Aldehyde Dehydrogenase 3B1: Characterization and Role in Oxidative Stress

描述（由申请人提供）：氧化应激和脂质过氧化是乙醇代谢的结果，与许多疾病有关。醛脱氢酶（ALDHs）是在这些过程中产生的活性醛解毒中起主要作用的酶。ALDH3B1属于ALDH家族，但其特性和功能意义尚不清楚。长期目标是全面表征ALDH3B1并研究其在氧化应激中的作用。具体的假设是，ALDH3B1在肝脏中具有功能性作用，代表了抗氧化应激和活性醛的重要细胞防御系统。这一假设是基于以下的初步观察：1)ALDH3B1在肝脏中高表达，2)ALDH3B1对脂质过氧化衍生的醛具有催化活性，3)ALDH3B1保护细胞免受这些醛诱导的细胞毒性。具体目标：1；纯化重组人ALDH3B1并进行生化表征。ALDH3B1将通过亲和层析从杆状病毒感染的Sf9细胞中纯化。酶活性，抑制剂敏感性和辅助因子偏好将在各种醛底物的存在下进行分光光度评价。2：测定ALDH3B1在C57BU6J小鼠中的组织和细胞特异性表达及亚细胞定位。采用Western blot和RT-PCR技术分别分析ALDH3B1蛋白和mRNA的组织表达。ALDH3B1的细胞特异性表达将通过免疫组织化学在器官切片中进行评估。ALDH3B1的亚细胞定位将通过器官亚细胞分离、Western blot和免疫组织化学以及共聚焦显微镜来确定。3：体外评价ALDH3B1对氧化应激的保护作用。稳定的ALDH3B1和模拟转染的Hepa-1和HEK293细胞系将用各种醛处理，细胞毒性、凋亡、蛋白质氧化和脂质过氧化将通过细胞分析来测量。在表达内源性ALDH3B1的细胞中使用siRNA将被用来评估ALDH3B1的保护作用。我们将在表达内源性ALDH3B1的细胞中检测乙醇对ALDH3B1的调节。用乙醇和ALDH3B1蛋白处理细胞，用Western blot和RT-PCR分析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发挥重要作用。我们将研究ALDH3B1的性质及其在这些过程中的功能作用。

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.