Ethanol Effects on Hepatic Proteolytic Systems

描述（由申请人提供）：过量的乙醇摄入会导致肝损伤，导致脂肪变性（脂肪肝）、酒精性肝炎、纤维化，最终导致肝硬化。乙醇引起的肝损伤部分是由氧化应激引起的，由于氧化剂的形成增强。一种氧化剂，过氧亚硝酸盐（PN），是乙醇代谢的副产物，通过诱导一氧化氮合酶产生一氧化氮，一氧化氮与超氧化物反应形成PN。过氧亚硝酸盐在蛋白质的酪氨酸残基上形成3-硝基酪氨酸加合物，在蛋白质的半胱氨酸残基上形成s -亚硝基，从而引起蛋白质的硝化作用。这些加合物可以改变蛋白质的生物活性。在细胞的两个主要的蛋白质水解系统——溶酶体和蛋白酶体上，氧化剂的压力也改变了蛋白质的降解。我的研究目标是探索乙醇诱导的氧化和/或乙醇诱导的硝化作用，以及蛋白质的s -亚硝基化如何影响整体活性，以及这些修饰蛋白质对降解的易感性，以及这些改变如何最终影响肝细胞的活力。我们将使用体内和体外方法来验证我们的假设，即乙醇诱导的氧化应激通过氧化剂与蛋白质反应在蛋白质上形成加合物而导致细胞损伤。在Aim 1中，通过使用模型蛋白——鸡蛋溶菌酶，我们将确定过氧亚硝酸盐和/或过氧亚硝酸盐供体对该蛋白的修饰将如何影响分离系统中蛋白酶体对蛋白质功能、结构和降解的易感性。接下来使用肝细胞中的蛋白质环境，其中包括蛋白酶体，我们将确定修饰的效果。在目标2中，我们将采用动物模型来确定乙醇诱导的氧化和硝化如何引起蛋白质的改变，并确定是否有任何修复系统（如通过蛋白酶快速消除改变的蛋白质）能够帮助细胞控制蛋白质损伤。酒精对蛋白质的损害会对细胞造成损害，从而导致酒精性脂肪性肝病（AFLD）。AFLD会降低肝脏为身体排毒的能力，并导致进一步的健康问题。通过学习如何帮助修复或促进细胞中因乙醇诱导氧化而受损的蛋白质的清除，有可能增强肝脏修复，并制定实验和治疗策略，以尽量减少酒精诱导的细胞死亡。

DESCRIPTION (provided by applicant): Excessive ethanol consumption causes liver injury, resulting in steatosis (fatty liver), alcoholic hepatitis, fibrosis, and ultimately cirrhosis. Ethanol-induced liver injury is, in part, caused by oxidative stress, due to enhanced formation of oxidants. One oxidant, peroxynitrite (PN), a by-product of ethanol metabolism, is generated by the induction of nitric oxide synthase, generating nitric oxide, which reacts with superoxide to form PN. Peroxynitrite causes the nitration of proteins by forming 3-nitrotyrosine adducts on their tyrosine residues as well as S-nitroso groups on their cysteine residues. Such adducts can alter the biological activities of proteins. Degradation of proteins is also altered by the stress of oxidants on the lysosome and proteasome, the two principal proteolytic systems of the cell. The goal of my research is to explore how ethanol-induced oxidation and/or ethanol-elicited nitration, and S-nitrosylation of proteins affect the overall activity, and susceptibility of these modified proteins to degradation, and how these alterations may ultimately affect liver cell viability. We will use in vivo and in vitro methods to test our hypothesis that ethanol-induced oxidative stresscauses cell damage through formation of adducts on proteins by reactions of oxidants with proteins. In Aim 1, through the use of a model protein, hen egg lysozyme, we will determine how modification of this protein by peroxynitrite and/or peroxynitrite donors will affect protein function, structure and susceptibility to degradation by the proteasome in an isolated system. Next using a protein milieu from liver cells, which includes the proteasome, we will determine the effects of modification. In Aim 2 we will employ an animal model to determine how ethanol-induced oxidation and nitration causes alteration of proteins and determine what if any repair systems (such as rapid elimination of altered proteins by proteases) are able to help the cell control protein damage. Damage' i to proteins caused by alcohol consumption can cause damage to the cell leading to alcoholic fatty liver disease (AFLD). AFLD can reduce the ability of the liver to detoxify the body and cause further health issues. By learning how to help repair or facilitate removal of damaged proteins in the cell due to ethanol-induced oxidation it may be possible to enhance liver repair, and develop experimental and therapeutic strategies to minimize alcohol induced cell death.