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

慢性 ALD 模型中活性醛对 L-FABP 的修饰

• 批准号: 8130541
• 负责人: Rebecca LeAnne Smathers McCullough
• 金额: $ 3.22万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8130541
• 关键词: 4 hydroxynonenal; 4-oxo-2-nonenal; Address; Alcoholic Fatty Liver; Alcoholic Liver Diseases; Alcohols; Aldehydes; Binding; Binding Proteins; Biological Assay; CYP2E1 gene; Cell Nucleus; Cell Respiration; Cells; Chronic; Complex; DNA; Data; Disease; Ethanol; Exposure to; FABP1 gene; Fatty Acid-Binding Protein 1; Fatty Acids; Free Radicals; Genes; Genetic Transcription; Glutathione Disulfide; Hepatic; Hepatocyte; Histology; Homeostasis; Hydrogen Peroxide; Immunoblotting; Immunohistochemistry; In Situ; In Vitro; Inflammatory; Injury; Iron; Knock-out; Ligands; Link; Lipid Binding; Lipid Peroxidation; Lipid Peroxides; Lipids; Liver; Liver diseases; MALDI-TOF Mass Spectrometry; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Metabolism; Modeling; Modification; Molecular Chaperones; Monitor; Mus; Nonesterified Fatty Acids; Oxidation-Reduction; Oxidative Stress; PPAR alpha; Pathogenesis; Pathway interactions; Phase; Physiological; Play; Post-Translational Protein Processing; Production; Protein Binding; Protein Denaturation; Proteins; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Side; Simulate; Site; Site-Directed Mutagenesis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Survival Rate; System; Techniques; Testing; Time; Toxic effect; Transgenic Organisms; Triglycerides; Western Blotting; Work; chronic alcohol ingestion; covalent bond; design; dinitrophenylhydrazine; feeding; human FABP4 protein; indexing; insight; interest; lipid metabolism; lipid transport; oxidation; peroxidation; research study; response; stable isotope; trafficking; transcription factor; uptake

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.

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，并且已知有助于这些脂质的摄取和运输的脂质结合蛋白尚未被研究。所提出的实验旨在检验以下假设：长期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，最终导致脂质代谢紊乱和ALD脂肪变性发病机制。研究计划的第一阶段解决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是什么。使用体外孵育测定、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种群中使用慢性乙醇喂养模型，其中将使用 L-FABP 敲除小鼠和 L-FABP 转基因小鼠。使用肝损伤的标准指标（ALT、甘油三酯含量、GSH:GSSG 比率、CYP2E1 活性和组织学），该模型将阐明 L-FABP 是否归因于 ALD 早期阶段肝脏中脂质的积累。此外，还将利用 RT-PCR、免疫印迹和免疫组织化学等技术评估脂质摄取和运输的途径，以表征该模型。该应用提供的数据将进一步深入了解涉及 4-HNE 和 4-ONE 毒性的机制，以及它们对早期 ALD 脂质稳态的影响。