Modifications of L-FABP by Reactive Aldehydes in a Model of Chronic ALD

描述（由申请人提供）：酒精性肝病（ALD）的早期阶段以肝细胞内脂质积累为特征，统称为脂肪变性。众所周知，脂质稳态的改变发生在慢性乙醇条件下，脂质结合蛋白已知有助于这些脂质的摄取和运输，但尚未研究。本实验旨在验证慢性乙醇消耗导致肝脏脂肪酸结合蛋白（L-FABP）被4-HNE和4-ONE共价修饰，最终导致ALD中脂质代谢紊乱和脂肪变性的发病机制。研究计划的第一阶段解决了L-FABP是否被4-HNE和4-ONE共价修饰，以及这种修饰的生理和结构后果。使用体外培养试验、免疫印迹、lc -MS/MS和MALDI-TOF-TOF质谱法，将鉴定共价修饰的蛋白质侧链。还将进行活性测定，以评估这些修饰对脂质结合的生理影响。此外，将在氧化应激原位模型中研究蛋白质结合脂质在脂质氢过氧化物形成中的作用。研究的第二阶段涉及在转基因小鼠中使用慢性乙醇喂养模型，其中将使用L-FABP敲除和L-FABP转基因小鼠。利用肝损伤的标准指标（ALT、甘油三酯含量、GSH:GSSG比值、CYP2E1活性和组织学），该模型将阐明L-FABP是否与ALD早期肝脏脂质积累有关。此外，脂质摄取和运输的途径将通过使用RT-PCR、免疫印迹和免疫组织化学等技术来评估模型的特征。该应用程序提供的数据将进一步深入了解4-HNE和4-ONE毒性的机制，以及它们对早期ALD中脂质稳态的影响。

DESCRIPTION (provided by applicant): Early stages of Alcoholic Liver Disease (ALD) are characterized by the accumulation of lipid within hepatocytes and is collectively referred to as steatosis. It is known that alterations in lipid homeostasis occur in chronic ethanol conditions, and lipid binding proteins that are known to aid in the uptake and trafficking of these lipids have not been investigated. The experiments proposed are designed to test the hypothesis that chronic ethanol consumption leads to the covalent modification of liver fatty acid binding protein (L-FABP) by 4-HNE and 4-ONE, which ultimately leads to the disruption of lipid metabolism and pathogenesis of steatosis in ALD. The first phase of the research plan addresses if L-FABP is covalently modified by 4-HNE and 4-ONE, and what are the physiological and structural consequences ofthis modification. Using in vitro incubation assays, immunoblotting, and-LC-MS/MS and MALDI-TOF-TOF mass spectrometry, covalently modified protein side-chains will be identified. Activity assays will also be conducted to assess the physiological consequences of these modifications on lipid binding. In addition, protein bound lipid will be investigated in the formation of lipid hydroperoxides in an in situ model of oxidative stress. The second phase of the research involves the use of chronic ethanol feeding models in genetically modified stocks of mice, where L-FABP knockout and L-FABP transgenic stocks will be used. Using standard indices of liver injury (ALT, triglyceride content, GSH:GSSG ratios, CYP2E1 activity, and histology), the model will elucidate if L-FABP attributes to the accumulation of lipid in the liver during early phases of ALD. Also, pathways in lipid uptake and trafficking will be evaluated to characterize the model by utilizing techniques involving RT-PCR, immunoblotting, and immunohistochemistry. The data provided by this application will provide further insight into the mechanisms involving 4-HNE and 4-ONE toxicity, and the effects they have on lipid homeostasis in early ALD.