Alcohol Metabolism, Functional Consequence And Signaling

氧化应激是乙醇介导的细胞和组织损伤的主要因素之一。在酒精暴露的细胞中，大多数活性氧和活性氮（ROS/RNS）是通过直接抑制线粒体呼吸链和诱导或激活乙醇诱导的细胞色素P450 2E1 （CYP2E1）、诱导型一氧化氮合酶和nadph氧化酶产生的。尽管ROS/RNS在酒精诱导的细胞功能障碍和损伤中的作用已经确立，但对于哪些蛋白质被升高的ROS/RNS氧化修饰以及它们的功能是否被改变，我们知之甚少。为了解决这些问题，我们最近开发了一种使用生物素- n-马来酰亚胺（生物素- nm）作为特异性探针的敏感方法，以阳性识别酒精暴露的肝癌细胞或动物组织中的氧化蛋白。在本财政年度，我们已经应用这种靶向蛋白质组学方法来鉴定暴露于酒精的小鼠或大鼠肝脏的细胞质和线粒体部分中的氧化蛋白质。生物素- nm标记的氧化蛋白用链亲和素-琼脂糖珠纯化，用二维凝胶电泳分离。与成对喂养的对照组相比，在酒精喂养的小鼠或大鼠肝脏中显示出不同丰度的蛋白质点，从二维凝胶中切除，用胰蛋白酶在凝胶中消化并进行质谱分析。质谱数据显示，在酒精喂养的小鼠肝脏中，许多参与伴侣活性、抗氧化防御、包括转甲基化途径在内的中间代谢和细胞骨架蛋白的细胞质蛋白被氧化。我们目前的研究结果可能解释了一些酶失活的潜在机制，这些酶的失活导致抗氧化剂（如s -腺苷蛋氨酸和谷胱甘肽）水平降低，同时在酒精暴露的动物组织和酗酒的人类中观察到同型半胱氨酸水平升高。过氧化氢酶和过氧化物酶等抗氧化酶的氧化失活，通过亚亚磺酸形成其活性位点Cys，可能有助于在含有cyp2e1的E47 HepG2肝癌细胞和酒精暴露动物中观察到过氧化物水平升高。在暴露于酒精的大鼠肝脏中，许多参与线粒体电子转移、能量产生、脂肪酸β氧化和伴侣活性的线粒体蛋白也被氧化修饰。例如，与成对喂养的对照大鼠相比，酒精喂养的大鼠线粒体中参与脂肪酸β -氧化的3-酮酰基辅酶a硫酶被氧化修饰并失活。3-酮酰基辅酶a硫酶和线粒体β -氧化途径中的其他三种酶的抑制与生化和组织学方法确定的甘油三酯积累增加一致。我们对3-硝基Tyr抗体的免疫印迹分析还显示，在酒精暴露的动物中，线粒体ATP合成酶（复合物V）的酪氨酸（Tyr）残基被硝化，导致其抑制并随后减少ATP的产生。质谱分析进一步证实了ATP合酶活性位点Tyr残基的硝化作用。此外，参与乙醛和有毒脂质过氧化物代谢的线粒体醛脱氢酶（ALDH2）被氧化，导致其活性受到抑制。为了直接证明酒精喂养大鼠ALDH2的氧化修饰，我们从配对喂养的对照大鼠肝脏或酒精喂养的大鼠中免疫纯化线粒体ALDH2蛋白。两种样品均鉴定出一种免疫纯化的ALDH2蛋白。然而，抗s -亚硝基化- cys抗体检测到的s -亚硝基化蛋白仅在酒精处理的大鼠中检测到，而在对照组动物中没有检测到。添加DTT等还原剂可使酒精喂养大鼠s -亚硝基化- cys条带消失。这些数据有力地表明，ALDH2的活性位点Cys302在酒精喂养的大鼠中发生了可逆的s -亚硝基化。在乙醇暴露的肝癌细胞系中也观察到类似的氧化修饰结果。这些关于ALDH2 s -亚硝基化的结果也可以解释ALDH2活性水平降低的潜在机制，这种降低通常在酗酒者和暴露于各种有毒化学物质后观察到。总的来说，基于目前的结果，许多氧化线粒体蛋白的失活可能导致酒精诱导的线粒体功能障碍和对乙醇介导的氧化组织损伤的敏感性增加。

Oxidative stress is one of the major contributing factors in ethanol (alcohol)-mediated cell and tissue damage. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells is being produced through direct inhibition of the mitochondrial respiratory chain and induction or activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase, and NADPH-oxidase. Despite the well-established roles of ROS/RNS in alcohol-induced cellular dysfunction and injury, it is poorly understood which proteins are oxidatively-modified by elevated ROS/RNS and whether their functions are altered. To address these questions, we recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to positively identify oxidized proteins in alcohol-exposed hepatoma cells or animal tissues. During this fiscal year, we have applied this targeted proteomics approach to identify oxidized proteins in cytosolic and mitochondrial fractions of alcohol-exposed mouse or rat livers. The biotin-NM labeled oxidized proteins were purified with streptavidin-agarose beads and resolved by 2-D gel electrophoresis. Protein spots, that displayed differential abundances in alcohol-fed mouse or rat livers compared to those in the pair-fed controls, were excised from the 2-D gels, in-gel digested with trypsin and subjected to mass spectrometry. Mass spectrometric data revealed that many cytosolic proteins involved in chaperone activities, anti-oxidant defense, intermediary metabolism including the transmethylation pathway and cytoskeletal proteins were oxidized in alcohol-fed mouse livers. Our current results are likely to explain the underlying mechanisms for the inactivation of some of these enzymes leading to the reduced levels of antioxidants such as S-adenosylmethionine and glutathione with the increased levels of homocysteine observed in alcohol-exposed animal tissues and alcoholic human subjects. Oxidative inactivation of anti-oxidant enzymes such as catalase and peroxiredoxin through sulfinic/sulfonic acid formation of its active site Cys may contribute to the elevated levels of peroxides observed in CYP2E1-containing E47 HepG2 hepatoma cells and alcohol-exposed animals. Many mitochondrial proteins involved in mitochondrial electron transfer, energy production, beta-oxidation of fatty acids, and chaperone activities were also oxidatively-modified in alcohol-exposed rat livers. For instance, mitochondrial 3-ketoacylCoA thiolase involved in the beta-oxidation of fatty acids was oxidatively-modified and inactivated in alcohol-fed rats, compared to that in the pair-fed control rats. Inhibition of 3-ketoacylCoA thiolase and three other enzymes in the mitochondrial beta-oxidation pathway is consistent with increased accumulation of triglycerides determined by biochemical and histological methods. Our immunoblot analysis with the antibody against 3-nitroTyr also showed that tyrosine (Tyr) residues of mitochondrial ATP synthase (complex V) were nitrated in alcohol-exposed animals, resulting in its inhibition and subsequently reduced ATP production. Nitration of the active site Tyr residues of ATP