Structure/Function of Complex II Oxidoreductase

描述(申请人提供)：复合II酶是具有两个空间分离的活性中心的整膜异构体，它们将琥珀酸和富马酸的氧化还原与对苯二酚和对苯二酚的氧化还原结合在一起。在人类中，复合体II是一种线粒体酶，通过参与TCA循环(通过琥珀酸氧化)和氧化磷酸化(通过苯醌还原)来促进能量生成，其突变与肿瘤形成和神经退化有关。这项研究计划的长期目标是揭示催化、组装和生理功能的机制。在这项拟议的研究中，我们将确定复杂II组装因子的作用，揭示复杂II如何在体内信号转导中发挥作用，并展示疾病相关突变如何改变复杂II的功能。目的1：直到最近，复合体II的组装还被认为是自动催化进行的，但现在已经在人类身上发现了两个组装因子。两者都被认为是辅助辅助因子插入的，但已发表的支持这一点的证据很少，也没有一致的组装机制。了解这些组装因子的作用将有助于深入了解这种重要蛋白质复合体的组装的基本机制。我们将使用大肠杆菌复合体II来揭示每个组装因子的作用，确定它们是否直接或间接发挥作用，并通过基因缺失和互补、突变、体内位点特异性交联、结合分析和结晶学来鉴定是否有额外的组装因子。目的2：在人类和细菌中，生物能量学以外的功能都需要复杂的II活性。例如，在大肠杆菌中，鞭毛的组装和对富马酸的趋化反应需要复合体II同系物QFR的活性。由于未来的抗菌药可能会破坏细菌的运动，因此揭示QFR如何支持鞭毛组装将是重要的。我们将结合位点特定的交联、结合分析、运动性分析、结晶学和计算建模来确定QFR如何影响鞭毛扭矩环的构象以促进组装和影响趋化性。目的3：编码人类复合体II的基因突变与多效性临床表现有关。文献中已经报道了与这些突变相关的细胞变化，但关于这些突变对酶活性的生化后果几乎一无所知。确定与复杂II突变相关的生化变化对于开发未来的线粒体疾病治疗方法非常重要。因此，我们将开发一个人类复合体II的表达系统，并评估疾病相关突变对酶组装、动力学和结构的影响。

DESCRIPTION (provided by applicant): Complex II enzymes are integral-membrane heterotetramers with two spatially separated active sites that couple the oxidoreduction of succinate and fumarate to the oxidoreduction of quinone and quinol. In humans, Complex II is a mitochondrial enzyme that contributes to energy generation by participating in both the TCA cycle (via succinate oxidation) and oxidative phosphorylation (via quinone reduction), and its mutation is associated with tumor formation and neurodegeneration. The long-term objective of this research program is to reveal mechanisms of catalysis, assembly, and physiological function. In the proposed research, we will identify roles of Complex II assembly factors, reveal how Complex II contributes to in vivo signaling, and demonstrate how disease-associated mutations alter Complex II function. Aim 1: Until very recently, Complex II assembly was thought to proceed autocatalytically, but two assembly factors have now been identified in humans. Both are proposed to assist in cofactor insertion, but the published evidence supporting this is tenuous and there is no consensus mechanism of assembly. Understanding the roles of these assembly factors will provide insight into fundamental mechanisms of assembly of this essential protein complex. We will use Escherichia coli Complex II to reveal the role of each assembly factor, identify if they work directly or indirectly, and identify whether thre are additional assembly factors using gene deletion and complementation, mutagenesis, in vivo site-specific cross-linking, binding assays, and crystallography. Aim 2: In both humans and bacteria, Complex II activity is required for functions outside of bioenergetics. For example, in E coli, assembly of the flagellum and the chemotactic response to fumarate requires activity of the Complex II homolog QFR. Since future antimicrobials could be directed toward disruption of bacterial motility, it will be important to reveal how QFR supports flagellar assembly. We will combine site-specific cross- linking, binding assays, motility assays, crystallography, and computational modeling to identify how QFR influences the conformation of the flagellar torque ring to promote assembly and influence chemotaxis. Aim 3: Mutations in the genes encoding human Complex II are associated with pleiotropic clinical presentations. Cellular changes associated with these mutations have been reported in the literature, but almost nothing is known about the biochemical consequences of these mutations on enzyme activity. Identifying the biochemical changes associated with Complex II mutation is important for developing future therapies for mitochondrial disease. We will therefore develop an expression system for human Complex II and assess the consequences of disease-associated mutations on enzyme assembly, kinetics, and structure.