Alcohol Metabolism, Functional Consequence And Signaling

酒精代谢、功能后果和信号传导

• 批准号: 6982862
• 负责人: BYOUNG-JOON SONG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6982862
• 关键词: Bax gene /protein; JUN kinase; apoptosis; biotin; cell line; cytochrome P450; cytotoxicity; detoxification; drug metabolism; ethanol; immunocytochemistry; intracellular transport; kinase inhibitor; laboratory mouse; liver metabolism; mitochondria; oxidative stress; protein degradation; protein quantitation /detection; protein transport; proteomics

Despite well established roles of ROS/RNS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS/RNS are poorly characterized. We hypothesized that certain cysteine residues of the target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in alcohol-mediated mitochondrial dysfunction and cell damage. A targeted proteomics approach using biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized proteins has been developed at our laboratory and used to identify oxidized mitochondrial proteins during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 50 and 100 mM ethanol for 4 and 8 hours, increased levels of biotin-NM-labeled proteins were detected and oxidized proteins subsequently purified with agarose-streptavidin or agarose-monoclonal antibody against biotin. The purified oxidized proteins were resolved by 2-D gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and subjected to mass spectrometry. The mass spectrometric analysis revealed that many mitochondrial proteins including mitochondrial aldehyde dehydrogenases were oxidized after alcohol exposure. Because of the time differences in protein modification (observed at early times) and apoptosis (at later times) after alcohol exposure, we believe that inactivation or functional loss of some of these oxidized proteins may contribute toward ethanol-mediated oxidative injury. This method was also used to identify oxidized mitochondrial and cytosolic proteins isolated from alcohol-fed mouse liver to demonstrate the utility of our detection method for in vivo tissue samples. Our unpublished results of mass spectrometric analysis show that many cytosolic proteins, involved in the efficient removal of peroxides (e.g. peroxiredoxin isozymes), glutathione biosynthesis and utilization, and cellular protective enzymes (heat shock proteins, aldehyde dehydrogenase, isocitrate dehydrogenase, etc), were oxidized after chronic alcohol exposure. The mechanism of inactivation of peroxiredoxin isozymes and its reactivation by sulfiredoxin or sestrins is being investigated in E47 cells after acute ethanol exposure. We plan to measure the activities of individual enzymes, since potential inactivation of these enzymes through oxidation of certain critical cysteine residues may provide the molecular basis for the well-established results of reduced glutathione levels and increased peroxide levels after alcohol exposure. Our current detection method has clear advantages over other existing methods for detecting oxidized proteins and can also be applied to identification of oxidized proteins in another type of cultured cells, subcellular fractions, or animal tissues exposed to various chemicals known to produce ROS/RNS or under pathophysiological conditions. We have recently reported persistent activation of c-Jun N-terminal protein kinase (JNK) and p38 protein kinase (p38 kinase) by many substrates of CYP2E1 such as ethanol, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids as well as a non-CYP2E1 substrate such as troglitazone, which causes liver damage. Our unpublished results also showed that ethanol caused time- and dose-dependent cell death in E47 HepG2 cells and two other types of cells in culture. Ethanol increased the activities of JNK and p38 kinase and caused translocation of proapoptotic Bax to mitochondria in a time-dependent manner. Activation of both JNK and p38 kinase seemed important in ethanol-induced cell death, because pretreatment with a respective inhibitor of JNK or p38 kinase significantly reduced the activity of each kinase and the rate of ethanol-induced apoptosis.. Based on these results, we hypothesized that proapoptotic Bax protein is retained by its anchoring proteins in the cytosol under physiological resting states but Bax may be unleashed from its anchoring proteins and translocate to mitochondria after exposure to cell death stimuli, most of which activate JNK and p38 kinase. We have so far identified several cytosolic proteins that could bind Bax and thus potentially prevent Bax translocation to mitochondria prior to cytochrome c release, caspase activation, and apoptosis. We are currently determining the binding and dissociation kinetics between Bax and its anchoring proteins. We have started investigating the direct relationship between activated JNK or p38 kinase and Bax or its anchoring proteins in alcohol-treated E47 HepG2 cells. Our results so far seem to represent new data for better understanding about the role of Bax anchoring proteins in Bax-mediated apoptosis. Our results also reflect the true Bax anchoring proteins present under normal physiological states. These proteins are different from those recently identified under non-physiological systems with artificially transfected DNA coding for a specific protein of interest.

