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

CPT-I和CD36在线粒体脂肪酸转运调节中的分子控制

• 批准号: RGPIN-2019-05113
• 负责人: Holloway, Graham
• 金额: $ 3.42万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715293
• 关键词: Molecular; control; CPT; CD36; regulation

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.

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稳态的关键细胞器。 影响线粒体生物学的一个基本事件是长链脂肪酸穿过线粒体外/内膜的运动。虽然我们对调节这一过程的基本机制的理解仍然不完整，但肉碱棕榈酰转移酶-I (CPT-I) 和 CD36 发挥着关键作用。 虽然 CPT-I 是线粒体膜脂质转运所必需的，但我们的目标是确定细胞应激是否会改变 CPT-I 对各种底物的敏感性、CPT-I 的调节方式以及通过该酶的催化通量变化的生物学后果。 我们计划通过确定细胞应激增加（例如急性运动）期间 CPT-I 潜在的翻译后修饰来解决这些知识差距。我们预计各种丝氨酸残基在运动过程中会被磷酸化（即 S330、S572、S401、S402 和 S610），与 CPT-I 生化特性的变化相关，因此计划瞬时表达突变的 CPT-I 构建体（即丙氨酸）在肌肉特异性基因敲除动物中替代丝氨酸以防止磷酸化），以研究对各种线粒体和细胞参数的影响。这种方法将使我们能够确定 CPT-I 磷酸化、酶通量、线粒体生物能学和细胞稳态变化之间的因果关系。 我们还发现细胞应激可以诱导蛋白质 (CD36) 从细胞内区域移动到线粒体膜。 CD36似乎位于CPT-I的上游，其中的积累影响脂质向CPT-I的递送，从而间接影响膜运输，然而，这种亚细胞运输的分子基础仍然未知。我们计划确定 CD36 的 C 端 YCACR 基序是否与各种信号事件相互作用以影响该细胞过程。为了实现这一目标，我们将野生型和 C 端突变体（C 端氨基酸删除）瞬时转染到 CD36 敲除小鼠的骨骼肌中。此后，我们将提供代谢挑战（例如肌肉收缩）并确定 CD36 在线粒体膜上积累的能力及其功能后果。为了确定特定信号事件在介导线粒体 CD36 运输中的必要性，将在转基因小鼠中或使用药物抑制剂进行类似的实验。 结合起来，拟议的研究将提供有关调节线粒体脂质转运机制的基础知识，这一过程对理解细胞稳态具有广泛的影响。