Protein Phosphatase Regulation of the Principal Renal Sodium Transporter NHE3

主要肾钠转运蛋白 NHE3 的蛋白磷酸酶调节

• 批准号: 8329702
• 负责人: Ion Alexandru Bobulescu
• 金额: $ 8.68万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8329702
• 关键词: Acute; Address; Affect; Amino Acids; Animal Model; Area; Basic Science; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Blood Pressure; Catalytic Domain; Cell Culture Techniques; Cell Nucleus; Cells; Clinical; Collaborations; Data; Development; Development Plans; Disease; Dominant-Negative Mutation; Dopamine; Electric Capacitance; Endocytosis; Environment; Excretory function; Exposure to; Faculty; Fostering; Functional disorder; Funding; Goals; Half-Life; Human; Hypertension; Image; Immunohistochemistry; In Vitro; Institute of Medicine (U.S.); Institution; Internal Medicine; Intravenous; Intravenous infusion procedures; Ions; Kidney; Label; Laboratories; Laboratory Animal Production and Facilities; Laboratory Research; Lead; Learning; Link; Liquid substance; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Medical center; Membrane; Membrane Protein Traffic; Mentors; Methods; Micropuncture; Microscopy; Mission; Modeling; Molecular; Mutate; National Institute of Diabetes and Digestive and Kidney Diseases; Natriuresis; Nobel Prize; Outcome; Pathogenesis; Pharmacology; Phospho-Specific Antibodies; Phosphopeptides; Phosphorylation; Physicians; Physiologic pulse; Physiological; Physiology; Positioning Attribute; Postdoctoral Fellow; Prevention; Protein Dephosphorylation; Protein phosphatase; Protons; Public Health; Publications; Publishing; Rattus; Recycling; Regulation; Research; Research Project Grants; Research Support; Research Training; Rodent Model; Role; Running; Scientist; Signal Pathway; Signal Transduction; Site; Sodium; Stimulus; System; Techniques; Testing; Texas; Time; Training; Tube; United States; United States National Academy of Sciences; Universities; Vesicle; Wistar Rats; Work; autocrine; base; blood pressure regulation; brush border membrane; career; career development; fluorescence imaging; gludopa; human disease; in vivo; innovation; instructor; kidney cell; member; monolayer; multi-photon; mutant; novel; paracrine; professor; programs; protein phosphatase 2A regulatory subunit 65 kDa; research study; response; scaffold; skills; square foot; success; teacher; trafficking

DESCRIPTION (provided by applicant): Dr. Ion Alexandru Bobulescu is a physician-scientist recently appointed to a full-time faculty position at the instructor level at the University of Texas Southwestern Medical Center at Dallas (UT Southwestern). His short-term career goals (next 4 years) are to rigorously pursue the training and career development plan outlined in this K01 application, perform the proposed experiments using both established and highly innovative techniques to define the role of Protein Phosphatase 2A (PP2A) in the regulation of renal sodium/proton exchange, and then apply for funding under the R01 mechanism to explore one or more of the scientific opportunities derived from this work. Dr. Bobulescu's long-term career goal is to be a leader and innovator in the field of renal regulation of sodium and blood pressure homeostasis, using pioneering approaches and techniques to address fundamental unanswered questions from molecule to model organism to human disease. To achieve these goals, a thorough and detailed Career Development Plan is proposed to address the following training requirements: (1) COURSEWORK. Additional formal training in several areas is considered critical for the advancement of the candidate's research. (2) ADVANCED TECHNIQUES. Working with his mentor and expert collaborators on a hands-on basis to address the Aims of this proposal, Dr. Bobulescu will learn several novel, state-of-the-art methods, and will gain the ability to apply these techniques for his own research questions. (3) MENTORED CAREER DEVELOPMENT. Dr. Bobulescu's chances of achieving relevant results in the new and understudied field of signal transduction via protein phosphatases will greatly