Impact of ethanol-induced protein nitration on the histone modification code

描述（由申请人提供）：蛋白酪氨酸硝化（PTN）的增加与许多疾病相关，越来越多的证据表明这种修饰可能是一种致病因素，而不仅仅是疾病的一般结果。PTN的产生与小鼠中早期酒精诱导的肝炎有关，其中诱导型一氧化氮合酶（iNOS）和一氧化氮衍生产物过氧亚硝酸盐（一种在细胞中引起PTN的强效剂）已被确定为肝脏这种病理生理学状况发展的主要参与者。在试图确定潜在的肝PTN目标，初步蛋白质组学研究进行了在我们的实验室和结果表明，组蛋白进行选择性硝化后，在原代人肝细胞培养急性乙醇暴露。因此，委员会认为，这个R21项目的目标是通过测试以下假设来检查乙醇诱导的PTN对组蛋白修饰密码的影响：1）蛋白质硝化作为一种新的组蛋白修饰，可能导致随后的基因转录过程的改变; 2）直接调节组蛋白修饰水平的酶的氧化/硝化修饰可能对各种乙醇-诱导的表观遗传标记（例如，组蛋白H3在Lys 9处的乙酰化）。在本提案的具体目标1中，我们将描述组蛋白H2 A、H2 B、H3和H4亚型中乙醇诱导的酪氨酸硝化过程。在具体目标2中，我们将表征乙醇暴露后的核硝基蛋白质组，以鉴定与改变组蛋白修饰密码（例如，HAT或HDAC酶）相关的硝化蛋白质。此外，我们的体外研究结果将在慢性乙醇暴露的体内动物模型中得到验证。该项目的结果将有助于阐明氧化/硝化应激在乙醇诱导的组蛋白修饰密码改变中的作用，并为PTN与疾病病因的联系提供进一步的证据。核硝化的目标和酶的过程，影响PTN水平的鉴定可能会提供新的信息，在乙醇诱导的组织损伤，PTN与损伤发作或严重程度的药物治疗的潜在目标。

DESCRIPTION (provided by applicant): Increase in protein tyrosine nitration (PTN) has been associated with numerous diseases and mounting evidence suggests this modification could be a causative factor rather than just a general outcome of disease. The generation of PTN has been implicated in early alcohol-induced hepatitis in mice where inducible nitric oxide synthase (iNOS) and the nitric oxide-derived product, peroxynitrite (a potent agent that causes PTN in the cell), have been identified as major players in the development of this pathophysiological condition of the liver. In an attempt to identify potential hepatic PTN targets, preliminary proteomic studies were performed in our laboratory and the results suggest that histone proteins undergo selective nitration in primary human hepatocyte culture after acute ethanol exposure. Accordingly, the goal of this R21 project is to examine the impact of ethanol-induced PTN on the histone modification code by testing the hypotheses that 1) protein nitration serves as a novel histone modification that could result in subsequent alteration of gene transcription processes and 2) oxidative/nitrative modifications of enzymes that directly modulate histone modification levels could have an impact on various ethanol-induced epigenetic markers (for example, acetylation of histone H3 at Lys9). In Specific Aim 1 of this proposal, we will characterize the process of ethanol-induced tyrosine nitration in histone H2A, H2B, H3, and H4 isoforms. In Specific Aim 2, we will characterize the nuclear nitroproteome after ethanol exposure to identify nitrated proteins associated with altering the histone modification code (for example, HAT or HDAC enzymes). Additionally, our findings from in vitro studies will be validated in an in vivo animal model of chronic ethanol exposure. The results from this project will help shed light on the role of oxidative/nitrative stress in ethanol-induced alterations of the histone modification code and provide further evidence to the link of PTN and disease causation. Identification of nuclear nitration targets and enzyme processes that affect PTN levels will likely provide new information on potential targets for drug therapy in ethanol-induced tissue injury where PTN is linked to injury onset or severity.