synthase was further confirmed by mass spectral analysis. Furthermore, mitochondrial aldehyde dehydrogenase (ALDH2) involved in the metabolism of acetaldehyde and toxic lipid peroxides were oxidized, leading to inhibition of its activity. To directly demonstrate oxidative modification of ALDH2 in alcohol-fed rats, we immunopurified the mitochondrial ALDH2 protein from pair-fed control rat livers or alcohol-fed rats. One immunopurified ALDH2 protein was identified for both samples. However, S-nitrosylated protein, detected with the anti-S-nitrosylated-Cys antibody, was observed only in the alcohol-treated rats but not in control animals. Addition of a reducing agent such as DTT caused disappearance of the S-nitrosylated-Cys band in alcohol-fed rats. These data strongly suggest that the active site Cys302 of ALDH2 was reversibly S-nitrosylated in alcohol-fed rats. Similar results about oxidative modification of ALDH2 were also observed in ethanol-exposed hepatoma cell lines. These results on S-nitrosylation of ALDH2 may also explain the underlying mechanism for the reduced levels of ALDH2 activity often observed in alcoholic individuals and after exposure to various toxic chemicals. Collectively, based on the current results, inactivation of many of these oxidized mitochondrial proteins is likely to contribute to alcohol-induced mitochondrial dysfunction and increased sensitivity toward ethanol-mediated oxidative tissue injury. In addition, we have investigated the signaling mechanism during cellular damage caused by many toxic compounds. Our earlier results showed selective and persistent activation of c-Jun N-terminal protein kinase (JNK) by many toxic substrates of CYP2E1 such as acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids. In contrast, ethanol, another substrate of CYP2E1, and non-CYP2E1 substrates such as troglitazone, hydrogen peroxide, etoposide, and staurosporine (STS) activated JNK and p38 protein kinase (p38 kinase) simultaneously. Our results also showed that all these compounds caused translocation of proapoptotic Bax from cytoplasm to mitochondria in a time-dependent manner. Since the mechanism of Bax activation and its mitochondrial translocation prior to apoptosis was unknown, we further investigated the mechanism for Bax activation after cells were treated with various cell death stimulants. Our results showed that stress-activated protein kinases stimulated after exposure to various toxic compounds directly phosphorylated Bax before it was translocated to mitochondria to initiate actual cell death observed at later time points. Phosphorylation of Bax was demonstrated by the shift of the pI value of 4.0 (phosphorylated Bax) from pI 5.1 (non-phosphorylated Bax) on 2-D gels and confirmed by metabolic labeling with 32P-inorganic phosphate. Important roles of JNK and/or p38 kinase in mitochondrial translocation of Bax and apoptosis were demonstrated by using a specific inhibitor of JNK or p38 kinase and a specific siRNA to MAPKK4, the upstream kinases of JNK and/or p38 kinase. Pretreatment with each agent significantly reduced the activity of each kinase and the rates of mitochondrial translocation of Bax and apoptosis. To determine the phosphorylated amino acid, several Bax mutants (Ser87Ala, Thr167Ala and others) were prepared, based on the fact that JNK and p38 kinase are proline-directed protein kinases. Critical roles of phosphorylation of Bax in its mitochondrial translocation were further confirmed by confocal microscopy of various Bax mutants and transfection into Bax/Bak double knockout mouse embryonic fibroblast cells. Our confocal microscopic results with various Bax mutants clearly show that Thr167 is a critical amino acid which is phosphorylated by stress-activated protein kinases. Taken together, our results suggest that JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation, which might disrupt the previous interaction between the N-terminal domain and the C-terminal transmembrane domain of Bax. Exposure of the C-terminal transmembrane domain is likely to lead to mitochondrial translocation of Bax to initiate mitochondria-dependent apoptosis. Our results demonstrate for the first time that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis. Taken together, our results are likely to explain the underlying mechanisms for the positive relationship between activation of JNK or p38 kinase and apoptosis caused by many distinct cell death stimulants or conditions, as previously reported by many scientists.