4-HNE and MDA adduction of SREBP-1 and PPAR-alpha in ALD

描述（由申请人提供）：重要的是表征酒精性肝病（ALD）的第一阶段肝骨化病，以便确定逆转肝骨化病和/或防止进展到更高级疾病阶段的治疗方法。肝细胞的脂质代谢受甾醇调节元件结合蛋白-la （SREBP-1a）和过氧化物酶体增殖激活受体- α (ppar - α)的调控，脂质醛4-羟基-2-壬烯醛（4-HNE）和丙二醛（MDA）是在慢性乙醇消耗的反应中产生的，并且能够共价修饰蛋白质。本申请提出的实验旨在验证一个普遍假设，即4-HNE和MDA对转录因子srebp -1 α和ppar α的共价修饰改变了它们与各自启动子序列的结合亲和力，以及它们的泛素依赖性降解速率，这两者都可能导致肝细胞中的脂质积累。加合物将通过串联质谱法（MS/MS）进行鉴定，并通过电泳迁移位移测定（EMSA）将修饰的srebp -1 α和ppar α的启动子结合活性与天然蛋白结合进行比较。泛素化和蛋白酶体降解将在兔网织细胞裂解物（RRL）系统中测量。

DESCRIPTION (provided by applicant): It is important to characterize hepatosteatosis, the first stage of alcoholic liver disease (ALD), in order to identify therapies to reverse hepatosteatosis and/or prevent progression to more advanced disease stages. Lipid metabolism in hepatocytes is regulated by sterol regulatory element-binding protein-la (SREBP-1a) and peroxisome proliferator-activated receptor-alpha (PPARalpha), The lipid aldehydes 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) are generated in response to chronic ethanol consumption, and are capable of covalently modifying proteins. The experiments proposed by this application are designed to test the general hypothesis that covalent modification of the transcription factors SREBP-1alpha and PPARalpha by 4-HNE and MDA alters binding affinity to their respective promoter sequences, as well as their rates of ubiquitin-dependent degradation, both of which may result in lipid accumulation in hepatocytes. Adducts will be identified by tandem mass spectrometry (MS/MS), and promoter binding activity of modified SREBP-1alpha and PPARalpha will be compared to native protein binding by electrophoretic mobility shift assays (EMSA). Ubiquitination and proteasomal degradation will be measured in a rabbit reticulocyte lysate (RRL) system.