FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

脂肪酸结合蛋白-配体特异性

• 批准号: 7391890
• 负责人: Friedhelm Schroeder
• 金额: $ 5.09万
• 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/7391890
• 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

Abnormal energy regulation may contribute to onset and progression of chronic metabolic conditions such as obesity, diabetes mellitus, cardiovascular disease, and cancer, which cause -60%of the world's mortality (rev. in (1). Our lab is interested in how long chain fatty acids (LCFAs) reach nuclei, bind Deroxisomal proliferator receptor a (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, hypertriglycerid- emia, 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.

能量调节异常可能导致慢性代谢疾病的发生和进展 如肥胖、糖尿病、心血管疾病和癌症，这些疾病造成了约60%的 世界的死亡率（rev. in（1）.我们的实验室对长链脂肪酸（LCFA）如何到达细胞核，结合 过氧化物酶体增殖物受体a（PPARa），并启动转录用于能量代谢或储存。我们 假设肝脂肪酸结合蛋白（L-FABP）将LCFA转移并引导至细胞核，结合至 并启动PPARa转录活性。使用纯化的L-FABP和PPARa，L-FABP过表达L- 细胞、L-FABP缺失小鼠和来自L-FABP缺失小鼠的培养肝细胞，我们建议： 目标1.确定L-FABP缺失小鼠的表型是否与异常PPARa调节一致。 L-FABP敲除小鼠共有许多PPARa敲除小鼠特征（LCFA氧化抑制、高脂血症抑制和高脂血症抑制）。 贫血、性别/年龄依赖性肥胖）。L-FABP敲除小鼠表现出SCP-2/SCP-x的上调， SCP-2/SCP-x缺失小鼠中L-FABP的上调。我们已经繁殖了SCP-x、SCP-2/SCP-x和L-FABP/SCP- 2/SCP-x缺失小鼠中进行的研究，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。物理和免疫技术 显示L-FABP在体外结合PPARa。我们将检查配体特异性，构象反应性， 和共激活物或共阻遏物结合，使用：（i）体外纯化的蛋白质;（ii）EYFP-L- 活细胞中的FABP/ECFP-PPARa或Cy 3-L-FABP/Cy 5-PPARa;（iii）免疫金EM;和（iv）荧光 活细胞（L细胞，原代培养的肝细胞）中的相关光谱。 目标4。确定L-FABP介导的LCFA转移至PPARa的机制。我们将确定是否 LCFA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的基因的转录， 存储.这种核受体/脂肪酸能量调节的差异可能有助于 肥胖症、胰岛素抵抗、2型糖尿病和高血脂症的发病机制。