NO production and S-nitrosylation in liver vasculature

描述(由申请人提供)： 该候选人有三年的博士后工作经验，目前正在被推荐为耶鲁大学医学院的副研究科学家。本次申请K01导师奖的目的是为候选人提供进一步的培训经验，使其成为一名在肝脏分子生物学和生物化学领域工作的独立研究员。该项目的长期目标是促进我们对一氧化氮(NO)信号在肝脏生理学和病理生理学中的理解。这对于开发治疗与肝脏疾病相关的并发症的新策略非常重要，因为内皮型一氧化氮合酶(ENOS)的异常NO产生在肝硬变门脉高压症的血管异常中起着关键作用。 蛋白质的S亚硝化是研究NO介导的生理反应的一个新兴领域。我们建议研究S亚硝化的机制，重点是eNOS和NO产生的亚细胞定位，并用蛋白质组学方法鉴定S亚硝化蛋白在血管内皮细胞和肝细胞中的分布。我们的初步结果表明，在高尔基山脉的eNOS局域区存在高浓度的NO气体。这使得我们推测，高尔基体上eNOS附近形成的NO梯度可能有利于蛋白质的S亚硝化。候选人建议1)阐明eNOS是否在活细胞中产生NO梯度；2)使用蛋白质组学方法鉴定高尔基复合体中/上的S亚硝化蛋白；3)检测肝窦内皮细胞和肝细胞中的S亚硝化蛋白。 耶鲁大学消化病学部、药理学系以及细胞生物学系是进行这些研究的理想选择，因为他们的教员质量、他们作为导师的经验以及耶鲁大学肝脏中心的核心设施。医学院承诺在担任独立调查员之前，在进一步培训期间为候选人提供受保护的时间。

DESCRIPTION (provided by applicant): The candidate has a three-year post-doctoral experience and is currently being proposed for an Associate Research Scientist appointment at the School of Medicine of Yale University. This application for a mentored K01 award is submitted with the goal of providing the candidate with the further training experience to function as an independent investigator working in the field of molecular biology and biochemistry of the liver. The project's long-term objective is to advance our understanding of nitric oxide (NO) signaling in liver physiology and pathophysiology. This is important for developing novel strategies to treat complications associated with liver diseases, since abnormal NO production by endothelial NO synthase (eNOS) plays a key role in vascular abnormalities observed in portal hypertension in liver cirrhosis. S-nitrosylation of proteins is an emerging area of investigation for NO-mediated physiological responses. We propose to investigate the mechanisms of S-nitrosylation, focusing on subcellular localization of eNOS and NO production, and identify the profiles of S-nitrosylated proteins in endothelial cells and liver cells using a proteomic approach. Our preliminary results suggest that there is a high concentration of NO gas at the eNOS localized area on the Golgi. This leads us to hypothesize that a gradient of NO formed in proximity to eNOS on the Golgi may be favorable to S-nitrosylation of proteins. The candidate proposes to 1) Elucidate if eNOS generates a gradient of NO in living cells, 2) Identify S-nitrosylated proteins in/on the Golgi complex using a proteomic approach, and 3) Examine protein S-nitrosylation in sinusoidal endothelial cells and hepatocytes. The Section of Digestive Diseases and the Department of Pharmacology as well as the Department of Cell Biology at Yale University are ideal for carrying out these studies because of the quality of their faculty, their experience as mentors, and the core facility at the Yale Liver Center. The School of Medicine has pledged protected time for the candidate during this further training period prior to functioning as an independent investigator.