FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

描述(由申请人提供)：能量调节异常可能导致肥胖、糖尿病、心血管疾病和癌症等慢性代谢疾病的发生和发展，这些疾病导致的死亡率约占世界死亡率的60%[in(1)修订版]。我们的实验室感兴趣的是，长链脂肪酸(LCFA)到达细胞核，与过氧化体增殖物受体α(PPARa)结合，并启动转录以进行能量代谢或储存。我们假设肝脏脂肪酸结合蛋白 (L-FABP)将LCFAs转导到细胞核，与PPARa结合并启动转录活性。用纯化的L-FABP和PPARa，L-FABP过表达L细胞，L-FABP缺失小鼠，以及培养L-FABP缺失小鼠的肝细胞，我们提出：目的1.确定 L-FABP基因缺失小鼠与PPARa调节异常一致。L-FABP基因缺失小鼠具有许多PPARA基因缺失小鼠的特征(抑制LCFA氧化、高甘油三酯血症、性别/年龄相关性肥胖)。 L-FABP基因敲除小鼠的SCP-2/SCP-x基因表达上调，而L-FABP基因敲除小鼠的表达上调。我们培育了SCP-x、SCP-2/SCP-x和L-FABP/SCP-2/SCP-x缺失小鼠，以阐明LCFA/LCFA-CoA结合蛋白在体内对PPARa调节的作用。目的2.研究L-FABP在将LCFAs靶向与PPARa相互作用的细胞核中的作用。L-FABP增强了饱和LCFA的核靶向性。我们将使用新的荧光多不饱和(n-3，n-5)和支链(植酸)LCFA，荧光-L-FABP(EYFP-，Cy3-，Cy5-)和免疫金EM，以表明：(1)L-FABP共转运蛋白将LCFA结合到细胞核中；(Ii)LCFA促进L-FABP在细胞核中的分布；(Iii)LCFA的核靶向性取决于L-FABP和PPARA的相对结合亲和力。目的3.解决L-FABP与PPARa的分子相互作用。物理和免疫学技术表明，L-FABP在体外能与PPARa结合。我们将使用：(I)体外纯化的蛋白质；(Ii)活细胞中EYFP-L-FABP/ECFP-PPARa或Cy3-L-FABP/Cy5-PPARa之间的FRET；(Iii)免疫金EM；以及(Iv)活细胞(L细胞、原代培养的肝细胞)中的荧光相关光谱，来检测配体的特异性、构象反应和共激活或共抑制物结合。 目的4.确定L-FABP介导的LCFA向PPARa转移的机制。我们将确定LCFA是通过直接分子相互作用还是通过LCFA扩散从L-FABP转移到PPARa。这些发现将有助于我们基本理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。

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.