ASS, NOS2, AND ALCOHOLIC LIVER DISEASE

描述(申请人提供)：酒精性肝损伤涉及严重的线粒体损伤和诱导型一氧化氮合酶(NOS2)的上调，导致过量产生影响细胞存活的活性氧(ROS)和活性氮(ROS)。了解NOS2导致病理性一氧化氮(NO)过度产生的分子机制对于潜在的治疗干预具有重要意义。我们的实验室结合使用了尖端的蛋白质组学技术和系统生物学方法来阐明参与ALD的线粒体蛋白可能影响NO的合成。我们发现长期饮酒可上调精氨酸琥珀酸合成酶(ASS)的表达。酒精性肝病(ALD)或肝细胞癌患者也显示肝ASS升高，提示ASS与ALD之间存在潜在联系。ASS是一种来自L-瓜氨酸/NO循环的酶，它可能通过NOS2对高产的NO合成起到限速作用。酒精如何调节ASS的表达，以及L-精氨酸“循环”途径如何影响NO的生成和肝脏损伤，目前几乎一无所知。我们推测，酒精衍生的物种上调ASS可能增加了NOS2合成NO的细胞内底物的可用性，从而参与了ALD的病理生理学。我们将验证这一假说，并沿着以下特定目的研究ASS诱导的机制及其生物学意义：1)为了剖析酒精介导的ASS上调是否在NOS2增加肝细胞合成NO中起作用，我们将探讨：a)酒精上调ASS是否增加细胞内L精氨酸对NOS2合成NO的利用率，以及b)酒精是否诱导L精氨酸内流在NO合成中发挥作用；2)为了确定酒精诱导ASS的机制，我们将考虑：a)增加的ROS是否作为感受器导致酒精上调ASS，以及b)ASS是否经历了S亚硝化来调节NO的产生，这表明了一种新的酒精相关的反馈机制，即NO通过调节底物再生来限制自己的合成，从而促进NO的高产量合成；3)为了评估酒精诱导ASS在体内合成NO的生物学相关性，野生型小鼠、ASS和注射对照EGFP-AAV8或ASS-EGFP-AAV8的小鼠将喂饲对照或酒精Lieber-DiCarli饲料和肝功能，并将氧化和亚硝化应激的生化指标作为ASS对肝脏损伤的贡献的读数。这些信息可能导致积极的药理靶向，以减轻酒精的肝毒性。

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.