Mechanism of Fatty Acid Uptake by CD36

我的研究计划的总体目标是了解CD36介导的脂肪酸(FA)跨细胞膜运输的机制和影响，从最基本的问题开始：CD36的物理结构如何促进FA的摄取？一直到体内的生理和病理生理变化。尽管如今被更广泛地接受，但蛋白质介导的FA摄取仍然存在争议，因为非电离FA可以快速地跨膜。然而，Flip/Flop并不导致FA有效地掺入三酰甘油酯和磷脂的细胞池中。相反，当膜上存在CD36时，超过90%的FA随后被发现存在于FA的细胞衍生品中。CD36缺陷的小鼠和人类都有表型变化，这可以用FA摄取缺陷来解释。最近发表的CD36的模型结构提供了重要的新信息，允许合理和战略性的结构：CD36的功能询问，并可能是对FA摄取争议的最终答案。我们假设，根据结构模型，CD36通道将FA隔离到膜上，在那里它们翻转/翻转。CD36定位于特定的结构域、脂筏、洗涤剂抗性结构域和小窝蛋白，这些结构域可能作为转运枢纽。其他辅助CD36的蛋白质可能存在物理上的相互作用，也可能只是简单地被浓缩了。因此，我们设想CD36将FA通向细胞质脂肪酸结合蛋白(FABP)和脂肪酰辅酶A合成酶，从而允许FA似乎单向流动并并入细胞池。然而，正是CD36的结构，我们认为这是结果的内在假设。本研究的目标是：1.在FA通道中建立CD36的突变体，使其能够在细胞表面正确表达，但不能摄取FA；2.检测该突变体(S)对巨噬细胞功能的影响；3.验证CD36通过信号转导影响FA酯化的假说。这些目标是对实验室中其他工作的补充和支持，在实验室中，我们通过小鼠模型研究了CD36与细胞质FABP和脂肪酰辅酶A合成酶的共定位，以及内皮细胞CD36对全身性FA摄取、肥胖、胰岛素抵抗和动脉粥样硬化的影响。这些项目还具有协同作用，为一个项目创造的试剂和技术可以融入其他项目，从一个项目获得的知识可以告知其他项目。了解细胞摄取FA的基本原理增加了我们的科学知识，并有可能影响肥胖、胰岛素抵抗/糖尿病和心血管疾病的治疗策略。

The overall objective of my research program is to understand the mechanism and impact of CD36 mediated transport of fatty acids (FA) across cell membranes, starting from the most basic question: how does the physical structure of CD36 facilitate FA uptake?, all the way to physiological and pathophysiological changes in vivo. Although more widely accepted today, protein mediated FA uptake remains controversial because un-ionized FA can rapidly flip/flop across membranes. Flip/flop, however, does not lead to productive incorporation of FA into cellular pools of triacylglycerides and phospholipids. In contrast, when CD36 is present on membranes, more than 90% of FA are subsequently found in cellular derivatives of FA. CD36-deficient mice and humans have phenotypic changes that could be explained by a defect in FA-uptake. The recently published model structure of CD36 provides important new information that allows rational and strategic structure:function interrogation of CD36 and perhaps a final answer to the FA uptake controversy. We hypothesize that, based on the structural model, a CD36 channel sequesters FA through to the membrane, where they flip/flop over. CD36 is localized in specific domains, lipid rafts, detergent resistant domains and caveolin, which may act as transport hubs. Other proteins which assist CD36 may interact physically, or may simply be enriched. Thus we imagine CD36 channels FA to cytoplasmic fatty acid binding proteins (FABP) and fatty acyl-CoA synthetase allowing for seemingly unidirectional FA flow and incorporation into cellular pools. It is the structure of CD36, however, that we hypothesize is intrinsic to the outcome. The objectives of this research proposal are 1. To create mutants of CD36 in the FA channel, such that they are FA-uptake dead, but otherwise competent, and expressed correctly on the cell surface, 2. To test the impact of this (these) mutant(s) on macrophage function and 3. To test an alternative hypothesis that CD36 affects FA esterification through signaling. These Aims complement and support other work in the lab in which we study CD36 co-localization with cytoplasmic FABPs and fatty acyl-CoA synthetase and the impact of endothelial cell CD36 on systemic FA uptake, obesity, insulin resistance and atherosclerosis using mouse models. The projects also synergize in that reagents and techniques created for one project can be incorporated into others, and knowledge gained from one informs the others. Understanding the fundamentals of FA uptake into cells increases our scientific knowledge and has the potential to effect strategies for obesity, insulin resistance/diabetes and cardiovascular diseases.