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.

环蛋白-D（CYP-D）是一种肽基类蛋白酶异构酶，可催化肽基的顺式传播异构化脯氨酰键，从而调节靶蛋白的构象变化。 cyp-d是唯一的环磷脂res- 在线粒体中的识别，直到最近，它以对线粒体通透性的影响而闻名孔。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钻RNA聚合酶。分离的细胞中CYP-D的遗传沉默破坏了线粒体基因的表达减少线粒体O2消耗量（VO2）。最近，使用构成CYP-D 敲出（CYP-D KO）小鼠，我们还显示了VO2（休息和运动）中的体内减少。同时，呼吸道交换比率提高，表明代谢转移有利于利用碳氢胖的。有趣的是，运动能力提高了指向自适应反应，从而使用了O2利用率效率提高。在使用硅建模中，我们已经确定了能够不稳定的小分子 CYP-D和TFB2M之间的相互作用。这就是我们假设CYP-D在氧气中起关键作用通过调节线粒体基因表达和信号表达氧气的自适应增加，可以利用效率。我们建议基于两个特定目标进一步进一步假设。特定目标1：签名以完成使用心脏的CYP-D效应对线粒体基因表达的表征相关的组织和检查其他下游过程（也是由CYP-D介导的）可能会影响的细胞呼吸（以4个亚目标结构）。 sub-aim 1，旨在完全表征互动线粒体转录机械的主要参与者和评估他们对CYP-D PPI的依赖 ASE活动； Sub-aim 2，旨在确认CYP-D对线粒体基因表达的影响是特异流并影响呼吸复合物的核编码蛋白的表达； Sub-aim 3，设计为了确认CYP-D沉默减少了呼吸链复合物的线粒体编码亚基 F1FOATP合酶和检查CYP-D过表达是否会产生相反的作用；和子 - 目的4，研究CYP-D是否也充当伴侣辅助呼吸复合物组装的伴侣。在成年小鼠原发性心肌细胞，成纤维细胞和组织中将进行特定目标1的研究相关时，来自CYP-D KO和野生型（WT）小鼠。 CYP-D将通过使用慢病毒向量调节使用环孢菌素A携带构造以使CYP-D沉默或过表达CYP-D和药物抑制（CSA）和新鉴定的小分子。特定目的2：旨在确定功能性结合查询小鼠中CYP-D的遗传消融或药物抑制，并检查了基础适配器导致O2利用效率提高的机制。 CYP-D活性将通过使用构造来降低 Tive和条件CYP-D KO小鼠，并通过使用环孢菌素A和小分子进行药物抑制与组成型CYP-D KO小鼠，我们将评估急性CYP-D消融后细胞系统中的发现是否也发生在体内并检查自适应反应导致O2利用效率提高。使用有条件的CYP-D KO小鼠，我们将 SESS CYP-D消融的直接影响以及是否发生适应性反应，随后检查 - 是否可以通过药物抑制引起类似的影响。心肌能量影响具有功能性心肌效应将使用31p磁共振光谱和磁性评估共振成像。了解增加O2利用效率的潜在机制可能具有对人类生理和疾病的重要意义，并计划还检查急性条件下的影响使用心脏骤停模型和出血性休克降低O2的可用性。