Understanding the Mechanisms of Respiratory Supercomplexes and mitochondrial Complex I

项目摘要/摘要 线粒体氧化磷酸化电子传递链(ETC)由五个大分子组成 膜蛋白复合体(CI、CII、CIII2、CIV和CV)，负责大多数的产生 细胞内的三磷酸腺苷。因此，ETC对生物能量代谢是必不可少的。ETC缺陷是 最常见的诊断为先天性代谢缺陷，CI缺陷约占三分之一 这些诊断。尽管~50%的CI缺乏症患者在生命的头两年内死亡，而且只有 ~25%达到10岁，CI仍然是所有ETC复合体中机制了解最少的。 此外，尽管有大量的医疗需求，但目前还没有有效的治疗CI或其他ETC的方法 不足之处。这种差异在一定程度上是由于对其分子机制的不完全理解。 单独的络合物及其更高阶的组装形成超络合物(SC)。在哺乳动物的心脏 线粒体大多数CI与CIII2和CIV(SC I+III2+IV，呼吸系统)或 与CIII2(SC I+III2)的联系。最近的生化和结构研究已经产生了第一个原子-- 哺乳动物线粒体CI的分辨结构和单个复合体的排列 在呼吸系统和SC I+III2中。然而，在功能、机制方面仍然存在重大问题 以及对ETC复合体和SC的调节。为了解决我们理解中的这些差距，并发展 支持ETC缺陷潜在治疗策略的基础科学，我们将建立两个主要 我实验室里的研究方向。将详细的生化和酶分析与单颗粒结合使用 冷冻电子显微镜的结构表征，我们将阐明其机制、功能和 对1)分离CI和2)呼吸SCs的调节。为了实现这一目标，我们建议进行系统的 从哺乳动物线粒体纯化的呼吸CI和SCs的功能和结构比较 HeLa细胞培养和猪心组织培养)、反硝化副杆菌a-蛋白杆菌和真菌 模式系统为粗糙脉孢菌。脱氮假单胞菌是与原始生物最接近的生物体之一。 内生共生事件后形成线粒体的变形杆菌。克拉沙藻是一种成熟的 生物能量学强大的遗传和生化系统，但没有高分辨率等 结构是可用的。比较这些分化和遗传上易驯化的生物的CI和SCs 将使我们能够在该领域测试几个关键的机械假说，并 识别CI和SC的保守特征及其机制和调控。这将为我们提供对 CI的能量转换机制和SC形成的生理作用将定义科学的 为制定针对CI和进一步的ETC缺陷的治疗策略所需的基础。

PROJECT SUMMARY/ABSTRACT The mitochondrial oxidative phosphorylation electron transport chain (ETC) is composed of five large membrane protein complexes (CI, CII, CIII2, CIV and CV) and is responsible for the production of the majority of cellular ATP. Consequently, the ETC is essential to bioenergetic metabolism. ETC defects are one of the most commonly diagnosed congenital metabolic defects, with CI deficiencies representing roughly a third of these diagnoses. Although ~50% of patients with CI deficiencies die within the first 2 years of life and only ~25% reach 10 years of age, CI remains the least well mechanistically understood of all the ETC complexes. Furthermore, despite the large medical need, there are currently no effective treatments for CI or other ETC deficiencies. This discrepancy stems in part from an incomplete understanding of the molecular mechanisms of the individual complexes and their higher-order assemblies into supercomplexes (SCs). In mammalian heart mitochondria the majority of CI is found in association with CIII2 and CIV (SC I+III2+IV, the respirasome) or in association with CIII2 (SC I+III2). Recent biochemical and structural work has produced the first atomic- resolution structures of mammalian mitochondrial CI and defined the arrangement of the individual complexes within the respirasome and SC I+III2. However, significant questions remain regarding the function, mechanism and regulation of the ETC complexes and SCs. To address these gaps in our understanding and to develop the basic science that will underpin potential treatment strategies of ETC defects, we will establish two major research directions in my lab. Using detailed biochemical and enzymatic analyses together with single particle cryo-electron microscopy structural characterizations, we will elucidate the mechanisms, functions and regulation of 1) isolated CI and 2) respiratory SCs. To achieve this, we propose to perform systematic functional and structural comparisons of respiratory CI and SCs purified from mammalian mitochondria (from both HeLa cell culture and porcine heart tissue), the a-proteobacteria Paracoccus denitrificans and the fungal model system Neurospora crassa. P. denitrificans is one of the closest living organisms to the ancestral a- proteobacteria that originated mitochondria after the endosymbiotic event. N. crassa is an established, powerful genetic and biochemical system for bioenergetics, for which nonetheless no high-resolution ETC structures are available. Comparing the CI and SCs from these divergent and genetically tractable organisms to their mammalian counterparts will allow us to test several key mechanistic hypotheses in the field and to identify the conserved features of CI and SC mechanism and regulation. This will provide deep insights into the energy-converting mechanism of CI and the physiological roles of SC formation, which will define the scientific foundation needed for the development of therapeutic strategies against CI and further ETC deficiencies.