Biochemical, Structural and Polymorphic Characterization of Human ALDH1B1

描述（由申请人提供）：作为酒精（乙醇）代谢的主要毒性代谢物之一，乙醛在分子水平上引起蛋白质加合物和细胞功能障碍，乙醛积累在生理水平上引起面部潮红、心动过速、头痛和恶心以及避酒。乙醛主要由醛脱氢酶清除，即ALDH2、ALDH1B1和ALDH1A1。ALDH2是一种众所周知的失活多态性，大约50%的亚洲人群存在ALDH2*2。ALDH1B1最近已被部分表征，但还有很多需要了解。最近的流行病学研究发现ALDH1B1基因多态性与酒精回避和酒精超敏反应有关；这是在高加索人群中报道的第一个乙醛清除变异。除了酒精代谢途径外，我们还假设ALDH1B1可能在硝化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，我们建议使用广泛的技术，包括分子建模，酶动力学，代谢组学，抑制剂和激活剂的高通量筛选，以及位点定向诱变来全面表征ALDH1B1，以更好地了解这种潜在关键酶的多个动态方面。首先，将测试ALDH1B1的更完整的底物谱，包括我们从以前的证据（硝化甘油和视黄醛）中假设的底物，以及通过无偏倚代谢组学方法确定的底物。为了更好地操纵实验系统中的ALDH1B1水平，将首先使用已知的ALDH抑制剂，然后使用高通量筛选技术确定抑制剂和活化剂的配置文件。ALDH1B1很可能被磷酸化，我们将使用光谱法结合激酶和磷酸酶来确定ALDH1B1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，ALDH1B1的三种非同义且在人群中出现频率至少为1%的变体将通过定点诱变产生，在杆状病毒系统中表达，并以酶活性为特征，以更好地了解这些潜在失活酶对携带它们的个体的影响。这些实验将有助于更好地理解不同的乙醛代谢可能对高加索人群的影响，以及澄清ALDH1B1的其他不同作用。

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. PUBLIC HEALTH RELEVANCE: In this study we propose to characterize the effects of an enzyme (ALDH1B1) which modulates the extent of alcohol symptoms and toxicity. Similar to ALDH2*2, a variant in Asian populations which causes facial flushing and increased nausea and headaches after consuming alcohol, ALDH1B1 has polymorphisms which may affect the alcohol sensitivity of Caucasian populations. Better characterization of this critical enzyme will improve public health by allowing a more complete understanding of alcohol sensitivity and susceptibility to alcoholism.