Molecular control of CPT-I and CD36 in the regulation of mitochondrial fatty acid transport

线粒体影响多个过程，包括能量供应、细胞凋亡、钙处理和氧化还原平衡，因此现在被认为是影响几乎每个细胞的动态平衡的关键细胞器。影响线粒体生物学的一个基本事件是长链脂肪酸在线粒体外/内膜上的移动。虽然我们对调控这一过程的基本机制的了解还不完全，但肉碱棕榈酰转移酶-I(CPT-I)和CD36发挥了关键作用。 虽然CPT-I是线粒体膜脂运输所必需的，但我们的目标是确定细胞应激是否可以改变CPT-I对各种底物的敏感性，CPT-I是如何调节的，以及通过这种酶的催化通量的变化会产生什么生物学后果。我们计划通过确定在细胞应激增加(例如，急性运动)期间CPT-I的潜在翻译后修饰来解决这些知识差距。我们预计，随着CPT-I生化性质的变化，运动过程中各种丝氨酸残基(即S330、S572、S401、S402和S610)将被磷酸化，因此计划在肌肉特异性敲除动物中瞬时表达突变的CPT-I结构(即通过丙氨酸替代丝氨酸来防止磷酸化)，以研究其对各种线粒体和细胞参数的影响。这一方法将使我们能够确定CPT-I磷酸化、酶通量、线粒体生物能量学和细胞内稳态变化之间的因果关系。 我们还发现，细胞应激可以诱导一种蛋白质(CD36)从细胞内区域移动到线粒体膜。CD36似乎位于CPT-I的上游，在那里聚集影响脂质向CPT-I的转运，从而间接影响膜转运，然而，这种亚细胞转运的分子基础仍不清楚。我们计划确定CD36的C-末端YCACR基序是否与各种信号事件相互作用来影响这一细胞过程。为了实现这一点，我们将把野生型和C末端突变体(C末端氨基酸缺失)瞬时导入CD36基因敲除小鼠的骨骼肌。此后，我们将提供代谢挑战(例如肌肉收缩)，并确定CD36在线粒体膜上堆积的能力和功能后果。为了确定特定信号事件在介导线粒体CD36运输中的必要性，将在转基因小鼠身上或使用药物抑制剂进行类似的实验。 总而言之，拟议的研究将提供关于调控线粒体脂质运输的机制的基础知识，这一过程对理解细胞内稳态具有广泛的意义。

Mitochondria influence several processes, including energy provision, apoptosis, calcium handling and redox balance, and as result are now recognized as a key organelle that influences homeostasis in almost every cell. A fundamental event influencing mitochondrial biology is the movement of long-chain fatty acids across the outer/inner mitochondrial membranes. While our understanding of the basic mechanisms regulating this process remains incomplete, carnitine palmitoyltransferase-I (CPT-I) and CD36 play key roles. While CPT-I is required mitochondrial membrane lipid transport, we aim to establish if cellular stresses can alter the sensitivity of CPT-I to various substrates, how CPT-I is regulated and what the biological consequence is to changes in catalytic flux through this enzyme. We plan to address these knowledge gaps by determining potential post-translational modifications of CPT-I during increased cellular stress (e.g. acute exercise). We anticipate that various serine residues will be phosphorylated during exercise (i.e. S330, S572, S401, S402 and S610) in association with changes in the biochemical properties of CPT-I, and therefore plan to transiently express mutated CPT-I constructs (i.e. alanine substitutions for serine to prevent phosphorylation) in a muscle specific knock out animal to study the impact on various mitochondrial and cellular parameters. This approach will enable us to determine cause-and-effect relationships between alterations in CPT-I phosphorylation, enzymatic flux, mitochondrial bioenergetics and cellular homeostasis. We have also identified that cellular stress can induce the movement of a protein (CD36) from an intracellular region to mitochondrial membranes. CD36 appears to be located upstream of CPT-I, where an accumulation influences the delivery of lipids to CPT-I to indirectly influence membrane transport, however, the molecular basis for this subcellular trafficking remains unknown. We plan to determine if the C-terminal YCACR motif of CD36 interacts with various signaling events to influence this cellular process. To achieve this, we will transiently transfect wild type and C-terminal mutants (C-terminal amino acids deleted) into the skeletal muscle of CD36 knock out mice. Thereafter we will provide metabolic challenges (e.g. muscle contraction) and determine the ability of CD36 to accumulate on mitochondrial membranes and the functional consequence. To determine the necessity of specific signalling events in mediating mitochondrial CD36 trafficking, similar experiments in genetically modified mice or with pharmacological inhibitors will be employed. Combined, the proposed studies will provide fundamental knowledge on the mechanisms regulating mitochondrial lipid transport, a process that has broad implications to understanding cellular homeostasis.