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

PHPT1 在乙醇氧化应激诱导的表观遗传修饰中的作用

• 批准号: 8445959
• 负责人: Stanley M Stevens
• 金额: $ 17.41万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445959
• 关键词: 3-nitrotyrosine; ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Acetylation; Active Sites; Acute; Affect; Alcohol consumption; Alcoholic Liver Diseases; Amino Acids; Antibodies; Area; Back; Binding; Biochemical; Biological Assay; Cell Culture Techniques; Cells; Cessation of life; Code; Confocal Microscopy; Data; Dose; Drug Targeting; Edetic Acid; Epigenetic Process; Ethanol; Eukaryotic Cell; Event; Exposure to; Gene Expression; Goals; Health; Hepatic; Hepatocyte; Histidine; Histone Acetylation; Histone H3; Histones; Human; Hydrogen Peroxide; Immunoprecipitation; Incubated; Investigation; Label; Lead; Link; Liver; Liver diseases; Mass Spectrum Analysis; Measures; Mediating; Metabolic; Metabolism; Methionine; Methods; Modification; Molecular; Molecular Profiling; Molecular Target; Monitor; Mus; Mutate; Nuclear; Oxidative Stress; Pathway interactions; Peroxonitrite; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Play; Prevention; Process; Proteins; Proteomics; Reactive Oxygen Species; Reagent; Recombinants; Research; Resolution; Risk Factors; Role; Signal Transduction; Site; Small Interfering RNA; Stable Isotope Labeling; Surveys; Techniques; Testing; Therapeutic Intervention; Transcriptional Activation; Validation; Western Blotting; alcohol effect; alcohol exposure; base; chronic alcohol ingestion; histone modification; insight; methionine sulfoxide; mortality; mutant; nitration; novel; oxidation; phosphohistidine; protein expression; public health relevance; response; stable isotope; tool; trafficking

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.

描述（由申请人提供）：酒精性肝病是一种严重的健康状况，每年在美国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。从最初的蛋白质组学为基础的调查，我们已经确定了一个肝细胞的蛋白质目标的氧化修饰，被发现被氧化到更大程度后，急性，高剂量的乙醇暴露。具体而言，这种蛋白质，磷酸组氨酸磷酸酶1（PHPT 1），通过乙醇诱导的氧化应激在一个氨基酸残基，这是重要的底物结合到其活性位点的差异氧化。假设该氧化事件将对磷酸组氨酸 PHPT 1底物如ATP-柠檬酸裂解酶（ACL）的水平，其然后将影响乙醇诱导的表观遗传修饰（组蛋白H3乙酰化）的水平，所述修饰可能通过ACL产生的乙酰辅酶A池介导。这一假设将通过1）小鼠永生化肝细胞中乙醇诱导的氧化修饰的表征来检验（AML 12细胞）和原代肝细胞在全球范围内，PHPT 1氧化修饰的验证和表征以及对PHPT 1活性改变的肝细胞蛋白质组学应答的评估，以及2）乙醇-ACL的磷酸组氨酸水平和细胞定位介导的改变，以及这些发现与肝细胞组蛋白H3乙酰化状态以及其他ACL介导的组蛋白乙酰化位点的相关性。在目标1中，除了常规的生物化学工具之外，还将采用质谱法来鉴定和定量PHPT 1的各种氧化修饰以及由此产生的活性变化。然后将使用全球规模的蛋白质表达谱来评估PHPT 1活性改变的影响。在目标2中，将使用基于蛋白质组学和质谱的新方法来准确定量乙醇暴露后ACL和其他含磷酸组氨酸蛋白质的磷酸组氨酸水平。ACL和其他蛋白质的定位将通过乙醇暴露后核质运输的全球规模研究，然后通过共聚焦显微镜和生化验证来确定。将通过稳定同位素代谢示踪监测由ACL生成的乙酰辅酶A介导的组蛋白H3乙酰化。该项目的结果可以揭示乙醇诱导的氧化应激，细胞代谢和肝细胞表观遗传修饰的后续变化之间的新联系。此外，这项研究的结果可能导致识别蛋白质或途径，这些蛋白质或途径可以用于基于表观遗传学的治疗干预，以治疗人类过度饮酒引起的肝脏途径改变。