Cyclophilin-D: A Regulator of Mitochondrial Oxidative Phosphorylation

亲环蛋白-D：线粒体氧化磷酸化的调节剂

基本信息

批准号：
10265322
负责人：
Raul Jaime Gazmuri
金额：
--
依托单位：
EDWARD HINES JR VA HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10265322
关键词：
Ablation Acute Adult Affect Aging Animals Awareness Carbohydrates Cardiac Cardiac Myocytes Cell Respiration Cell physiology Cells Complex Consumption Cyclophilins Cyclosporine DNA-Directed RNA Polymerase Data Dependence Disease Duct (organ) structure EFRAC Electron Transport Exercise Fatty acid glycerol esters Fibrinogen Fibroblasts Gene Expression Genes Genetic Genetic Transcription Grant Heart Arrest Heart failure Hemorrhagic Shock Human In Vitro Ischemia Kinetics Knock-out Knockout Mice Left Lentivirus Vector Life Magnetic Resonance Imaging Magnetic Resonance Spectroscopy Malignant Neoplasms Measures Mediating Metabolic Metabolism Mitochondria Mitochondrial Proteins Mitochondrial RNA Modeling Molecular Chaperones Molecular Conformation Mus Myocardial Neurodegenerative Disorders Nuclear Nutrient Organ Oxidative Phosphorylation Oxygen Oxygen saturation measurement Peptidylprolyl Isomerase Pharmacology Physiology Play Process Production Proteins Reperfusion Therapy Resistance Respiration Respiratory Chain Rest Role Signal Transduction Stream Structure System Testing Tissues Vascular Diseases Ventricular Veterans Wild Type Mouse Work base cis trans isomerization cyclophilin D design exercise capacity experimental study in silico in vivo indexing inhibitor/antagonist mitochondrial genome mitochondrial permeability transition pore overexpression promoter respiratory respiratory protein response small molecule stressor transcription factor treadmill

项目摘要

Cyclophilin-D (Cyp-D) is a peptidyl prolyl isomerase which catalyzes the cis-trans isomerization of peptidyl prolyl bonds and thereby regulates conformational changes of target proteins. Cyp-D is the only cyclophilin res- ident in mitochondria, and until recently it was best known for its effect on the mitochondrial permeability transi- tion pore. We recently discovered that Cyp-D interacts with two of the three mitochondrial transcription factors (i.e., TFB1M and TFB2M but not TFAM) and subsequently found that Cyp-D also interacts with the mitochon- drial RNA polymerase. Genetic silencing of Cyp-D in isolated cells disrupted mitochondrial gene expression re- sulting in a reduction in mitochondrial O2 consumption (VO2). More recently, working with a constitutive Cyp-D knock out (Cyp-D KO) mice, we showed also in vivo reductions in VO2 (at rest and exercise). Concomitantly, the respiratory exchange ratio increased suggesting a metabolic shift favoring utilization of carbohydrates over fat. Intriguingly, the exercise capacity was increased pointing to an adaptive response whereby O2 utilization efficiency was increased. Using in silico modeling, we have identified small molecules able to destabilize the interaction between Cyp-D and TFB2M. Thus, we hypothesize that Cyp-D plays a key role in oxygen me- tabolism by regulating mitochondrial gene expression and signaling an adaptive increase in oxygen utilization efficiency. We propose to further this hypothesis based on two specific aims. Specific Aim 1: De- signed to complete the characterization of the Cyp-D effects on mitochondrial gene expression using cardiac relevant tissue and examine additional downstream processes – also mediated by Cyp-D – that may impact cell respiration (structured in 4 Sub Aims). Sub-Aim 1, designed to completely characterize the interactions among key players of the mitochondrial transcription machinery and assess their dependency on Cyp-D PPI- ase activity; Sub-Aim 2, designed to confirm that the effects of Cyp-D on mitochondrial gene expression are HSP2 specific (as suggested by preliminary data) and to assess whether Cyp-D silencing could signal up- stream and affect the expression of nuclear-encoded proteins of respiratory complexes; Sub-Aim 3, designed to confirm that Cyp-D silencing reduces mitochondrial encoded subunits of the respiratory chain complexes and F1FoATP synthase and examine whether Cyp-D overexpression results in the opposite effect; and Sub- Aim 4, to investigate whether Cyp-D also acts as chaperone aiding in the assembly of respiratory complexes. Studies under Specific Aim 1 will be conducted in adult mouse primary cardiomyocytes, fibroblasts, and tissues from Cyp-D KO and wild-type (WT) mice when relevant. Cyp-D will be modulated by using lentiviral vectors harboring constructs to silence or overexpress Cyp-D and pharmacological inhibition using cyclosporine A (CsA) and newly identified small molecules. Specific Aim 2: Designed to determine the functional conse- quences of genetic ablation or pharmacological inhibition of Cyp-D in mice and examine the underlying adap- tive mechanisms leading to increased O2 utilization efficiency. Cyp-D activity will be reduced by using constitu- tive and conditional Cyp-D KO mice and by pharmacological inhibition with cyclosporine A and small molecules able to selectively destabilize the interaction between Cyp-D and TFB2M. With constitutive Cyp-D KO mice, we will assess whether the findings in cell systems after acute Cyp-D ablation also occur in vivo and examine the adaptive responses leading to increased O2 utilization efficiency. With conditional Cyp-D KO mice, we will as- sess the immediate effects of Cyp-D ablation and whether an adaptive response occurs, subsequently examin- ing whether similar effects can be elicited through pharmacological inhibition. Myocardial energy effects along with functional myocardial effects will be assessed using 31P magnetic resonance spectroscopy and magnetic resonance imaging. Understanding the underlying mechanisms of increase O2 utilization efficiency may have important implications for human physiology and disease and plan to also examine effects in acute conditions of reduced O2 availability using models of cardiac arrest and hemorrhagic shock.

