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

ALD 中 SREBP-1 和 PPAR-α 的 4-HNE 和 MDA 内合

• 批准号: 7135648
• 负责人: BENJAMIN J STEWART
• 金额: $ 2.65万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7135648
• 关键词: Kupffer' s cell; adduct; alcoholism /alcohol abuse; aldehydes; computer assisted sequence analysis; gel mobility shift assay; genetic promoter element; laboratory rat; lipid metabolism; liquid chromatography mass spectrometry; liver disorder; malonaldehyde; molecular pathology; peroxisome proliferator activated receptor; predoctoral investigator; protein binding; recombinant proteins; reticulocytes; transcription factor; ubiquitin

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.

描述（由申请方提供）：重要的是表征脂肪肝（酒精性肝病（ALD）的第一阶段），以确定逆转脂肪肝和/或预防进展至更晚期疾病阶段的治疗方法。肝细胞中的脂质代谢受固醇调节元件结合蛋白-Ia（SREBP-1a）和过氧化物酶体增殖物激活受体-α（PPARalpha）调节。脂质醛4-羟基-2-壬烯醛（4-HNE）和丙二醛（MDA）响应于慢性乙醇消耗而产生，并且能够共价修饰蛋白质。本申请提出的实验旨在检验以下一般假设：4-HNE和MDA对转录因子SREBP-1 α和PPARalpha的共价修饰改变了与其各自启动子序列的结合亲和力，以及其泛素依赖性降解速率，这两者都可能导致肝细胞中的脂质蓄积。将通过串联质谱法（MS/MS）鉴定加合物，并通过电泳迁移率变动试验（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。将在兔网织红细胞裂解物（RRL）系统中测量泛素化和蛋白酶体降解。