Role of PHPT1 in oxidative stress-induced epigenetic modifications by ethanol

描述(申请人提供)：酒精性肝病是一种严重的健康问题，在美国每年导致大量与肝病相关的死亡。许多途径改变与乙醇对肝脏的深刻影响有关，包括氧化应激和组蛋白的表观遗传修饰；然而，连接这些过程的分子机制尚不清楚。从最初的基于蛋白质组学的调查中，我们已经确定了一个氧化修饰的肝细胞蛋白靶标，发现在急性高剂量乙醇暴露后，该蛋白被更大程度地氧化。具体地说，这种蛋白质，磷酸组氨酸磷酸酶1(PHPT1)，通过乙醇诱导的氧化应激在一个氨基酸残基上被差异氧化，该氨基酸残基对于底物与其活性位点的结合是重要的。据推测，这种氧化事件将对磷酸组氨酸产生重大影响 PHPT1底物的水平，如三磷酸腺苷-柠檬酸裂解酶(ACL)，将影响乙醇诱导的表观遗传修饰的水平，组蛋白H3乙酰化，这一修饰可能通过ACL产生的乙酰辅酶A池介导。这一假说将通过以下方面得到验证：1)全球范围内乙醇诱导的永生化肝细胞(AML12细胞)和原代肝细胞中氧化修饰的特征，PHPT1氧化修饰的验证和特征，以及肝细胞蛋白质组学对PHPT1活性变化的响应；2)量化乙醇介导的组氨酸水平的变化和ACL的细胞定位，以及这些发现与肝细胞组蛋白H3乙酰化状态和其他ACL介导组蛋白乙酰化位点的相关性。在目标1中，除了传统的生化工具外，还将使用质谱仪来识别和量化PHPT1的各种氧化修饰以及由此产生的活性变化。然后，将使用全球范围的蛋白质表达谱来评估PHPT1活性变化的影响。在目标2中，新的蛋白质组学和基于质谱学的方法将被用来准确地定量酒精暴露后ACL和其他含磷酸组氨酸的蛋白质的磷酸组氨酸水平。ACL和其他蛋白质的定位将通过全球范围的酒精暴露后核质运输的调查，然后是共聚焦显微镜和生化验证来确定。组蛋白H3由ACL产生的乙酰辅酶A介导的乙酰化将通过稳定的同位素代谢示踪进行监测。该项目的结果可能揭示乙醇诱导的氧化应激、细胞代谢和随后的肝细胞表观遗传修饰变化之间的新联系。此外，这项研究的结果可能导致识别出可作为基于表观遗传学的治疗干预的靶向的蛋白质或途径，以治疗因人类过度饮酒而发生的肝脏途径改变。

DESCRIPTION (provided by applicant): Alcoholic liver disease is a serious health condition that results in a significant amount of deaths related to liver disease each year in the U.S. A number of pathway alterations are associated with the profound effects of ethanol on the liver including oxidative stress and epigenetic modification of histones; however, the molecular mechanisms linking these processes are unknown. From an initial proteomics-based survey, we have identified a hepatocellular protein target of oxidative modification that was found to be oxidized to a greater extent after acute, high-dose ethanol exposure. Specifically, this protein, phosphohistidine phosphatase 1 (PHPT1), was differentially oxidized via ethanol-induced oxidative stress at an amino acid residue that is important for substrate binding to its active sit. It is hypothesized that this oxidation event will have a significant impact on the phosphohistidine levels of PHPT1 substrates such as ATP-citrate lyase (ACL) which will then influence the level of the ethanol-induced epigenetic modification, histone H3 acetylation, a modification that is potentially mediated through the acetyl-CoA pool generated by ACL. This hypothesis will be tested by 1) characterization of ethanol-induced oxidative modifications in mouse immortalized hepatocytes (AML12 cells) and primary hepatocytes on a global-scale, validation and characterization of PHPT1 oxidative modifications and assessment of hepatocellular proteomic response to alteration in PHPT1 activity and 2) quantification of ethanol-mediated alterations in the phosphohistidine level and cell localization of ACL and correlation of these findings to hepatocellular histone H3 acetylation status as well as other ACL-mediated histone acetylation sites. In Aim 1, mass spectrometry in addition to conventional biochemical tools will be employed to identify and quantify various oxidative modifications of PHPT1 as well as resultant activity changes. The effect of alteration of PHPT1 activity will then be assessed using global-scale protein expression profiling. In Aim 2, novel proteomics and mass spectrometry-based methods will be used to accurately quantify the phosphohistidine level of ACL and other phosphohistidine-containing proteins after ethanol exposure. Localization of ACL and other proteins will be determined through global-scale investigation of nucleocytoplasmic trafficking after ethanol exposure followed by confocal microscopy and biochemical validation. Acetylation of histone H3 that is mediated by ACL-generated acetyl-CoA will be monitored via stable isotope metabolic tracing. The results from this project could reveal a novel link between ethanol-induced oxidative stress, cellular metabolism and subsequent changes in hepatocellular epigenetic modification. Additionally, the results from this study could lead to the identificationof proteins or pathways that can be targeted for epigenetic-based therapeutic intervention in order to treat hepatic pathway alterations that have occurred through excessive alcohol use in humans.