Respiratory Complex III: Supercomplexes and ROS, from Bacteria to Human

描述(由申请人提供)：生物有机体的一个重要特征是它们有效地产生生物能量的能力。三磷酸腺苷是许多细胞功能所必需的，它的合成依赖于电子传递链(ETC)的呼吸复合体(RC)。复合体III(CIII)是ETC的中心，以单独的RC或与其他RC结合形成超复合体(SC)存在。含有CIII的活性干细胞以及复合体I和IV(呼吸体)被认为在呼吸过程中提供稳定性、通道底物和屏蔽有毒中间体。RCS和SCs功能异常降低细胞的能量效率，增加有毒的活性氧(ROS)，导致氧化损伤。CIII是线粒体膜间隙产生超氧化物(O2.-)的主要部位，CIII功能障碍会导致肌肉和神经疾病。因此，CIII研究对人类健康具有重要意义。我们的目标是了解含有CIII的SCs的形成、稳定和调控，它们对能量转导、ROS产生的影响，以及对线粒体疾病的后果。最近，我们发现了一种细胞色素b突变，使CIIIO2敏感，增加了细菌和人类体内ROS的产生，并改变了线粒体中的SCs组织。这种突变在人类中引起多系统紊乱，其同源的胶囊红杆菌氧化分解CIII的2Fe2S辅因子。使用细菌和人类囊胞虫模型，我们将研究位于Cytb关键区域的类似的与疾病相关的Cytb突变，该突变影响CIII QO位点ROS的产生并改变SCs组织。我们假设RCS的一些“辅助”(即非催化)亚基作为SC促进器(SCF)，可能受到O2/ROS的调节，以调节ETC的动态可塑性。新出现的细菌SCF候选蛋白是小的“单一跨膜-酸性结构域”(STMD)拓扑蛋白。它们包括线粒体Rcf1/HIG2A、牛CIII亚基VII和MCJ/DNAJ15的CcoH、FbcQ和Dna JL同源物。具体地说，我们将1-研究一组产生ROS和改变SCs组织的疾病相关线粒体Cytb突变体，以阐明CIIIO2敏感性和SCs改变的机制基础；2-构建相应线粒体Cytb突变的细菌同源物，并表征它们对O2/ROS的QO位点ROS产生和SCs组织的响应；以及3-确定线粒体RCS附属亚基的细菌同源物作为SCF的作用，并评估其对O2的调节，以更好地了解SCF在SCs形成和调控中的生理作用。我们将从我们已经生产的CIII~CIV开始，构建和表征活性融合干细胞，并评估扰动ETC可塑性的后果。这些研究将有助于我们理解SCs形成、稳定和调控的分子基础，建立CIII QO位点ROS产生、SCs改变及其疾病相关后果之间的相互联系，最终改善线粒体疾病的诊断和治疗。

DESCRIPTION (provided by applicant): A vital characteristic of living organisms is their ability to produce biological energy (ATP) efficiently. ATP is required for a myriad of cellular functions, and its synthesis relies on respiratory complexes (RCs) of the electron transport chain (ETC). The Complex III (CIII) is central to ETC, and exists as an individual RC or in association with other RCs to form supercomplexes (SCs). Active SCs that contain CIII together with the Complexes I and IV (respirasomes) are thought to provide stability, channel substrates, and shield toxic intermediates during respiration. Improper functions of RCs and SCs decrease energetic efficiency of cells, and increase toxic reactive oxygen species (ROS), leading to oxidative damages. CIII is a major site of superoxide (O2.-) production on the inter membrane space of mitochondria, and CIII malfunctions lead to muscular and neurological diseases. Hence, CIII studies are significant for human health. Our goal is to understand the formation, stability and regulation of CIII containing SCs, their impact on energy transduction, ROS production, and consequences for mitochondrial diseases. Recently, we found a cyt b mutation that renders CIII O2-sensitive, enhances ROS production in both bacteria and humans, and alters SCs organizations in mitochondria. This mutation causes multisystem disorder in humans, and its Rhodobacter capsulatus homologue oxidatively disintegrates the 2Fe2S cofactor of CIII. Using bacterial and human cybrids models, we will investigate similar disease-associated cyt b mutations located at a critical region of cyt b that affects CIII Qo site ROS production and alter SCs organizations. We hypothesize that some of the "accessory" (i.e., non-catalytic) subunits of RCs act as SC facilitators (SCFs) that might be regulated by O2/ROS to modulate the dynamic plasticity of the ETC. Emerging bacterial SCF candidates are small, "single trans membrane-acidic domain" (STMD) topology proteins. They include CcoH, FbcQ and DnaJL homologues of mitochondrial Rcf1/HIG2A, bovine CIII subunit VII and MCJ/DNAJ15. Specifically, we will 1- study a group of disease-associated mitochondrial cyt b mutants that produce ROS and alter SCs organizations to elucidate the mechanistic basis of CIII O2 sensitivity and SCs alterations; 2- construct bacterial homologues of the corresponding mitochondrial cyt b mutations, and characterize them for Qo site ROS production and SCs organizations in response to O2/ROS; and 3- determine the role of bacterial homologues of mitochondrial RCs accessory subunits as SCFs, and evaluate their O2 regulation to better understand the physiological roles of SCFs in SCs formation and regulation. We will construct and characterize active fused-SCs, starting with CIII~CIV that we already produced, and evaluate the consequences of perturbing the plasticity of ETC. These studies will contribute to our understanding of the molecular basis of SCs formation, stability and regulation, to establish the interconnections between the CIII Qo site ROS production, SCs alterations, and their disease- associated consequences to eventually improve the diagnostic and treatment of mitochondrial diseases.