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

描述(由申请人提供)：重要的是要确定酒精性肝病(ALD)的第一阶段--肝骨病的特征，以便确定逆转肝骨病和/或防止进展到更严重的疾病阶段的治疗方法。肝细胞的脂质代谢受固醇调节元件结合蛋白-1a(SREBP-1a)和过氧化物酶体增殖物激活受体-α(PPARpha)的调节，在慢性酒精摄入后产生脂醛4-羟基-2-壬烯醛(4-HNE)和丙二醛(MDA)，并能共价修饰蛋白质。本申请提出的实验旨在检验这样一个普遍假设，即4-HNE和MDA对转录因子SREBP-1α和PPARpha的共价修饰会改变与它们各自启动子序列的结合亲和力，以及它们依赖泛素的降解率，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仪(MS/MS)进行鉴定，改良的SREBP-1α和PPARpha的启动子结合活性将通过凝胶迁移率改变分析(EMSA)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红细胞裂解物(RRL)系统中进行测量。

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.