Aldehyde Dehydrogenase 3B1: Characterization and Role in Oxidative Stress

描述（由申请人提供）：乙醇代谢导致氧化应激和脂质过氧化，并与许多疾病相关。醛脱氢酶（ALDH）是在这些过程中产生的反应性醛的解毒中起主要作用的酶。ALDH 3B 1属于ALDH家族，但其性质和功能意义尚不清楚。长期目标是全面表征ALDH 3B 1并研究其在氧化应激中的作用。具体的假设是ALDH 3B 1在肝脏中具有功能性作用，并且代表了针对氧化应激和反应性醛的重要细胞防御系统。该假设基于以下初步观察：1）ALDH 3B 1在肝脏中高度表达，2）ALDH 3B 1对脂质过氧化物衍生的醛具有催化活性，3）ALDH 3B 1保护细胞免受这些醛诱导的细胞毒性。具体目标：1。纯化并生化表征重组人ALDH 3B 1。将使用亲和层析从杆状病毒感染的Sf 9细胞中纯化ALDH 3B 1。在各种醛底物的存在下，将通过荧光定量法评价酶活性、底物敏感性和辅因子偏好性。2：确定C57 BU 6 J小鼠中ALDH 3B 1的组织和细胞特异性表达和亚细胞定位。分别采用Western blot和RT-PCR技术分析ALDH 3B 1蛋白和mRNA的组织表达。将通过免疫组织化学在器官切片中评估ALDH 3B 1的细胞特异性表达。ALDH 3B 1亚细胞定位将通过器官的亚细胞分级分离，然后进行蛋白质印迹和免疫组织化学，然后进行共聚焦显微镜检查来确定。3：评估ALDH 3B 1在体外氧化应激中的保护作用。将用各种醛处理稳定的ALDH 3B 1转染和模拟转染的Hepa-1和HEK 293细胞系，并使用细胞测定法测量细胞毒性、细胞凋亡、蛋白质氧化和脂质过氧化。在表达内源性ALDH 3B 1的细胞中使用siRNA将用于评估ALDH 3B 1的保护作用。将在表达内源性ALDH 3B 1的细胞中检查乙醇对ALDH 3B 1的调节。将用乙醇处理细胞，并通过Western印迹和RT-PCR分析ALDH 3B 1蛋白和mRNA表达。与公共卫生的相关性：酒精消费与许多人类疾病的病因有关，乙醇代谢导致许多有毒产物的形成。醛脱氢酶（ALDH）酶通过解毒这些产物在细胞存活中起主要作用。ALDH 3B 1的特性及其在这些过程中的功能作用将被研究。

DESCRIPTION (provided by applicant): Oxidative stress and lipid peroxidation occur as a result of ethanol metabolism and have been linked to many diseases. Aldehyde dehydrogenases (ALDHs) are enzymes that play major roles in detoxifying reactive aldehydes produced during these processes. ALDH3B1 belongs to the ALDH family, however, its properties and funcitonal significance remain unknown. The long term goal is to comprehensively characterize ALDH3B1 and investigate its role in oxidative stress. The specific hypothesis is that ALDH3B1 has a functional role in the liver and represents an important cellular defense system against oxidative stress and reactive aldehydes. This hypothesis is based on the preliminary observations that 1) ALDH3B1 is highly expressed in the liver, 2) ALDH3B1 is catalytically active with lipid peroxidaiton-derived aldehydes and 3) ALDH3B1 protects cells from cytoxicity induced by these aldehydes. Specific Aims: 1. Purify and biochemically characterize recombinant human ALDH3B1. ALDH3B1 will be purified from baculovirus-infected Sf9 cells using affinity chromatography. Enzymatic activity, inhibitor-sensitivity and cofactor preference will be evaluated spectrophotometrically in the presence of various aldehyde substrates. 2: Determine the tissue- and cell-specific expression and subcellular localization of ALDH3B1 in C57BU6J mice. The tissue expression of ALDH3B1 protein and mRNA will be analyzed by Western blot and RT-PCR techniques, respectively. Cell-specific expression of ALDH3B1 will be assessed in organ sections by immunohistochemistry. ALDH3B1 subcellular localization will be determined by subcellular fractionation of organs followed by Western blot and immunohistochemistry followed by confocal microscopy. 3: Evaluate the protective role of ALDH3B1 in oxidative stress in vitro. Stable ALDH3B1- and mocktransfected Hepa-1 and HEK293 cell lines will be treated with various aldehydes and cytotoxicity, apoptosis, protein oxidation and lipid peroxidation will be measured using cellular assays. The use of siRNA in cells expressing endogenous ALDH3B1 will be utilized to assess the protective role of ALDH3B1. The regulation of ALDH3B1 by ethanol will be examined in cells expressing endogenous ALDH3B1. Cells will be treated with ethanol and ALDH3B1 protein and mRNA expression will be analyzed by Western blot and RT-PCR. Relevance to Public Health: Alcohol consumption is implicated in the etiology of many human diseases and ethanol metabolism results in the formation of many toxic products. Aldehyde dehydrogenase (ALDH) enzymes play a major role in cell survival by detoxifying these products. The properties of ALDH3B1 and its functional role in these processes will be investigated.