ASS, NOS2, AND ALCOHOLIC LIVER DISEASE

描述（由申请人提供）：酒精性肝损伤包括显著的线粒体损伤和诱导型一氧化氮合酶（NOS2）的上调，导致活性氧（ROS）和活性氮的过量产生，影响细胞存活。了解NOS2引起病理性一氧化氮（NO）过量产生的分子机制对潜在的治疗干预具有重要意义。我们的实验室结合了尖端的蛋白质组学技术和系统生物学方法来阐明ALD中可能影响NO合成的线粒体蛋白。我们已经确定精氨酸琥珀酸合成酶（ASS）被慢性酒精喂养上调。酒精性肝病（ALD）或肝细胞癌患者也显示肝脏ASS增加，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，可能对通过NOS2合成高产量NO具有限速作用。实际上，对于酒精如何调节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，我们一无所知。我们推测，醇源物种对ASS的上调可能会增加NOS2合成NO的细胞内底物的可用性，从而促进ALD的病理生理。我们将验证这一假设，并沿着以下具体目标研究ASS诱导的机制方面和生物学相关性：1)解剖酒精介导的ASS上调是否在肝细胞NOS2合成NO增加中起作用，我们将解决：a)酒精对ASS的上调是否增加了肝细胞内NOS2合成NO所需的l -精氨酸可用性，b)酒精是否诱导l -精氨酸内流参与NO合成；2)为了确定酒精诱导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对ASS的上调，b)如果ASS经过s -亚硝基化来调节NO的产生，这表明一种新的酒精相关的反馈机制，即NO通过控制底物再生来限制其自身的合成，从而实现高产量的NO合成；3)为了评估酒精介导的ASS在体内诱导NO合成的生物学相关性，野生型小鼠、ASS和注射对照EGFP-AAV8或ASS-EGFP-AAV8的小鼠将被喂食对照组或酒精Lieber-DiCarli饲料，并评估肝功能和氧化应激和亚硝化应激的生化指标，作为ASS对肝损伤的贡献。这些信息可能导致积极的药物靶向，以改善酒精肝毒性。

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.