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经验，目前正在申请耶鲁大学医学院的副研究科学家职位。提交K 01指导奖申请的目的是为候选人提供进一步的培训经验，以作为在肝脏分子生物学和生物化学领域工作的独立研究者。该项目的长期目标是促进我们对肝脏生理学和病理生理学中一氧化氮（NO）信号传导的理解。这对于开发治疗与肝脏疾病相关的并发症的新策略是重要的，因为内皮型NO合酶（eNOS）的异常NO产生在肝硬化门脉高压症中观察到的血管异常中起关键作用。 蛋白质的S-亚硝基化是一个新兴的研究领域，NO介导的生理反应。我们建议调查的S-亚硝基化的机制，专注于eNOS和NO生产的亚细胞定位，并确定在内皮细胞和肝细胞中的S-亚硝基化蛋白质的配置文件，使用蛋白质组学方法。我们的初步研究结果表明，有一个高浓度的NO气体在eNOS定位区域的高尔基体。这使我们假设，在高尔基体上的eNOS附近形成的NO梯度可能有利于蛋白质的S-亚硝基化。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-亚硝基化蛋白，3）检查窦状内皮细胞和肝细胞中的蛋白质S-亚硝基化。 耶鲁大学的消化疾病科和药理学系以及细胞生物学系是开展这些研究的理想选择，因为他们的教师素质，他们作为导师的经验以及耶鲁肝脏中心的核心设施。医学院已承诺在此进一步培训期间为候选人提供保护时间，然后再担任独立调查员。

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.