NHE3 Polymorphisms and Chronic Diarrheal Diseases

描述(由申请人提供)： 这份提案描述了一项为期5年的培训计划，旨在发展胃肠病学和医学的学术研究生涯。该提案建立在候选人的优势和先前的研究技能的基础上，并利用了约翰·霍普金斯大学充满活力和激励的科学环境。首席研究员将得到Mark Donowitz博士的支持和指导，他是Na/H交换生物学和腹泻疾病方面的领先者。此外，一个由有成就和有地位的科学家组成的小组将定期向首席调查员提供科学和职业建议。其目标是促进国际腹泻研究所发展一个成功的、独立资助的研究项目，并获得必要的经验，以保持与腹泻疾病研究相关的富有成效的科学事业。本研究的目的是鉴定和表征导致钠氢交换蛋白3(NHE3)功能障碍的分子和细胞机制。以前的研究表明，NHE3基因敲除小鼠的主要表型是腹泻，这表明NHE3可能参与了腹泻疾病。单核苷酸多态(SNPs)是许多遗传性疾病的致病因素。我们的初步研究1)在NHE3细胞质尾部发现了三个错义SNPs，即NHE3的调节域；2)证明了这些突变体R474Q、V567M和R799C导致NHE3基础活性降低；3)发现这些SNPs在NHE3中的异常功能似乎是由于R474Q的内在NHE3功能的改变，或者是V567M的异常运输以及NHE3的内在功能和R799C的运输的缺陷；4)证明R474Q和R799C突变体对急性地塞米松刺激没有反应，提示NHE3在餐后对刺激的反应可能存在缺陷；5)在3例先天性钠腹泻(CSD)所致慢性腹泻患者中，2例检测到NHE3基因R474K突变，该突变对地塞米松无效。这些发现表明，功能不良的NHE3基因多态可能是导致慢性腹泻疾病的基因决定因素。然而，已识别的三个NHE3基因多态导致钠吸收减少的分子机制仍不清楚。在这项建议中，我们将确定NHE3蛋白复合体大小是否在基础条件和地塞米松刺激条件下发生多态改变。我们还将研究在基础和地塞米松刺激的条件下，NHE3突变体和NHE3已知调节蛋白的相互作用是否发生变化。最后，我们将使用蛋白质组学方法来鉴定与NHE3基因多态相关的新蛋白。总之，在这三个具体目标中提出的实验将提供对慢性腹泻疾病的遗传基础的洞察，最终目标是建立NHE3和相关蛋白质作为治疗这些腹泻疾病的新的治疗目标。 这笔赠款还将使我能够在慢性腹泻疾病方面建立坚实的知识基础，并具备独立研究人员的资格。为了实现这一目标，授勋期间的正规教育将通过JHU研究生院提供的招生和审计课程获得。与我的培训相关的具体课程有：生物光子学(585.634)和共焦显微镜基础(110.807)、生物信息学(410.639)和色谱学和质谱学在生物医学研究中的作用(330.804)。 公共卫生相关性： 发生在小肠和结肠的钠和氢的吸收主要是通过调节NHE3的活性来实现的。在多态NHE3中存在NHE3活性抑制。然而，导致这些功能低下的NHE3基因多态的机制仍不清楚。目前的研究将为S理解NHE3在慢性腹泻疾病中的作用的遗传成分提供新的见解。

DESCRIPTION (provided by applicant): This proposal describes a 5-year training program for the development of an academic research career in Gastroenterology and Medicine. The proposal builds upon the candidate's strengths and prior research skills, and takes advantage of a vibrant and stimulating scientific environment at The Johns Hopkins University. The principal investigator will be supported and mentored by Dr. Mark Donowitz, a leader in Na/H exchanger biology and diarrheal diseases. In addition, a panel of accomplished and well-established scientists will regularly provide the principal investigator with scientific and career advice. The goal is to foster the PI to develop a successful, independently funded research program and to gain the experience necessary to maintain a productive scientific career relevant to research on diarrheal disease. The objective of the proposed research is to identify and characterize the molecular and cellular mechanisms responsible for the dysfunction of sodium/hydrogen exchanger 3 (NHE3) we identified are present in NHE3 polymorphisms. Previous studies revealed that diarrhea is the major phenotype in NHE3 knockout mice, suggesting a potential involvement of NHE3 in diarrheal diseases. Single nucleotide polymorphisms (SNPs) account for many inherited diseases. Our preliminary studies 1) identified three missense SNPs in the NHE3 cytoplasmic tail, the regulatory domain for NHE3; 2) demonstrated that these mutants, R474Q, V567M and R799C, caused reduced basal NHE3 activity; 3) revealed the abnormal function of these SNPs in NHE3 appears to be due to either a change in intrinsic NHE3 function in R474Q, or abnormal trafficking in V567M and defect in both intrinsic NHE3 function and trafficking in R799C; 4) demonstrated that R474Q and R799C mutants failed to respond to acute dexamethasone stimulation, suggesting a possible defect of NHE3 in response to stimulation postprandially; and 5) identified a mutation of NHE3 at a polymorphic site R474K in 2 of 3 patients with chronic diarrhea due to congenital sodium diarrhea (CSD) and this mutant failed to respond to dexamethasone. These findings suggest that poorly functioning NHE3 polymorphisms may act as genetic determinants that contribute to chronic diarrheal diseases. However, the molecular mechanisms by which the identified three NHE3 polymorphisms cause reduced sodium absorption remain ill defined. In this proposal, we will determine whether NHE3 protein complex size is altered in polymorphisms under basal and dexamethasone-stimulated conditions. We will also investigate whether the interactions of NHE3 mutants and NHE3 known regulatory proteins are changed under basal and dexamethesone- stimulated conditions. Finally, we will use proteomic approaches to identify novel proteins associated with NHE3 polymorphisms. Together, experiments proposed in these three specific aims will provide insight into the genetic basis of chronic diarrheal disease with the ultimate goal of establishing NHE3 and related proteins as novel therapeutic targets for treatment of these diarrheal diseases. This grant will also allow me to develop a solid foundation of knowledge in chronic diarrheal diseases and the qualifications of an independent investigator. To achieve this goal, formal education during the award period will be obtained through enrolling and auditing courses offered by the JHU Graduate School. Specific courses that are relevant to my training, but not limited to, are: BIophotonics (585.634) and Fundamentals of confocal Microscopy (110.807), Bioinformatics (410.639) and Role of chromatography and Mass Spectrometry in Biomedical research (330.804). PUBLIC HEALTH RELEVANCE: Sodium and hydrogen absorption that occurs in small intestine and colon is mainly by regulation of NHE3 activity. Inhibition of NHE3 activity is present in polymorphic NHE3. However, the mechanisms responsible for these poorly functioning NHE3 polymorphisms remain unknown. The current study will provide novel insight s into understanding genetic component accounts for the role of NHE3 in chronic diarrheal diseases.