Mitochondrial OXPHOS regulation and the Rcf1/Hig1 protein family - a functional link between the ADP/ATP carrier and cytochrome c oxidase enzymes

 描述(由申请人提供)：线粒体通过氧化磷酸化(OXPHOS)消耗氧气(O2)的过程为细胞的新陈代谢需求产生ATP。因此，许多生物的生存依赖于O2的可用性，O2直接被OXPHOS系统的细胞色素c氧化酶(COX)酶利用。因此，了解线粒体如何调节OXPHOS系统的活动，以响应不同的代谢环境，或限制氧气条件(缺氧)，是至关重要的。线粒体生物能能力的降低是许多不同的神经和肌肉疾病的基础。线粒体O2失调和氧化应激也是许多疾病的特征，如癌症、缺血性心脏病、糖尿病和衰老。OXPHOS系统由电子转移复合体(ETC)(称为复合体I-IV)和F1Fo-ATP合成酶(也称为复合体V)组成，底物上的电子通过NADH和FADH2通过ETCs传递，将O2还原为水，这一步骤由COX酶(复合体IV)催化。ATP是由线粒体基质中的F1Fo-ATP合成酶合成的，由穿过内膜(IM)的pH梯度提供动力，并由ETC建立。ADP/ATP载体(AAC)蛋白将ATP运输出线粒体，分配给细胞的其余部分，并输入ADP，再循环回ATP。在IM中，OXPHOS酶相互作用形成超复合体，如细胞色素Bc1-COX-AAc超复合体(III-IV-AAc)。OXPHOS酶的超复杂组织被认为是为了使它们能够共同调节，然而，这是如何实现的分子细节尚不清楚。我们已经确定了一种名为Rcf1的蛋白质，它与COX酶的一个调节亚单位Cox3结合。我们发现RCF1也存在于与AAc蛋白非常接近的物理位置，特别是在H2-L-H3结构域，这是AAc蛋白的一个动态区域，对其功能至关重要。RCF1是低氧诱导基因家族1(Hig1)的成员，Hig1是一个高度保守的蛋白质家族，存在于所有真核生物中。Hig1蛋白家族包括结构性表达和应激诱导的异构体(如低氧、低糖应激或癌症)，表明它们在代谢环境改变的条件下调节OXPHOS活性。我们证明Rcf1/Hig1蛋白支持COX复合体的酶活性，同时与AAC蛋白相互作用并影响其功能。我们提出的实验旨在探索Rcf1是否在细胞色素Bc1-COX-AAc超复合体中作为生物能量构象传感器/调节器连接COX和AAC蛋白的活性。

 DESCRIPTION (provided by applicant): Mitochondria produce ATP for the cell's metabolic needs through an oxygen (O2) consuming process called oxidative phosphorylation (OXPHOS). The survival of many organisms is therefore dependent on the availability of O2, which is directly utilized by the cytochrome c oxidase (COX) enzyme of the OXPHOS system. Understanding how mitochondria regulate the activity of the OXPHOS system, in response to different metabolic environments, or limiting O2 conditions (hypoxia), is therefore of fundamental importance. Reduction in the bioenergetic capacity of mitochondria underlies numerous and diverse neurological and muscular diseases. Mitochondrial O2 dysregulation and oxidative stress are also hallmarks of many conditions such as cancers, ischemic heart disease, diabetes and aging. The OXPHOS system is composed of electron transfer complexes (ETC), referred to as complexes I-IV, and the F1Fo-ATP synthase enzyme, also termed complex V. Electrons from substrates are passed via NADH and FADH2 through the ETCs, to reduce O2 to water, a step catalyzed by the COX enzyme (complex IV). ATP is synthesized in the mitochondrial matrix by the F1Fo-ATP synthase, powered by a pH gradient across the inner membrane (IM) and established by the ETC's. The ADP/ATP carrier (AAC) protein transports the ATP out of the mitochondria for distribution to the rest of the cell and also imports ADP, for recycling back to ATP. The OXPHOS enzymes physically associate with each other to form supercomplexes in the IM, e.g. the cytochrome bc1-COX-AAC supercomplex (III-IV-AAC). The supercomplex organization of OXPHOS enzymes is proposed to enable their co-regulation, however, the molecular details of how this is achieved, are not clear. We have identified a protein, termed Rcf1, which binds to Cox3, a regulatory subunit of the COX enzyme. We found that Rcf1 also exists in close physical proximity to the AAC proteins, and in particular to the H2-L-H3 domain, a dynamic region of the AAC protein, critical for its function. Rcf1 is a member of the hypoxia-induced gene family 1 (Hig1), a highly conserved protein family found through all eukaryotic taxa. The Hig1 protein family includes both constitutively-expressed and stress-induced isoforms (e.g. hypoxic, low glucose stress or carcinomas), suggesting their role in modulating OXPHOS activity under conditions of altered metabolic environments. We demonstrate that Rcf1/Hig1 protein supports the enzyme activity of the COX complex, while at the same time interacts with, and influences the function of, AAC proteins. Our proposed experiments are designed to explore if Rcf1 functions as a bioenergetic conformational sensor/regulator coupling the activities of the COX and the AAC proteins, and within context of the cytochrome bc1-COX-AAC supercomplex.