项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人Jeff Brodsky博士和Tom Kleyman博士的指导下完成这项培训,他们不仅是成熟的调查人员,而且是熟练的教育工作者,我相信我将实现这个奖项中概述的目标。除了这项建议的技术方面,我将充分利用这个职业奖将通过参加期刊俱乐部,地方和国家会议,并定期与我的咨询委员会会面,为我的专业发展提供培训机会。我相信,匹兹堡大学生物科学系和Renal-Electrolyte部门提供的培训环境将帮助我实现成为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,运输和降解事件。

项目成果

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Teresa M Buck其他文献

Teresa M Buck的其他文献

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{{ truncateString('Teresa M Buck', 18)}}的其他基金

Role of GRP170 in ENaC Biogenesis and Renal Physiology
GRP170 在 ENaC 生物发生和肾脏生理学中的作用
  • 批准号:
    9886238
  • 财政年份:
    2019
  • 资助金额:
    $ 9.94万
  • 项目类别:
Role of GRP170 in ENaC Biogenesis and Renal Physiology
GRP170 在 ENaC 生物发生和肾脏生理学中的作用
  • 批准号:
    10133059
  • 财政年份:
    2019
  • 资助金额:
    $ 9.94万
  • 项目类别:
Role of GRP170 in ENaC Biogenesis and Renal Physiology
GRP170 在 ENaC 生物发生和肾脏生理学中的作用
  • 批准号:
    10382327
  • 财政年份:
    2019
  • 资助金额:
    $ 9.94万
  • 项目类别:
Role of GRP170 in ENaC Biogenesis and Renal Physiology
GRP170 在 ENaC 生物发生和肾脏生理学中的作用
  • 批准号:
    10609834
  • 财政年份:
    2019
  • 资助金额:
    $ 9.94万
  • 项目类别:
Investigating the role of GRP170 in ENaC biogenesis
研究 GRP170 在 ENaC 生物发生中的作用
  • 批准号:
    9087782
  • 财政年份:
    2016
  • 资助金额:
    $ 9.94万
  • 项目类别:
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:
    8607544
  • 财政年份:
    2011
  • 资助金额:
    $ 9.94万
  • 项目类别:
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:
    8803787
  • 财政年份:
    2011
  • 资助金额:
    $ 9.94万
  • 项目类别:
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:
    8028610
  • 财政年份:
    2011
  • 资助金额:
    $ 9.94万
  • 项目类别:
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:
    8234160
  • 财政年份:
    2011
  • 资助金额:
    $ 9.94万
  • 项目类别:
Identification and Characterization of Factors Involved in ENaC Biogenesis
ENaC 生物发生中涉及的因素的鉴定和表征
  • 批准号:
    7589811
  • 财政年份:
    2008
  • 资助金额:
    $ 9.94万
  • 项目类别:

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