Cyclophilin-D (Cyp-D) 是一种肽基脯氨酰异构酶，可催化肽基的顺反异构化 Cyp-D 是唯一的亲环蛋白抑制剂。直到最近，它因其对线粒体通透性转运的影响而闻名。我们最近发现 Cyp-D 与三个线粒体转录因子中的两个相互作用。（即 TFB1M 和 TFB2M，但不是 TFAM），随后发现 Cyp-D 也与线粒体相互作用分离细胞中 Cyp-D 的基因沉默破坏了线粒体基因表达。最近，与组成型 Cyp-D 一起工作，导致线粒体 O2 消耗 (VO2) 减少。敲除 (Cyp-D KO) 小鼠，我们还发现体内摄氧量 (VO2) 降低（同时在休息和运动时）。交换呼吸比增加表明代谢转变有利于碳水化合物的利用有趣的是，运动能力的增加表明了氧气利用的适应性反应。使用计算机模拟，我们已经确定了能够破坏稳定的小分子。 Cyp-D 和 TFB2M 之间的相互作用因此，我们认为 Cyp-D 在氧代谢中起着关键作用。通过调节线粒体基因表达和发出适应性氧增加信号来进行代谢我们建议基于两个具体目标进一步推进这一假设：De-。签署以使用心脏完成 Cyp-D 对线粒体基因表达的影响的表征相关组织并检查其他下游过程（也由 Cyp-D 介导），这可能会影响细胞呼吸（由 4 个子目标 1 组成），旨在完全表征相互作用。线粒体转录机制的关键参与者之一，并评估它们对 Cyp-D PPI 的依赖性酶活性；子目标 2，旨在确认 Cyp-D 对线粒体基因表达的影响 HSP2 特异性（如初步数据所示）并评估 Cyp-D 沉默是否可以发出上行信号流并影响呼吸复合物核编码蛋白的表达，设计；确认 Cyp-D 沉默减少了呼吸链复合物的线粒体编码亚基和 F1FoATP 合酶并检查 Cyp-D 过度表达是否会导致相反的效果；目标 4，研究 Cyp-D 是否也充当协助呼吸复合物组装的伴侣。具体目标 1 下的研究将在成年小鼠原代心肌细胞、成纤维细胞和组织中进行来自 Cyp-D KO 和野生型 (WT) 小鼠的 Cyp-D 在相关时将通过使用慢病毒载体进行调节。含有沉默或过表达 Cyp-D 的构建体以及使用环孢菌素 A 的药理学抑制 (CsA) 和新发现的小分子具体目标 2：旨在确定功能性结果。小鼠体内 Cyp-D 的基因消除或药理学抑制的序列，并检查潜在的适应性通过使用本构，可以降低导致 O2 利用效率提高的 Cyp-D 活性的机制。主动和条件性 Cyp-D KO 小鼠以及通过环孢素 A 和小分子的药理抑制通过组成型 Cyp-D KO 小鼠，我们能够选择性地破坏 Cyp-D 和 TFB2M 之间的相互作用。将评估急性 Cyp-D 消融后细胞系统中的发现是否也发生在体内，并检查适应性反应导致 O2 利用效率提高。对于条件性 Cyp-D KO 小鼠，我们将：评估 Cyp-D 消融的直接影响以及是否发生适应性反应，随后检查研究是否可以通过药物抑制心肌能量效应来引发类似的效果。将使用 31P 磁共振波谱和磁力评估对功能性心肌的影响了解提高 O2 利用效率的潜在机制可能具有以下作用：对人类生理学和疾病的重要影响，并计划还研究在急性条件下的影响使用心脏骤停和失血性休克模型观察氧气可用性的降低。