Methionine Adenosyltransferase Alpha1 in Alcoholic Liver Disease

摘要 蛋氨酸腺苷转移酶(MAT)是催化S形成的一种必不可少的细胞酶。 腺苷蛋氨酸(SAME)是主要的生物甲基供体，在肝脏中是关键抗氧化剂的前体 谷胱甘肽(GSH)在哺乳动物中，两个不同的基因MAT1A和MAT2A编码两个同源的垫子 催化亚基分别为α1和α2。Mat1a主要在正常肝脏中表达。大多数患者 在慢性肝病患者中，MAT1A编码同工酶的表达和活性降低。我们发现 在酒精性肝炎患者中，Mat1a的表达和肝脏中的相同水平都降低了。而垫子 同工酶是公认的催化胞质相同生物合成的酶，我们最近的工作有 揭示了它们的功能的高度新奇的方面。除了胞质和细胞核，我们的初步数据 提示MATα1也存在于线粒体基质中，以调节线粒体功能。这事很重要 我们发现肝细胞缺乏线粒体相同的转运蛋白SLC25A26。使用免疫沉淀(IP) 随后通过质谱分析，我们鉴定了许多线粒体蛋白和细胞色素P450 2E1 (细胞色素P421)作为MATα1相互作用蛋白。我们的初步数据显示MATα1负性调节 CYP2E1主要通过甲基化在蛋白质水平表达，目前尚未见报道。重要的是 MATα1线粒体靶向性在小鼠和人酒精性肝病中受损，我们建议 导致这种损害的两种新机制可能是协同作用的。目前的提案考验着中央银行 假设MATα1在肝细胞的线粒体基质中提供相同的来源 肌萎缩侧索硬化症患者MAT-α-1线粒体靶向性受损在肌萎缩侧索硬化症的发病机制中起着关键作用。这个 推论认为MATα1部分通过抑制线粒体功能维持肝细胞线粒体功能。 细胞色素P4502E 1的表达。在这里，我们在三个具体目标上跟进这些新发现：1)研究MATα1如何 靶向线粒体及其在ALD中靶向受损的原因。我们将阐明MATα1是如何针对 肌萎缩侧索硬化症的线粒体及其靶向受损机制。我们将测试新的假设，即这是 由于1)MATα1苏甲基化增加和2)MATα1与PIN1(一种肽-脯氨基顺式... 识别特定磷酸化基序的反式异构酶)，2)检查MATα1如何调节细胞色素P450 蛋白质表达。我们将在分子水平上研究matα1如何调节cyp2e1蛋白的稳定性。 并确定参与这一过程的CYP2E1残基和相互作用蛋白，3)检测 线粒体MATα-1和MAT-α-1调节肌萎缩侧索硬化症的细胞色素P450_2我们将测试新的假设，即 阻止MATα1苏甲基化或与PIN1相互作用将保护线粒体MATα1减少 ALD中的含量和损伤。我们还将研究MATα1是否通过甲基化来预防ALD 位于R379的细胞色素2E1R379，以促进其降解。如果成功完成，这些研究将改变现有的 MAT-α-1生物学、细胞色素P450-1调控的范例，并可能发现ALD的可用药靶点。

ABSTRACT Methionine adenosyltransferase (MAT) is an essential cellular enzyme that catalyzes the formation of S- adenosylmethionine (SAMe), the principal biological methyl donor and in liver, precursor of the key antioxidant glutathione (GSH). In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, α1 and α2, respectively. MAT1A is primarily expressed in normal liver. Majority of patients with chronic liver disease have decreased expression and activity of MAT1A-encoded isoenzymes. We found MAT1A expression and hepatic SAMe levels are reduced in alcoholic hepatitis patients. While MAT isoenzymes are widely acknowledged for catalyzing cytosolic SAMe biosynthesis, our recent works have uncovered highly novel aspects of their functions. In addition to cytosol and nucleus, our preliminary data indicate MATα1 is also present in the mitochondrial matrix to regulate mitochondrial function. This is important as we found hepatocytes lack the mitochondrial SAMe transporter SLC25A26. Using immunoprecipitation (IP) followed by mass spectrometry, we have identified many mitochondrial proteins and cytochrome P450 2E1 (CYP2E1) as MATα1-interacting proteins. Our preliminary data show that MATα1 negatively regulates CYP2E1 expression mainly at the protein level via methylation, which has not been reported. Importantly, MATα1 mitochondrial targeting is impaired in murine and human alcoholic liver disease (ALD) and we propose two novel mechanisms that may cooperate in causing this impairment. The current proposal tests the central hypothesis that MATα1 provides the SAMe source within the hepatocyte's mitochondrial matrix and impairment in MATα1 mitochondrial targeting in ALD plays a key role in the pathogenesis of ALD. The corollary hypothesis is that MATα1 maintains hepatocyte mitochondrial function in part by suppressing CYP2E1 expression. Here we follow up these novel findings in three specific aims: 1) examine how MATα1 targets the mitochondria and why its targeting is impaired in ALD. We will elucidate how MATα1 targets the mitochondria and mechanisms of its impaired targeting in ALD. We will test the novel hypothesis that this is due to 1) increased MATα1 sumoylation and 2) increased interaction of MATα1 with PIN1 (a peptidyl-prolyl cis- trans isomerase that recognizes a specific phosphorylated motif), 2) examine how MATα1 regulates CYP2E1 protein expression. We will examine how MATα1 regulates CYP2E1 protein stability at the molecular level and identify CYP2E1 residues and interacting proteins that participate in this process, 3) examine the role of mitochondrial MATα1 and MATα1-regulated CYP2E1 in ALD. We will test the novel hypothesis that preventing MATα1 sumoylation or interaction with PIN1 will protect against reduced mitochondrial MATα1 content and injury in ALD. We will also examine whether MATα1 will protect against ALD by methylating CYP2E1 at R379 to enhance its degradation. If successfully accomplished, these studies will change existing paradigms on MATα1 biology, CYP2E1 regulation and may uncover druggable targets for ALD.