FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

脂肪酸结合蛋白-配体特异性

• 批准号: 7268743
• 负责人: Friedhelm Schroeder
• 金额: $ 35.32万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7268743
• 关键词: Ablation; Accounting; Address; Affinity; Binding; Binding Proteins; Breeding; Cardiovascular Diseases; Catabolism; Cell Nucleus; Cells; Chronic; Circular Dichroism; Co-Immunoprecipitations; Coenzyme A; Complex; Condition; Cultured Cells; Diabetes Mellitus; Diffusion; Electron Microscopy; Energy Metabolism; Enzymes; Event; Exhibits; Family; Fatty Acid-Binding Protein 1; Fatty Acids; Financial compensation; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Genetic Transcription; Hepatocyte; Homeostasis; Hypertriglyceridemia; Immunologic Techniques; In Vitro; Insulin Resistance; Knockout Mice; L Cells; Label; Life; Ligand Binding; Ligands; Lipids; Liver; Malignant Neoplasms; Mediating; Membrane; Metabolic; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Receptors; Nutrient; Obesity; Pathogenesis; Peroxisome Proliferator-Activated Receptors; Phenotype; Physiological; Phytanic Acid; Protein Binding; Protein Family; Protein Overexpression; Proteins; Recombinant Proteins; Regulation; Relative (related person); Role; Signal Pathway; Signal Transduction; Specificity; Spectrum Analysis; Transcription Initiation; Transcriptional Regulation; Up-Regulation; Water; age related; analog; cell fixing; cyanine dye 5; fatty acid binding protein; fatty acid oxidation; fatty acid-binding proteins; glucose metabolism; in vivo; insulin signaling; interest; liver fatty acid-binding protein; long chain fatty acid; member; mortality; non-specific lipid transfer protein; novel; oxidation; protein distribution; protein transport; receptor; research study; response; sensor; sex

DESCRIPTION (provided by applicant): Abnormal energy regulation may contribute to onset and progression of chronic metabolic conditions such as obesity, diabetes mellitus, cardiovascular disease, and cancer, which cause approximately 60% of the world's mortality [rev. in (1). Our lab is interested in how long chain fatty acids (LCFAs) reach nuclei, bind peroxisomal proliferator receptor alpha (PPARa), and initiate transcription for energy metabolism or storage. We hypothesize that liver fatty acid binding protein (L-FABP) transfers and channels LCFAs to nuclei, binding to and initiating PPARa transcriptional activity. Using purified L-FABP and PPARa, L-FABP overexpressed L- cells, L-FABP null mice, and cultured hepatocytes from L-FABP null mice, we propose to: Aim 1. Determine if the phenotype of L-FABP null mice is consistent with abnormal PPARa regulation. L-FABP null mice share many PPARa null mouse features (inhibition of LCFA oxidation, hypertriglyceridemia, sex/age-dependent obesity). L-FABP null mice exhibit upregulation of SCP-2/SCP-x, the converse of upregulation of L-FABP in SCP-2/SCP-x null mice. We have bred SCP-x, SCP-2/SCP-x, and L-FABP/SCP- 2/SCP-x null mice to clarify the in vivo role of LCFA/LCFA-CoA binding proteins in PPARa regulation. Aim 2. Examine the role of L-FABP in targeting LCFAs to the nucleus for interaction with PPARa. L-FABP enhances saturated LCFA targeting to nuclei. We will use novel fluorescent polyunsaturated (n-3, n-5) and branched-chain (phytanic acid) LCFAs, fluorescent-L-FABP (EYFP-, Cy3-, Cy5-) and immunogold EM, to show if: (i) L-FABP cotransports bound LCFAs into nuclei; (ii) LCFAs enhance L-FABP distribution into nuclei; (iii) nuclear targeting of LCFAs depend on relative binding affinities of L-FABP and PPARa. Aim 3. Resolve molecular interactions of L-FABP with PPARa. Physical and immunological techniques show that L-FABP binds PPARa in vitro. We will examine ligand specificity, conformational responsiveness, and co-activator or co-repressor binding, using: (i) purified proteins in vitro; (ii) FRET between EYFP-L- FABP/ECFP-PPARa or Cy3-L-FABP/Cy5-PPARa in living cells; (iii) immunogold EM; and (iv) fluorescence correlation spectroscopy in living cells (L-cells, primary cultured hepatocytes). Aim 4. Determine the mechanism of L-FABP-mediated LCFA transfer to PPARa. We will determine if LCFA transfers from L-FABP to PPARa by direct molecular interactions or by LCFA diffusion. These findings will contribute to our basic understanding of how different types of fatty acids may activate a nuclear receptor, and thereby induce transcription of genes directing their energy metabolism and storage. Differences in this nuclear receptor/fatty acid energy regulation could contribute to the pathogenesis of obesity, insulin resistance, type 2 diabetes mellitus, and hyperlipidemic conditions.

