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/RNS在酒精诱导的细胞损伤中的作用已得到充分证实，但被ROS/RNS选择性氧化的蛋白质的特征很差。我们推测，某些目标蛋白的半胱氨酸残基被氧化的ROS暴露于酒精，这些修饰的蛋白质可能在酒精介导的线粒体功能障碍和细胞损伤中发挥作用。一个有针对性的蛋白质组学方法，使用生物素-N-马来酰亚胺（生物素-NM）作为一个特定的探针标记氧化蛋白质已经在我们的实验室开发，并用于识别氧化线粒体蛋白在酒精暴露期间和之后。以转染CYP 2 E1的人肝癌HepG 2细胞（E47细胞）为模型，通过CYP 2 E1介导的乙醇代谢产生ROS。在暴露于50和100 mM乙醇4和8小时后，检测到生物素-NM标记的蛋白质水平增加，随后用琼脂糖-链霉亲和素或琼脂糖-抗生物素单克隆抗体纯化氧化蛋白质。通过2-D凝胶电泳解析纯化的氧化蛋白，并从凝胶中切下显示差异丰度的蛋白质点，用胰蛋白酶在凝胶中消化并进行质谱分析。质谱分析表明，许多线粒体蛋白质，包括线粒体乙醛脱氢酶氧化后，酒精暴露。由于酒精暴露后蛋白质修饰（早期观察到）和细胞凋亡（后期观察到）的时间差异，我们认为这些氧化蛋白的失活或功能丧失可能有助于乙醇介导的氧化损伤。 这种方法也被用来确定氧化线粒体和胞质蛋白分离的酒精喂养的小鼠肝脏，以证明我们的检测方法在体内组织样品的实用性。我们未发表的质谱分析结果表明，许多胞质蛋白，参与有效地清除过氧化物（如过氧化物氧还蛋白同工酶），谷胱甘肽的生物合成和利用，和细胞保护酶（热休克蛋白，醛脱氢酶，异柠檬酸脱氢酶等），被氧化后，慢性酒精暴露。在E47细胞中，急性乙醇暴露后，正在研究过氧化物氧还蛋白同工酶的失活及其被sulfiredoxin或sestrins重新激活的机制。我们计划测量单个酶的活性，因为通过某些关键半胱氨酸残基的氧化，这些酶的潜在失活可能为酒精暴露后谷胱甘肽水平降低和过氧化物水平升高的既定结果提供分子基础。我们目前的检测方法具有明显的优势，比其他现有的方法检测氧化蛋白质，也可以应用于另一种类型的培养细胞，亚细胞组分，或动物组织暴露于各种化学物质已知产生ROS/RNS或在病理生理条件下的氧化蛋白质的鉴定。 我们最近报道了c-Jun N-末端蛋白激酶（JNK）和p38蛋白激酶（p38激酶）的持续激活CYP 2 E1的许多底物，如乙醇，对乙酰氨基酚（APAP），4-羟基壬烯醛，四氯化碳和长链脂肪酸以及非CYP 2 E1底物，如曲格列酮，这会导致肝损伤。我们未发表的研究结果还表明，乙醇在培养的E47 HepG 2细胞和其他两种细胞中引起时间和剂量依赖性细胞死亡。乙醇增加JNK和p38激酶的活性，并引起促凋亡Bax易位线粒体中的时间依赖性的方式。JNK和p38激酶的激活似乎在乙醇诱导的细胞死亡中很重要，因为用JNK或p38激酶的各自抑制剂预处理显著降低了每种激酶的活性和乙醇诱导的细胞凋亡的速率。基于这些结果，我们假设促凋亡Bax蛋白在生理静息状态下被其锚定蛋白保留在胞质溶胶中，但Bax可能从其锚定蛋白释放并在暴露于细胞死亡刺激后易位到线粒体，其中大部分激活JNK和p38激酶。到目前为止，我们已经确定了几种细胞溶质蛋白，可以结合Bax，从而可能防止Bax易位到线粒体细胞色素c释放，半胱天冬酶激活，和凋亡。我们目前正在确定Bax和其锚定蛋白之间的结合和解离动力学。我们已经开始研究酒精处理的E47 HepG 2细胞中激活的JNK或p38激酶与Bax或其锚定蛋白之间的直接关系。到目前为止，我们的研究结果似乎代表了新的数据，更好地了解Bax锚定蛋白在细胞凋亡介导的细胞凋亡中的作用。我们的研究结果也反映了正常生理状态下存在的真正的Bax锚定蛋白。这些蛋白质不同于最近在非生理系统下用人工转染的编码特定感兴趣蛋白质的DNA鉴定的那些蛋白质。