benefit from having an expert mentor who will directly guide, supervise and evaluate his research. The mentor for this K01, Dr. Marc Mumby, is a world-renowned expert in protein phosphatase research. In addition, Dr. Bobulescu is aware of the fact that a successful independent career requires much more than ideas, technical skills, and a good track record of publications. Running a research laboratory, managing and nurturing people, fostering collaborations, being an effective mentor and teacher, and being able to maintain a strong and productive research program resulting in successful funding beyond the first R01, are all formidable endeavors- but vital for true academic success. The candidate is certain that he has a lot to learn in these areas from Dr. Mumby, a leading scientist and ideal mentor for this K01. (4) DEPARTMENT. The disease-oriented perspective offered by Dr. Bobulescu's previous postdoctoral research training in a clinical department has been very important for his formation as a physician scientist. However, his training would greatly benefit from a period of immersive exposure to the vastly different environment of a leading basic science department. ENVIRONMENT: UT Southwestern is one of the world's foremost research institutions, with four Nobel prize winners, 36 members of the National Academy of Sciences and/or its Institute of Medicine, more than 3,500 research projects under way with more than $400 million in annual funding, as well as more than 582,000 sq ft of research space with more under construction. Dr. Bobulescu will train primarily in the laboratory of Dr. Marc Mumby in the Department of Pharmacology, including over 2,000 sq ft of total laboratory space, well equipped with all necessary items needed to carry out biochemical, molecular biological, and cell biological experiments. Dr. Bobulescu will also retain his 250 sq ft laboratory space, private 90 sq ft office and faculty position in the Department of Internal Medicine. The candidate will have access to UT Southwestern's state of the art animal research facility, as well as to the UT Southwestern George M. O'Brien Kidney Research Core Center. UT Southwestern has a tradition of retaining the best postdoctoral fellows and fostering their independent careers. This tradition includes the candidate's former mentor, Dr. Orson Moe, who was a postdoctoral fellow at UT Southwestern until 1990 and has remained there ever since, rising to full Professor and Center Director. RESEARCH: This proposal will study the role of PP2A in the regulation of the principal renal sodium transporter NHE3, with special emphasis on the regulation of NHE3 by intra-renal dopamine. If the proposed hypotheses are correct, PP2A will be established as a major regulator of intrarenal dopamine action and of NHE3. This will have a direct impact on our understanding of the physiology and pathophysiology of blood pressure regulation, since impaired dopamine-mediated natriuresis has been described in hypertensive humans, has been attributed to defective intra-renal dopamine signaling in two rodent models of hypertension, and has been linked to defective renal PP2A function. The proposal includes three independent aims that will be pursued simultaneously using a variety of techniques. Aim 1 will use cell culture and in vitro experiments to test whether the inhibition of NHE3 by dopamine requires the direct action of PP2A on NHE3. Aim 2 will use cell culture, biochemistry and advanced electrophysiological techniques to test whether dephosphorylation of NHE3 by PP2A acts as an intracellular switch determining the fate of NHE3. Aim 3 will use advanced in vivo imaging and immunohistochemistry to test whether the inhibition of NHE3 in vivo by physiologic stimuli involves NHE3 dephosphorylation by PP2A. By proposing to uncover the molecular details of a signaling pathway that, when impaired, has been implicated in the pathogenesis of hypertension, this project is clearly relevant to the mission of NIDDK to support research on the most serious diseases affecting public health.

