Defining Molecular Interactions that Drive Mitochondrial Fission

项目概要（摘要） 线粒体是一种双膜细胞器，它的形状、大小和丰度会随着 具体的刺激。控制线粒体分裂的蛋白质相互作用受到严格调控，并直接影响 ATP产生、Ca 2+稳态和程序性细胞死亡的调节。线粒体 动力学最近成为几种退行性疾病的治疗靶点，包括 神经变性、癌症和心血管疾病。但缺乏对这一监管的洞察力， 过程是一个很大的限制。线粒体分裂的主要驱动力是细胞质GT3，动力蛋白相关的 蛋白1（Drp 1）。为了介导膜断裂，Drp 1的募集和自组装通过以下方式协调： 与线粒体表面的脂质、蛋白质和核苷酸的组合相互作用。这项建议 旨在确定线粒体分裂机制的关键属性，以及Drp 1的失调如何导致 细胞器损伤和细胞退化。这将通过多方面的方法来实现， 将分子研究与功能细胞实验相结合，以提供对Drp 1的全面评估 控制膜重塑的相互作用。根据更新的具体目标1，冷冻EM研究将 检查限制胞质状态中Drp 1寡聚化的自抑制相互作用。不同构象 将进行研究，以鉴定和表征Drp 1募集和组装过程中的中间结构 变成一个功能性的裂变复合体我们认为，受调控的重排“打开”了分子的功能， 在线粒体分裂的特定位点组装。对于特定目标2，复溶实验提供了 评估驱动线粒体膜重塑的大分子相互作用的手段。具体 线粒体的线索，包括脂质和伴侣蛋白，将被研究，以评估每个贡献 细胞膜重塑。将鼓励蛋白质-脂质小管的收缩，以评估 使用先进的结构方法测量收缩幅度。Liquid-EM将可视化动态缩小 真实的时间的Drp 1-脂质小管，并且冷冻ET将用于解析各种Drp 1收缩的3D结构 平行事件。在具体目标3中，将在细胞水平上检查线粒体分裂的缺陷， 确定Drp 1的有害变化如何直接影响线粒体生物能量学。的完整性 ETC复合物将被研究，以揭示改变细胞器形态如何通知代谢应激。 同时，将监测这种应激对ROS信号传导和线粒体自噬的影响。总之， 从该建议中获得的结构和功能见解将催化定向治疗策略， 在各种疾病状态中抵消线粒体损伤。

PROJECT SUMMARY (ABSTRACT) Mitochondria are double-membrane organelles that change shape, size and abundance in response to specific stimuli. Protein interactions that control mitochondrial division are tightly regulated and directly impact ATP production, Ca2+ homeostasis, and regulation of programmed cell death. Therefore, mitochondrial dynamics has recently come to the forefront as a therapeutic target in several degenerative diseases, including neurodegeneration, cancer, and cardiovascular disease. But the lack of insight into the regulation of this process is a major limitation. The major driver of mitochondrial division is a cytosolic GTPase, dynamin-related protein 1 (Drp1). To mediate membrane scission, Drp1 recruitment and self-assembly is coordinated through combinatorial interactions with lipids, proteins and nucleotides at the surface of mitochondria. This proposal seeks to identify key attributes of the mitochondrial division machinery and how dysregulation of Drp1 leads to organelle damage and cellular degeneration. This will be accomplished using a multifaceted approach that combines molecular studies with functional cell experiments to provide a comprehensive evaluation of Drp1 interactions that govern membrane remodeling. Under Specific Aim 1 of the renewal, cryo-EM studies will examine auto-inhibitory interactions that limit Drp1 oligomerization in a cytosolic state. Distinct conformations will be studied to identify and characterize intermediate structures during recruitment and assembly of Drp1 into a functional fission complex. We propose that regulated rearrangements “open” the molecule for functional assembly at defined sites of mitochondrial division. For Specific Aim 2, reconstitution experiments provide a means to evaluate macromolecular interactions that drive mitochondrial membrane remodeling. Specific mitochondrial cues, including lipids and partner proteins, will be studied to evaluate the contribution of each component to membrane remodeling. Constriction of protein-lipid tubules will be encouraged to evaluate the magnitude of constriction using advanced structural methods. Liquid-EM will visualize dynamic narrowing of Drp1-lipid tubules in real time, and cryo-ET will be used to resolve 3D structures of assorted Drp1 constriction events in parallel. In Specific Aim 3, defects in mitochondrial fission will be examined at the cellular level to establish how deleterious changes in Drp1 can directly influence mitochondrial bioenergetics. The integrity of ETC complexes will be studied to reveal how altered organelle morphology informs metabolic stress. Concurrently, the impact of this stress on ROS signaling and mitophagy will be monitored. In summary, the structural and functional insight gained from this proposal will catalyze directed therapeutic strategies that counteract mitochondrial damage in various disease states.