尽管ROS/RN在酒精诱导的细胞损伤中的作用良好，但被ROS/RN选择性氧化的蛋白质的特征很差。我们假设靶蛋白的某些半胱氨酸残基在酒精暴露后被ROS氧化，这些改良的蛋白可能在酒精介导的线粒体功能障碍和细胞损伤中起作用。使用Biotin-N-甲酰亚胺（Biotin-NM）作为针对氧化蛋白的特定探针的靶向蛋白质组学方法已在我们的实验室中开发出来，并用于鉴定酒精暴露期间和之后的氧化性线粒体蛋白。具有转导的CYP2E1（E47细胞）的人肝癌HEPG2细胞用作通过CYP2E1介导的乙醇代谢产生ROS的模型。暴露于50和100 mM乙醇4和8小时后，检测到生物素-NM标记的蛋白水平升高，并随后用琼脂糖 - 链霉亲和蛋白或琼脂糖单克隆抗体抗生物素纯化的蛋白质氧化蛋白。通过2-D凝胶电泳和蛋白质斑点解析纯化的氧化蛋白，从凝胶中切除显示差异丰度的蛋白质斑点，并用胰蛋白酶消化并进行质谱法。质谱分析表明，许多线粒体蛋白在内，包括线粒体醛醛脱氢酶在酒精暴露后被氧化。由于蛋白质修饰的时间差异（在早期观察到）和酒精暴露后的凋亡（在以后的时间），因此我们认为，这些氧化蛋白中某些的失活或功能丧失可能有助于乙醇介导的氧化损伤。 该方法还用于鉴定从酒精喂养的小鼠肝脏中分离出的氧化的线粒体和胞质蛋白，以证明我们的检测方法用于体内组织样品。我们未发表的质谱分析结果表明，许多细胞质蛋白参与有效去除过氧化物（例如过氧蛋白同工酶），谷胱甘肽生物合成和利用以及细胞保护酶（热休克蛋白，aldehyde dehydeyde脱氢酶）的氧化酶氧化酶，氧化酶氧化酶的氧化酶等氧化酶，等氧化酶异源性酶高，培育酶等。急性乙醇暴露后，正在研究E47细胞中过氧化物氧化毒素同工酶灭活的机理及其硫蛋白或sestrins的再活化的机制。我们计划测量单个酶的活性，因为通过氧化某些关键半胱氨酸残基的氧化可能失活这些酶可能会为良好的谷胱甘肽水平降低和酒精暴露后的过氧化物水平升高提供分子基础。我们当前的检测方法比检测氧化蛋白质的其他现有方法具有明显的优势，还可以应用于在另一种类型的培养细胞，亚细胞级分或暴露于已知产生ROS/RN或病理生理条件下已知的各种化学物质的动物组织中鉴定氧化蛋白。 We have recently reported persistent activation of c-Jun N-terminal protein kinase (JNK) and p38 protein kinase (p38 kinase) by many substrates of CYP2E1 such as ethanol, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids as well as a non-CYP2E1 substrate such as troglitazone，会造成肝脏损害。我们未发表的结果还表明，乙醇在E47 HEPG2细胞中引起了时间和剂量依赖性细胞死亡，在培养中引起了另外两种细胞。乙醇增加了JNK和p38激酶的活性，并以时间依赖性的方式使促凋亡Bax转移到线粒体上。 Activation of both JNK and p38 kinase seemed important in ethanol-induced cell death, because pretreatment with a respective inhibitor of JNK or p38 kinase significantly reduced the activity of each kinase and the rate of ethanol-induced apoptosis.. Based on these results, we hypothesized that proapoptotic Bax protein is retained by its anchoring proteins in the cytosol under physiological resting在暴露于细胞死亡刺激后，状态可能会从其锚定蛋白中释放到线粒体，并转移到线粒体上，其中大多数激活JNK和p38激酶。到目前为止，我们已经鉴定出几种可以结合BAX的胞质蛋白，从而有可能在细胞色素C释放，caspase激活和凋亡之前阻止Bax易位与线粒体。我们目前正在确定Bax及其锚定蛋白之间的结合和解离动力学。我们已经开始研究活化的JNK或p38激酶与BAX之间的直接关系，或其在酒精处理的E47 HEPG2细胞中的锚定蛋白。迄今为止，我们的结果似乎代表了新数据，以更好地了解Bax锚定蛋白在Bax介导的细胞凋亡中的作用。我们的结果还反映了正常生理状态下存在的真正的Bax锚定蛋白。这些蛋白质不同于在非物理学系统下鉴定出的蛋白质，该系统具有人为转染的DNA编码为特定感兴趣的蛋白质的蛋白质。