Cyclophilin-D: A Regulator of Mitochondrial Oxidative Phosphorylation

亲环素-D(Cyp-D)是一种催化肽基顺反异构化的肽基-脯氨酰异构酶 脯氨酸键，从而调节目标蛋白的构象变化。CYP-D是唯一的亲环素- 在线粒体中存在，直到最近，人们还知道它对线粒体通透性传递的影响。 喷气孔。我们最近发现，Cyp-D与三种线粒体转录因子中的两种相互作用 (即TFB1M和TFB2M，而不是TFAM)，随后发现Cyp-D也与有丝分裂粒相互作用- 干式RNA聚合酶。Cyp-D在分离细胞中的遗传沉默干扰线粒体基因表达 导致线粒体耗氧量(VO2)减少。最近，使用一个结构性的Cyp-D 在剔除(Cyp-D KO)小鼠后，我们还显示了体内VO2(休息和运动时)的减少。伴随而来的是， 呼吸交换比率增加，表明代谢转变有利于碳水化合物的利用。 胖的。有趣的是，运动能力的增加表明了一种适应性反应，即氧气的利用 提高了效率。在计算机模拟中，我们已经识别出能够破坏分子稳定的小分子 Cyp-D与TFB2M的相互作用。因此，我们假设Cyp-D在氧代谢中起关键作用-- 调节线粒体基因表达和信号适应性氧气增加的代谢 利用效率。我们建议基于两个特定的目标来进一步推进这一假设。具体目标1：消除 签署完成Cyp-D对线粒体基因表达影响的表征 并检查其他下游过程--也是由Cyp-D介导的--可能影响 细胞呼吸(结构为4个子目标)。子目标1，旨在完全描述相互作用 在线粒体转录机制的关键参与者之间，并评估他们对Cyp-D PPI的依赖- 酶活性；亚目标2，旨在确认Cyp-D对线粒体基因表达的影响是 HSP2特异性(如初步数据所示)，并评估Cyp-D沉默是否可以发出信号上调- 流动并影响呼吸复合体的核编码蛋白的表达；次级目标3，设计 证实Cyp-D沉默减少呼吸链复合体线粒体编码亚基 和F1FoATP合成酶，并检查Cyp-D过度表达是否会导致相反的效果； 目的4、探讨Cyp-D是否也作为伴侣参与了呼吸复合体的组装。 特定目标1下的研究将在成年小鼠的原代心肌细胞、成纤维细胞和组织中进行。 来自Cyp-D KO和野生型(WT)小鼠(如果相关)。CYP-D将通过慢病毒载体进行调节 含有沉默或过表达Cyp-D的构建体和使用环孢素A的药物抑制 (CsA)和新发现的小分子。具体目标2：旨在确定功能结构-- Cyp-D在小鼠体内的遗传消融或药物抑制的序列，并检测其潜在的adap-D。 提高氧气利用效率的积极机制。CYP-D活性将通过使用组织- 活动性和条件性Cyp-D KO小鼠及环孢素A和小分子的药理抑制作用 能够选择性地破坏Cyp-D和TFB2M之间的相互作用。对于构成Cyp-D KO的小鼠，我们 将评估急性Cyp-D消融后细胞系统的发现是否也发生在体内，并检查 适应性反应提高了氧气的利用效率。对于有条件的Cyp-D KO小鼠，我们将- 评估Cyp-D消融的即刻效果和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作用。心肌能量效应沿线 将使用31P磁共振波谱和磁共振仪评估有功能的心肌效应 磁共振成像。了解提高氧气利用效率的潜在机制可能会 对人类生理学和疾病的重要影响，并计划还检查急性情况下的影响 使用心脏骤停和失血性休克模型的氧气供应减少。

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.