FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

能量调节异常可能导致慢性代谢疾病的发生和发展 例如肥胖、糖尿病、心血管疾病和癌症，这些疾病导致了-60%的 世界死亡率(第(1)版)我们的实验室对长链脂肪酸(LCFA)到达细胞核有多长感兴趣，结合 去氧体增殖物受体a(PPARa)，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)将低分子脂肪酸转移和通道到细胞核，结合到 并启动PPARa转录活动。利用纯化的L-FABP和PPARa，L-FABP过表达L-FABP。 细胞，L-FABP缺失小鼠，以及L-FABP缺失小鼠的培养肝细胞，我们建议： 目的1.确定L-FABP基因缺失小鼠的表型是否与PPARa调节异常一致。 L-FABP基因缺失小鼠有许多PPARA基因缺失小鼠的特征(抑制LCFA氧化，高甘油三酯- 血症、性别/年龄依赖性肥胖)。L-FABP基因缺失小鼠SCP-2/SCP-x表达上调，而 L-FABP基因在SCP-2/SCP-x基因缺失小鼠中的表达我们培育出了SCP-x、SCP-2/SCP-x和L-FABP/SCP-1。 2/SCP-x缺失小鼠，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 增强饱和LCFA针对细胞核的靶向。我们将使用新型荧光多不饱和(n-3，n-5) 和支链(植酸)LCFA、荧光L FABP(EYFP-、Cy3-、Cy5-)和免疫金 EM，以表明是否：(I)L-FABP共转运蛋白将LCFA结合到细胞核中；(Ii)LCFA促进L-FABP的分布 LCFAs的核靶向取决于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 结果表明，L-FABP在体外可与PPARa结合。我们将检测配基的特异性，构象响应性， 和共激活子或共抑制子结合，使用：(I)体外纯化的蛋白质；(Ii)EYFP-L-FRET之间的FRET- 活细胞中的FABP/ECFP-PPARa或Cy3-L-FABP/Cy5-PPARa；(Iii)免疫金EM；和(Iv)荧光 活细胞(L细胞、原代培养的肝细胞)的相关光谱。 目的4.确定L-FABP介导的LCFA向PPARa转移的机制。我们将确定是否 LCFA从L-FABP通过直接分子相互作用或通过LCFA扩散向PPARa转移。 这些发现将有助于我们对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 储藏室。这种核受体/脂肪酸能量调节的差异可能有助于 肥胖、胰岛素抵抗、2型糖尿病和高脂血症的发病机制。

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.