描述（由申请人提供）：Ion Alexandru Bobulescu 博士是一位医师兼科学家，最近被任命为达拉斯德克萨斯大学西南医学中心（UT Southwestern）的全职讲师职位。他的短期职业目标（未来 4 年）是严格执行本 K01 申请中概述的培训和职业发展计划，使用既定且高度创新的技术进行拟议的实验，以定义蛋白磷酸酶 2A (PP2A) 在肾钠/质子交换的调节，然后在R01机制下申请资助，探索一项或多项源自这项工作的科学机会。 Bobulescu 博士的长期职业目标是成为钠和血压稳态肾脏调节领域的领导者和创新者，利用开创性的方法和技术来解决从分子到模型生物再到人类疾病的基本未解答问题。为了实现这些目标，提出了一个全面、详细的职业发展计划，以满足以下培训要求： (1) 课程。多个领域的额外正式培训被认为对于候选人研究的进步至关重要。 (2)先进技术。 Bobulescu 博士将与他的导师和专家合作者一起实践，以实现该提案的目标，他将学习几种新颖、最先进的方法，并将获得将这些技术应用于自己的能力研究问题。 (3) 指导职业发展。 Bobulescu 博士在通过蛋白磷酸酶进行信号转导这一新的、尚未得到研究的领域取得相关成果的机会将大大受益于有一位直接指导、监督和评估他的研究的专家导师。这款K01的导师Marc Mumby博士是世界著名的蛋白磷酸酶研究专家。此外，Bobulescu 博士意识到，成功的独立职业需要的不仅仅是想法、技术技能和良好的出版物记录。运营一个研究实验室、管理和培养人员、促进合作、成为一名有效的导师和老师，以及能够维持一个强大而富有成效的研究项目，从而在第一个 R01 之外获得成功的资助，这些都是艰巨的努力，但对于真正的学术至关重要成功。候选人确信他在这些领域有很多东西需要向 Mumby 博士学习，Mumby 博士是一位领先的科学家，也是 K01 的理想导师。 （4）部门。 Bobulescu 博士之前在临床科室接受的博士后研究训练所提供的以疾病为导向的观点对于他成为一名医师科学家非常重要。然而，他的训练将极大地受益于一段时间的沉浸式接触领先基础科学部门截然不同的环境。 环境： UT Southwestern 是世界上最重要的研究机构之一，拥有 4 名诺贝尔奖获得者、36 名美国国家科学院和/或其医学研究所成员，正在进行的研究项目超过 3,500 个，每年资助超过 4 亿美元，以及超过 582,000 平方英尺的研究空间，更多空间正在建设中。 Bobulescu 博士将主要在药理学系 Marc Mumby 博士的实验室接受培训，该实验室总面积超过 2,000 平方英尺，配备了进行生化、分子生物学和细胞生物学实验所需的所有必要物品。 Bobulescu 博士还将保留他 250 平方英尺的实验室空间、90 平方英尺的私人办公室以及在内科系的教职。候选人将可以使用德克萨斯大学西南医学中心最先进的动物研究设施，以及德克萨斯大学西南医学中心的乔治·M·奥布莱恩肾脏研究核心中心。德州大学西南分校有着留住最优秀博士后并培养他们独立职业生涯的传统。 这一传统包括该候选人的前导师 Orson Moe 博士，他在 UT 西南大学担任博士后研究员直至 1990 年，此后一直留在那里，并晋升为正教授和中心主任。 研究：本提案将研究 PP2A 在主要肾脏钠转运蛋白 NHE3 的调节中的作用，特别强调肾内多巴胺对 NHE3 的调节。如果所提出的假设正确，PP2A 将被确定为肾内多巴胺作用和 NHE3 的主要调节因子。这将直接影响我们对血压调节的生理学和病理生理学的理解，因为在高血压人群中已经描述了多巴胺介导的尿钠排泄受损，这归因于两种高血压啮齿动物模型中肾内多巴胺信号传导的缺陷，以及与肾 PP2A 功能缺陷有关。该提案包括三个独立的目标，将使用多种技术同时实现。目标1将利用细胞培养和体外实验来测试多巴胺对NHE3的抑制是否需要PP2A对NHE3的直接作用。目标 2 将使用细胞培养、生物化学和先进的电生理学技术来测试 PP2A 对 NHE3 的去磷酸化是否充当决定 NHE3 命运的细胞内开关。目标3将使用先进的体内成像和免疫组织化学来测试体内生理刺激对NHE3的抑制是否涉及PP2A对NHE3的去磷酸化。通过提议揭示信号通路的分子细节，该信号通路在受损时与高血压发病机制有关，显然与 NIDDK 支持研究影响公众健康的最严重疾病的使命相关。