STRUCTURE-FUNCTION ANALYSIS OF INTESTINAL FATTY ACID-BIN

肠脂肪酸仓的结构功能分析

• 批准号: 6188487
• 负责人: Judith Storch
• 金额: $ 4.03万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6188487
• 关键词: fatty acid binding protein; fluorescence resonance energy transfer; hydropathy; intestines; lipid bilayer membrane; membrane activity; membrane permeability; protein localization; protein structure function; site directed mutagenesis; tissue mosaicism

Intestinal enterocytes express high levels of two homologous fatty acid-binding proteins (FABP), liver FABP (LFABP) and intestinal FABP (IFABP). It is hypothesized that FABPs are important in intracellular transport of FA. Both I- and LFABP bind long chain fatty acids (FA) with high affinity, and it has recently been shown that they have similar tertiary structures. Nevertheless, it has been suggested that they are functionally distinct because they have different substrate specificities and stoichometry and different tissue distributions. Furthermore, recent work from our laboratory has shown that these proteins employ markedly different mechanisms to transfer FA to acceptor membranes. Whereas FA transfer from LFABP occurs via aqueous diffusion, FA transfer from IFABP occurs during direct collisional interactions of the protein with acceptor membranes. We have recently demonstrated the critical importance of the helix-turn helix domain in the interaction of IFABP with membranes, and in its FA transfer mechanism. In the present proposal we will use biochemical, molecular biological and biophysical techniques to provide a molecular level analysis of the specific domains and residues that are involved in these IFABP-membrane interactions, as well as a detailed understanding of the physicochemical nature of the IFABP-membrane association itself. Further, the structural basis for the absence of collisional transfer of FA from LFABP will also be elucidated. The specific aims of this proposal are: 1) To determine the importance of the alphaI helix and the alphaII helix in collisional transfer of fatty acids to membranes, by constructing chimeric I/LFABP; and 2) To elucidate the roles of specific charged and hydrophobic residues in collisional transfer of FA to membranes, employing site-directed mutagenesis techniques. This approach to the fatty acid transport function of FABPs should provide molecular level detail of the specific structural elements which lead to the unique transport properties of IFABP versus LFABP. Specifically, for all point mutant and chimeric proteins engineered, we will analyze the rate and mechanism of fatty acid transfer to model membranes using a fluorescence resonance energy transfer assay, and the interaction of the proteins with membranes using several complementary biochemical and biophysical techniques. Since both LFABP and IFABP are highly expressed in the small intestinal enterocyte, an understanding of their mechanisms of actions will enable the effective nutritional and/or pharmacological manipulation of dietary lipid assimilation.

肠上皮细胞表达高水平的两种同源脂肪酸结合蛋白 (FABP)：肝 FABP (LFABP) 和肠 FABP (IFABP)。 据推测，FABP 在 FA 的细胞内转运中很重要。 I-和LFABP都以高亲和力结合长链脂肪酸(FA)，最近表明它们具有相似的三级结构。然而，有人认为它们在功能上是不同的，因为它们具有不同的底物特异性和化学计量以及不同的组织分布。 此外，我们实验室最近的工作表明，这些蛋白质采用明显不同的机制将 FA 转移到受体膜。 LFABP 的 FA 转移是通过水扩散发生的，而 IFABP 的 FA 转移则发生在蛋白质与受体膜直接碰撞相互作用期间。我们最近证明了螺旋转角螺旋结构域在 IFABP 与膜的相互作用及其 FA 转移机制中的至关重要性。 在本提案中，我们将使用生物化学、分子生物学和生物物理技术对参与这些 IFABP-膜相互作用的特定结构域和残基进行分子水平分析，并详细了解 IFABP-膜关联本身的物理化学性质。 此外，还将阐明 LFABP 不存在 FA 碰撞转移的结构基础。该提案的具体目标是： 1) 通过构建嵌合 I/LFABP，确定 αI 螺旋和 αII 螺旋在脂肪酸碰撞转移至膜中的重要性； 2) 采用定点诱变技术阐明特定带电残基和疏水残基在 FA 碰撞转移至膜中的作用。 这种 FABP 脂肪酸转运功能的方法应提供特定结构元件的分子水平细节，从而导致 IFABP 相对于 LFABP 具有独特的转运特性。 具体来说，对于所有工程化的点突变和嵌合蛋白，我们将使用荧光共振能量转移测定分析脂肪酸转移到模型膜的速率和机制，并使用几种互补的生化和生物物理技术分析蛋白质与膜的相互作用。 由于 LFABP 和 IFABP 在小肠肠上皮细胞中高度表达，了解它们的作用机制将有助于对膳食脂质同化进行有效的营养和/或药理学操作。