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Biochemical, Structural and Polymorphic Characterization of Human ALDH1B1

人 ALDH1B1 的生化、结构和多态性表征

基本信息

批准号：
8712303
负责人：
Brian Christopher Jackson
金额：
$ 2.08万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8712303
关键词：
Acetaldehyde Acetates Affect Alcohol consumption Alcohol dehydrogenase Alcoholism Alcohols Asians Baculoviruses Behavior Biochemical Biological Assay Biological Markers Cancer Biology Caucasians Caucasoid Race Cell Line Cell physiology Cells Code Coupled Cytochrome P-450 CYP2E1 Data Disease Enzyme Kinetics Enzymes Epidemiologic Studies Ethanol Ethanol Metabolism Face Family Flushing Frequencies Functional disorder Gas Chromatography Genetic Polymorphism Headache Health Heterozygote Homozygote Human Human Cell Line Hypersensitivity Individual Insecta Intestines Laboratories Learning Life Liquid Chromatography Literature Liver Malignant Neoplasms Mass Spectrum Analysis Metabolic Metabolism Mitochondria Modification Molecular Molecular Models Names Nausea Nitroglycerin Pathway interactions Phosphoric Monoester Hydrolases Phosphotransferases Physiological Play Population Post-Translational Protein Processing Predisposition Proliferating Property Proteins Public Health Reaction Recombinants Reporting Research Retinaldehyde Role Signal Transduction Silent Mutation Site-Directed Mutagenesis Small Interfering RNA Spectrometry Stem cells Substrate Specificity Symptoms Syndrome System Tachycardia Techniques Testing Tissues Toxic effect Transgenic Organisms Treatment Efficacy Tretinoin Variant adduct alcohol sensitivity aldehyde dehydrogenases catalase drug metabolism enzyme activity fast protein liquid chromatography genetic variant high throughput screening improved inhibitor/antagonist insight liquid chromatography mass spectrometry metabolomics molecular modeling novel oxidation research study

项目摘要

DESCRIPTION (provided by applicant): One of the primary toxic metabolites of alcohol (ethanol) metabolism, acetaldehyde causes protein adducts and cellular dysfunction at the molecular level, and acetaldehyde accumulation causes facial flushing, tachycardia, headache and nausea, and alcohol avoidance at the physiological level. Acetaldehyde is primarily cleared by aldehyde dehydrogenases, namely ALDH2, ALDH1B1, and ALDH1A1. ALDH2 has been well characterized and a well-known inactivating polymorphism, ALDH2*2 has been found to affect approximately 50% of some Asian populations. ALDH1B1 has been partially characterized more recently, but there is much to learn. Recent epidemiological studies have identified polymorphisms of ALDH1B1 which are associated with alcohol avoidance and alcohol hypersensitivity reactions; this is the first such variant in acetaldehyde clearance reported in Caucasian populations. In addition to the alcohol metabolism pathway, we also hypothesize that ALDH1B1 may play a role in the bioactivation of nitroglycerin and retinoic acid; the latter could have significant implications in cancer biology. Given these roles, we propose a comprehensive characterization of ALDH1B1 using a broad range of techniques including molecular modeling, enzyme kinetics, metabolomics, high throughput screening of inhibitors and activators, and site directed mutagenesis to better understand multiple dynamic aspects of this potentially critical enzyme. First a more complete spectrum of substrates for ALDH1B1 will be tested including those which we hypothesize from previous evidence (nitroglycerin and retinaldehyde), and those which are determined by unbiased metabolomic approaches. To better allow manipulation of ALDH1B1 levels in experimental systems, an inhibitor and activator profile will be determined, first using known ALDH inhibitors, and later using high throughput screening techniques. It is likely that ALDH1B1 is phosphorylated, and we will use spectrometry combined with kinases and phosphatases to determine if ALDH1B1 is phosphorylated, and if so, whether these modifications alter enzyme activity levels. Finally, the three variants of ALDH1B1 which are nonsynonymous and present at a frequency of at least 1% in human populations will be created through site-directed mutagenesis, expressed in a baculovirus system, and characterized for enzyme activity to better understand what effects these potentially inactive enzymes will have in individuals who carry them. These experiments will help to better understand the effects that differential acetaldehyde metabolism may have on Caucasian populations, as well as clarifying the other, diverse roles of ALDH1B1.

性状（由申请人提供）：酒精（乙醇）代谢的主要毒性代谢产物之一，乙醛在分子水平上引起蛋白质加合物和细胞功能障碍，乙醛蓄积在生理水平上引起面部潮红、心动过速、头痛和恶心以及酒精回避。乙醛主要由醛脱氢酶，即ALDH 2、ALDH 1B 1和ALDH 1A 1清除。ALDH 2已被很好地表征，并且已发现ALDH 2 *2是一种众所周知的失活多态性，其影响约50%的一些亚洲人群。ALDH 1B 1最近已部分表征，但仍有很多东西需要学习。最近的流行病学研究已经确定了ALDH 1B 1的多态性与酒精回避和酒精超敏反应，这是第一个这样的变种，在高加索人群中报道的乙醛清除。除了酒精代谢途径，我们还假设ALDH 1B 1可能在硝酸甘油和视黄酸的生物活化中发挥作用;后者可能在癌症生物学中具有重要意义。鉴于这些作用，我们提出了一个全面的ALDH 1B 1的表征，使用广泛的技术，包括分子建模，酶动力学，代谢组学，高通量筛选的抑制剂和激活剂，和定点诱变，以更好地了解多个动态方面的这种潜在的关键酶。首先，将测试ALDH 1B 1底物的更完整谱，包括我们从以前的证据（硝酸甘油和视黄醇）中假设的底物，以及通过无偏代谢组学方法确定的底物。为了更好地允许在实验系统中操纵ALDH 1B 1水平，将首先使用已知的ALDH抑制剂，随后使用高通量筛选技术来确定抑制剂和激活剂谱。ALDH 1B 1很可能是磷酸化的，我们将使用光谱法结合激酶和磷酸酶来确定ALDH 1B 1是否被磷酸化，如果是，这些修饰是否改变了酶的活性水平。最后，ALDH 1B 1的三种变体是非同义的，并且在人群中以至少1%的频率存在，将通过定点诱变产生，在杆状病毒系统中表达，并表征酶活性，以更好地理解这些潜在的失活酶在携带它们的个体中将具有什么影响。这些实验将有助于更好地理解差异乙醛代谢可能对高加索人群的影响，以及阐明ALDH 1B 1的其他不同作用。