描述（由申请人提供）：能量调节异常可能导致慢性代谢疾病（如肥胖、糖尿病、心血管疾病和癌症）的发作和进展，这些疾病约占世界死亡率的60%[rev. in（1）.我们的实验室感兴趣的是长链脂肪酸（LCFA）如何到达细胞核，结合过氧化物酶体增殖物受体α（PPARa），并启动能量代谢或储存的转录。我们假设肝脏脂肪酸结合蛋白 （L-FABP）将LCFA转移并引导至细胞核，结合并启动PPARa转录活性。使用纯化的L-FABP和PPARa、L-FABP过表达的L-细胞、L-FABP缺失小鼠和来自L-FABP缺失小鼠的培养的肝细胞，我们提出：目的1。确定表型是否 L-FABP缺失小鼠与异常PPARa调节一致。L-FABP敲除小鼠共有许多PPARa敲除小鼠特征（抑制LCFA氧化、高脂血症、性别/年龄依赖性肥胖）。 L-FABP缺失小鼠表现出SCP-2/SCP-x的上调，与SCP-2/SCP-x缺失小鼠中L-FABP的上调匡威。我们饲养了SCP-x、SCP-2/SCP-x和L-FABP/SCP- 2/SCP-x缺失小鼠，以阐明LCFA/LCFA-CoA结合蛋白在PPARa调节中的体内作用。目标2.检查L-FABP在将LCFA靶向细胞核以与PPARa相互作用中的作用。L-FABP增强饱和LCFA靶向细胞核。我们将使用新的荧光多不饱和（n-3，n-5）和支链（植烷酸）LCFA，荧光-L-FABP（EYFP-，Cy 3-，Cy 5-）和免疫金EM，以显示：（i）L-FABP共转运结合的LCFA进入细胞核;（ii）LCFA增强L-FABP进入细胞核的分布;（iii）LCFA的核靶向依赖于L-FABP和PPARa的相对结合亲和力。目标3.解析L-FABP与PPARa的分子相互作用。物理和免疫学技术表明L-FABP在体外结合PPARa。我们将检查配体特异性、构象响应性和共激活物或共阻遏物结合，使用：（i）体外纯化的蛋白质;（ii）活细胞中EYFP-L- FABP/ECFP-PPARa或Cy 3-L-FABP/Cy 5-PPARa之间的FRET;（iii）免疫金EM;和（iv）活细胞（L细胞，原代培养的肝细胞）中的荧光相关光谱。 目标4。确定L-FABP介导的LCFA转移至PPARa的机制。我们将确定LCFA是否通过直接分子相互作用或通过LCFA扩散从L-FABP转移到PPARa。这些发现将有助于我们基本了解不同类型的脂肪酸如何激活核受体，从而诱导指导其能量代谢和储存的基因的转录。这种核受体/脂肪酸能量调节的差异可能导致肥胖、胰岛素抵抗、2型糖尿病和高脂血症的发病机制。