Understanding basic regulation of mitochondrial bioenergetics and adaptations to exercise

在肌肉内，线粒体影响几个过程，包括代谢稳态、细胞凋亡和氧化还原平衡。然而，线粒体的调节仍然不清楚。因此，更好地理解这些动态结构可以提供对许多细胞过程的洞察。通过结合分子、生物化学和生理学方法，我的长期目标是确定a）调节线粒体生物能量学的机制，B）线粒体生物发生，以及c）了解这些如何影响骨骼肌中的底物利用。我的具体假设和短期计划是：*1.线粒体FAT/CD 36易位的调节：我以前已经表明，运动将FAT/CD 36重新分配到线粒体膜，然而，这种亚细胞运输的分子基础仍然未知。我推测，FAT/CD 36的C-末端YCACR基序和AMP激活的蛋白激酶（AMPK）信号传导都是这个细胞过程所必需的。我将野生型和C-末端突变体（C-末端氨基酸缺失）导入FAT/CD 36敲除小鼠的骨骼肌中。此后，我将提供代谢挑战（例如。坐骨神经刺激收缩），并确定FAT/CD 36在线粒体膜上积累的能力和对脂肪酸氧化/呼吸速率的功能后果。在野生型和AMPK激酶死亡小鼠中的类似实验将确定AMPK信号传导在介导线粒体FAT/CD 36易位中的必要性。2.肉毒碱棕榈酰转移酶-I（CPTI）丙二酰辅酶A（M-CoA）动力学的调节：我之前已经证明运动可以增强M-CoA抑制CPTI的能力，但其分子基础仍未知。为了确定CPTI的直接调节，将进行底物动力学（PCoA和M-CoA），并将免疫沉淀CPTI，并在人体运动前后检查氧化还原修饰和乙酰化状态。此外，在存在和不存在细胞骨架网络（特别是β-微管蛋白）的情况下，将以各种方式确定CPTI底物动力学，以确定细胞骨架网络调节CPTI的能力。3.线粒体网的代谢作用：我以前已经表明，过表达mitofusin-2（MFN 2）独立不影响线粒体生物能量学。因此，我假设MFN 2的主要代谢作用是将线粒体锚在肌浆网上，为ADP从SERCA转运到线粒体创造一个有效的微域。因此，我将在大鼠骨骼肌中过度和不足表达MFN 2，并随后确定a）肌肉疲劳率，B）收缩期间的细胞内钙水平，和c）钙刺激的线粒体呼吸动力学。** 四、线粒体生物发生的调节：关于影响线粒体生物发生的转录后机制的信息很少。在真核细胞中，已经鉴定了多种调节mRNA稳定性的RNA结合蛋白（RBP）。这些RBP在哺乳动物肌肉中的作用仍然未知。因此，我将在大鼠肌肉中单独和组合过表达HuR（稳定剂），CUB-BP 1和AUF 1（均为去稳定剂），以确定对mRNA谱，全肌肉代谢，线粒体含量/呼吸能力以及对急性和慢性肌肉收缩的反应的影响。意义：所有要求的方法学都已在我的实验室建立。拟议的研究将提供不同的细胞过程的基础知识，因此，我的实验室内的HQP将得到全面的培训。

Within muscle, mitochondria influence several processes, including metabolic homeostasis, apoptosis and redox balance. However, the regulation of mitochondria remains poorly elucidated. Therefore, a better understanding of these dynamic structures could provide insight into many cellular processes. By incorporating molecular, biochemical and physiological approaches my long-term goals are to determine a) mechanisms regulating mitochondrial bioenergetics, b) mitochondrial biogenesis, and c) understand how these effect substrate utilization in skeletal muscle.**My specific hypotheses and short-term plans are:*1. Regulation of mitochondrial FAT/CD36 translocation: I have previously shown that exercise redistributes FAT/CD36 to mitochondrial membranes, however, the molecular basis for this subcellular trafficking remains unknown. I hypothesize that the C-terminal YCACR motif of FAT/CD36 and AMP activated protein kinase (AMPK) signaling are both required for this cellular process. I will transfect wild type and C-terminal mutants (C-terminal amino acids deleted) into the skeletal muscle of FAT/CD36 knock out mice. Thereafter I will provide metabolic challenges (eg. sciatic nerve stimulated contraction) and determine the ability of FAT/CD36 to accumulate on mitochondrial membranes and the functional consequence on rates of fatty acid oxidation/respiration. Similar experiments in wild type and AMPK kinase dead mice will determine the necessity of AMPK signaling in mediating mitochondrial FAT/CD36 translocation.**2. Regulation of carnitine palmitoyltransferase-I (CPTI) malonyl-CoA (M-CoA) kinetics: I have previously shown that exercise augments the ability of M-CoA to inhibit CPTI, but the molecular basis for this remains unknown. To ascertain direct regulation of CPTI, substrate kinetics (PCoA and M-CoA) will be performed and CPTI will be immunoprecipitated and examined for redox modifications and acetylation status before and after exercise in humans. In addition, CPTI substrate kinetics will be determined various ways in the presence and absence of the cytoskeletal network (specifically ß-tubulin) to determine the ability of the cytoskeletal network to regulate CPTI.**3. The metabolic role of the mitochondrial reticulum: I have previously shown that over-expressing mitofusin-2 (MFN2) independently does not influence mitochondrial bioenergetics. I therefore hypothesize that the primary metabolic role of MFN2 is to anchor mitochondria to the sarcoplasmic reticulum, creating an efficient micro domain for ADP transport from SERCA to mitochondria. Therefore, I will over-and-under express MFN2 in rat skeletal muscle and subsequently determine a) muscle fatigue rates, b) intracellular calcium levels during contraction, and c) calcium stimulated mitochondrial respiration kinetics. **4. Regulation of mitochondrial biogenesis: Scarce information exists regarding post-transcriptional mechanisms that influence mitochondrial biogenesis. In eukaryotic cells a variety of RNA binding proteins (RBPs) have been identified which regulate mRNA stability. The role of these RBPs in mammalian muscle remains unknown. Therefore, I will over-express HuR (stabilizer), CUB-BP1 and AUF1 (both destabilizers) alone and in combination in rat muscle to determine the effect on mRNA profiles, whole muscle metabolism, mitochondrial content/respiratory capacity, and responses to acute and chronic muscle contraction.**SIGNIFICANCE; All required methodologies have been established in my laboratory. The proposed studies will provide fundamental knowledge on diverse cellular processes, and therefore, HQP within my laboratory will be comprehensively trained.