ASS, NOS2, AND ALCOHOLIC LIVER DISEASE

描述（由申请人提供）：酒精诱导的肝损伤涉及显著的线粒体损伤和诱导型一氧化氮合酶（NOS 2）的上调，导致影响细胞存活的活性氧（ROS）和活性氮物质的过度产生。了解病理性一氧化氮（NO）过度生产的NOS 2的分子机制是潜在的治疗干预非常相关。我们的实验室已经使用了一个尖端的蛋白质组学技术沿着与系统生物学方法的组合，以阐明线粒体蛋白参与ALD可能会影响NO的合成。我们已经确定了乙酰氨基琥珀酸合成酶（ASS）的上调慢性酒精喂养。酒精性肝病（ALD）或肝细胞癌患者也表现出肝ASS增加，表明ASS和ALD之间存在潜在联系。ASS是一种来自L-瓜氨酸/NO循环的酶，可能对通过NOS 2的高输出NO合成具有限速作用。事实上，酒精如何调节ASS表达以及L-精氨酸“再循环”途径如何影响NO生成和肝损伤还不清楚。我们推测，上调ASS的酒精衍生的物种可能会增加细胞内底物的可用性NO合成的NOS 2有助于ALD的病理生理。我们将检验这一假设，并研究ASS诱导中涉及的机制方面和生物学相关性，沿着以下具体目的：1）为了剖析酒精介导的ASS上调是否在肝细胞中通过NOS 2增加NO合成中起作用，我们将解决：a）酒精对ASS的上调是否增加了细胞内L-精氨酸对肝细胞中NOS 2合成NO的可用性，和B）酒精是否诱导在NO合成中起作用的L-精氨酸内流; 2）为了鉴定酒精诱导ASS的机制，我们将考虑：a）如果增加的ROS作为传感器导致酒精上调ASS，和B）如果ASS经历S-亚硝基化以调节NO产生，则表明新的醇-相关的反馈机制，由此NO通过控制高输出NO合成的底物再生来限制其自身的合成;和3）为了评估酒精介导的ASS诱导体内NO合成的生物学相关性，野生型小鼠，Ass，并且用对照EGFP-AAV 8或ASS-EGFP-AAV 8注射的小鼠将被喂食对照或酒精Lieber-DiCarli饮食，并且肝功能和氧化和亚硝化应激的生物化学测量将被评价为ASS对肝损伤的贡献的读数。这些信息可能导致积极的药理学靶向改善酒精肝毒性。

DESCRIPTION (provided by applicant): Alcohol-induced liver injury involves significant mitochondrial damage and up-regulation of inducible nitric oxide synthase (NOS2) leading to excessive generation of reactive oxygen species (ROS) and reactive nitrogen species affecting cell survival. Understanding the molecular mechanisms of pathological nitric oxide (NO) overproduction by NOS2 is of great relevance for potential therapeutic intervention. Our laboratory has used a combination of a cutting edge proteomics technique along with a Systems Biology approach to elucidate mitochondrial proteins involved in ALD that could impact NO synthesis. We have identified argininosuccinate synthase (ASS) as up-regulated by chronic alcohol feeding. Patients with alcoholic liver disease (ALD) or with hepatocellular carcinoma also showed increased hepatic ASS suggesting a potential link between ASS and ALD. ASS is an enzyme from the L-citrulline/NO cycle which could have a rate-limiting role for high-output NO synthesis via NOS2. Virtually nothing is known on how alcohol modulates ASS expression and how the L-arginine "recycling" pathway may impact NO generation and liver injury. We hypothesize that up-regulation of ASS by alcohol-derived species may increase the availability of intracellular substrate for NO synthesis by NOS2 contributing to the pathophysiology of ALD. We will test this hypothesis and study the mechanistic aspects involved in ASS induction and the biological relevance along the following Specific Aims: 1) To dissect whether the alcohol-mediated up-regulation of ASS plays a role in increased NO synthesis by NOS2 in hepatocytes, we will address: a) whether the up-regulation of ASS by alcohol increases intracellular L-arginine availability for NO synthesis by NOS2 in hepatocytes, and b) whether alcohol induces L-arginine influx playing a role in NO synthesis; 2) To identify the mechanism by which alcohol induces ASS, we will consider: a) if increased ROS act as sensors leading to up-regulation of ASS by alcohol, and b) if ASS undergoes S-nitrosylation to regulate NO production indicating a novel alcohol-related feedback mechanism whereby NO limits its own synthesis by governing substrate regeneration for high-output NO synthesis; and 3) To assess the biological relevance of the alcohol-mediated induction of ASS for NO synthesis in vivo, wild-type mice, Ass, and mice injected with either control EGFP-AAV8 or ASS-EGFP-AAV8 will be fed the control or the alcohol Lieber-DiCarli diets and liver function and biochemical measures of oxidative and nitrosative stress will be evaluated as a read-out for the contribution of ASS to liver injury. Such information could lead to positive pharmacological targeting to ameliorate alcohol hepatotoxicity.