Defining Molecular Interactions that Drive Mitochondrial Fission

摘要 线粒体分裂的分子机制尚不清楚。这一过程是必不可少的，而且密切相关 通过对线粒体分裂复合体的主要蛋白质因子进行各种修饰来调节， Drp1.这项提议的目标是确定线粒体分裂复合体中的关键分子相互作用。 并了解影响这些相互作用并最终影响线粒体的调控变化 裂变。首先，将研究膜模板上较大的螺旋Drp1低聚物的结构，以确定 促进线粒体表面自组装的Drp1蛋白的构象变化。 特别是，冷冻-EM和线粒体分离研究将并行进行，以揭示相互作用 在邻近膜的线粒体分裂复合体内。同时，详细地 将使用蛋白质组学和突变方法来鉴定Drp1复合体内的相互作用。影响因素 扰动的Drp1自组装也会破坏细胞器的形态，因此Drp1与基本伙伴的相互作用 蛋白质，MFF，也将被检测。具体地说，蛋白质共聚体将使用结构表征 方法揭示Drp1靶向线粒体表面的机制以及MFF是如何 有助于进行高效的线粒体分裂。为了模仿与人类疾病相关的变化，Drp1 序列将使用定点突变来重述由以下原因引起的自然序列变化 翻译后修饰。这些变化的全部影响将使用结构和 生化分析，以及健康和患病线粒体分裂复合体之间的显著差异 将会被确认。这些相同的变化将被引入细胞培养中，以关联线粒体的变化 在体外观察到形态和功能的变化。为了进一步描述这些变化的影响，我们 计划评估稳定的细胞系表达仿磷突变体的生物能量能力。此外， Drp1及其伙伴蛋白的组装特性将在翻译后背景下进行分析 修饰以揭示改变线粒体网络的细胞信号改变的潜在影响。 总而言之，这个项目试图确定线粒体分裂复合体中的详细特征 对特定的疾病状态有贡献。这些差异为未来的发展提供了新的目标 防止神经系统疾病中细胞死亡的治疗方法，这些疾病与Drp1活性增加有关 和过度的线粒体分裂。

ABSTRACT The molecular mechanism of mitochondrial fission is not known. This process is essential and closely regulated through a variety of modifications to the major protein factor of the mitochondrial fission complex, Drp1. The goal of this proposal is to identify key molecular interactions within the mitochondrial fission complex and to understand regulatory changes that influence these interactions and ultimately affect mitochondrial fission. To begin, structures of larger, helical Drp1 oligomers on membrane templates will be studied to identify conformational changes in the Drp1 protein that promote self-assembly at the surface of mitochondria. Specifically, cryo-EM and mitochondrial isolation studies will be performed in parallel to reveal interactions within the mitochondrial fission complex adjacent to the neighboring membrane. In parallel, detailed interactions within Drp1 complexes will be identified using proteomics and mutagenesis methods. Factors that perturb Drp1 self-assembly also disrupt organelle morphology, so Drp1 interactions with the essential partner protein, Mff, will also be examined. Specifically, protein co-polymers will be characterized using structural methods to uncover the mechanism by which Drp1 is targeted to the surface of mitochondria and how Mff contributes to productive mitochondrial fission. To mimic changes associated with human disease, Drp1 sequence will be altered using site-directed mutagenesis to recapitulate natural sequence changes caused by post-translational modifications. The full effects of these changes will be assessed using structural and biochemical assays, and noticeable differences between healthy and diseased mitochondrial fission complexes will be identified. These same changes will be introduced in cell culture to correlate alterations in mitochondrial morphology to functional alterations observed in vitro. To further characterize the impact of these changes, we plan to assess the bioenergetic capacities of stable cell lines expressing phosphomimetic mutants. Moreover, the assembly properties of Drp1 and its partner proteins will be assayed in the context of post-translational modifications to reveal the underlying effects of cell signaling alterations that modify the mitochondrial network. Collectively, this project seeks to identify detailed features within the mitochondrial fission complex that contribute to specific disease states. These differences provide novel targets for the development of future therapeutics that prevent cell death in neurological disorders, which are associated with increased Drp1 activity and excessive mitochondrial fission.