Role of Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism and Toxicity

描述（由申请人提供）：慢性酒精滥用具有多种病理后果，包括对肝脏、大脑和其他器官的损害。这些病理学效应中的一些可归因于乙醇代谢。乙醇通过乙醇脱氢酶（ADH）、乙醇诱导型细胞色素P450（CYP 2 E1）和过氧化氢酶代谢为乙醛。乙醇的许多毒性作用是由于乙醛及其对分子靶点（即蛋白质、脂质和线粒体DNA）的作用。因此，乙醛代谢的作用可能是至关重要的了解组织损伤的发病机制，由酒精消费。乙醛代谢受损与人类乙醛脱氢酶（ALDH）酶的多态性有关;这些可能会影响饮酒行为和发生酒精性肝病的风险。线粒体ALDH 2和细胞溶质ALDH 1A 1酶在较小程度上已知在乙醛解毒中起主要作用。人ALDH 2具有多态性，具有2个不同的等位基因ALDH 2 *1和ALDH 2 *2。ALDH 2 *2纯合个体完全没有ALDH 2活性，而ALDH 2 *1/2*2杂合个体仅表现出正常ALDH活性的30-50%。这种改变的酶活性具有生理学后果。例如，在消耗等量的酒精后，ALDH 2 *2纯合个体的血液乙醛水平比几乎检测不到乙醛的ALDH 2 *1/2*1个体的血液乙醛水平高6-20倍。ALDH 1B 1是一种线粒体酶，与ALDH 2有75%的相同性，也可能参与乙醛代谢。虽然关于ALDH 2和ALDH 1A 1的信息相当多，但关于ALDH 1B 1的信息却很少。我们对R21赠款申请的工作假设是双重的。首先，我们假设线粒体ALDH 1B 1是一种功能酶，通过代谢乙醛和脂质过氧化相关的醛来保护细胞。其次，我们推测ALDH 2 *2蛋白可能通过与其他ALDHs相互作用而抑制它们。在本申请的2年期间，我们建议表征人和小鼠ALDH 1B 1蛋白与以下方面的关系：（a）酶功能（乙醛和脂质过氧化醛的代谢），（B）小鼠和人组织中的表达模式，以及（c）其对甘草酸诱导的毒性和细胞凋亡的保护作用。这些将使用重组ALDH 1B 1蛋白和表达、过表达或缺乏该蛋白的细胞系进行研究。此外，我们将确定ALDH 2 *2蛋白是否使ALDH 2 *1酶和ALDH 1B 1失活。这将通过使用重组蛋白和表达这些蛋白的细胞系研究异源四聚体的形成来实现。

DESCRIPTION (provided by applicant): Chronic alcohol abuse has a variety of pathological consequences including damage to liver, brain and other organs. Some of these pathological effects can be attributed to ethanol metabolism. Ethanol is metabolized to acetaldehyde by the alcohol dehydrogenase enzymes (ADHs), ethanol-inducible cytochrome P450 (CYP2E1) and catalase. Many of the toxic effects of ethanol are due to acetaldehyde and its actions on molecular targets viz. proteins, lipids and mitochondrial DNA. As a result, the role of acetaldehyde metabolism may be crucial for understanding the pathogenesis of tissue injury caused by alcohol consumption. Impaired acetaldehyde metabolism is associated with polymorphisms in the human aldehyde dehydrogenase (ALDH) enzymes; these may influence drinking behavior and risk for developing alcoholic liver disease. Mitochondrial ALDH2 and, to a lesser extent, cytosolic ALDH1A1 enzymes are known to play a major role in acetaldehyde detoxification. Human ALDH2 is polymorphic with 2 distinct alleles, ALDH2*1 and ALDH2*2. ALDH2*2 homozygous individuals are completely devoid of ALDH2 activity, whereas ALDH2*1/2*2 heterozygous individuals exhibit only 30-50% of the normal ALDH activity. This altered enzyme activity has physiological ramifications. For example, after equivalent amounts of alcohol are consumed, blood acetaldehyde levels in ALDH2*2 homozygous individuals are 6-20 times higher than those in ALDH2*1/2*1 individuals in whom acetaldehyde is hardly detectable. ALDH1B1 is a mitochondrial enzyme that is 75% identical to ALDH2 and also may be involved in acetaldehyde metabolism. Although there is a considerable amount of information about ALDH2 and ALDH1A1, very little is known about ALDH1B1. Our working hypothesis for this R21 grant application is two-fold. First, we hypothesize that the mitochondrial ALDH1B1 is a functional enzyme that protects cells by metabolizing acetaldehyde and lipid peroxidation-associated aldehydes. Second, we hypothesize that ALDH2*2 protein may inactivate other ALDHs by interacting with them. For the 2-year period of this application, we propose to characterize the human and mouse ALDH1B1 proteins in relation to: (a) enzymatic function (metabolism of acetaldehyde and lipid peroxidation aldehydes), (b) expression pattern in both mouse and human tissues, and (c) its protective role against aldehyde-induced toxicity and apoptosis. These will be studied using recombinant ALDH1B1 proteins and cell lines expressing, over-expressing or lacking this protein. In addition, we will determine whether ALDH2*2 protein inactivates both ALDH2*1 enzymes and ALDH1B1. This will be accomplished by studying the formation of heterotetramers using recombinant proteins and cell lines expressing these proteins.