INTESTINAL FATTY ACID BINDING PROTEINS

肠道细胞高水平表达两种同源脂肪酸结合蛋白(FABP)，即肝脏FABP(LFABP)和肠道FABP(IFABP)。推测FABP在FA的胞内转运中起重要作用。I-和LFABP都以高亲和力结合长链脂肪酸，最近的研究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同源性以及不同的组织分布。此外，我们实验室最近的工作表明，这些蛋白质使用明显不同的机制将FA转移到受体膜上。LFABP的FA转移是通过水扩散进行的，而IFABP的FA转移是通过蛋白质与受体膜的直接碰撞作用进行的。我们最近已经证明了螺旋-转角螺旋结构域在IFABP与膜的相互作用及其FA转移机制中的关键作用。在本提案中，我们将使用生化、分子生物学和生物物理技术对IFABP-膜相互作用中涉及的特定结构域和残基进行分子水平分析，并详细了解IFABP-膜结合本身的物理化学性质。此外，还将阐明LFABP中没有FA碰撞转移的结构基础。这一建议的具体目的是：1)通过构建嵌合的I/LFABP，确定αI螺旋和αII螺旋在脂肪酸碰撞转移到膜中的重要性；2)利用定点突变技术，阐明特定的带电和疏水残基在FA碰撞转移到膜中的作用。这种脂肪酸转运功能的方法应该提供特定结构元素的分子水平细节，从而导致IFABP与LFABP的独特转运特性。具体来说，对于所有的点突变和嵌合蛋白，我们将使用荧光共振能量转移分析来分析脂肪酸转移到模型膜的速度和机制，并使用几种互补的生化和生物物理技术来分析蛋白质与膜的相互作用。由于LFABP和IFABP在小肠上皮细胞中都高度表达，了解它们的作用机制将有助于有效的营养和/或药物调控饮食中的脂肪同化。

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.