Defining Molecular Interactions that Drive Mitochondrial Fission

项目摘要(摘要) 线粒体是双膜细胞器，可以改变形状、大小和丰度 特定的刺激。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 三磷酸腺苷的产生、钙离子的稳态和程序性细胞死亡的调节。因此，线粒体 动力学最近作为几种退行性疾病的治疗靶点走到了前列，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 流程是一个主要限制。线粒体分裂的主要驱动力是一种与动力蛋白相关的胞浆GTP酶 蛋白1(Drp1)。为了介导膜的断裂，Drp1的募集和自组装是通过 与线粒体表面的脂类、蛋白质和核苷酸的组合作用。这项建议 旨在确定线粒体分裂机制的关键属性以及DRp1的失调如何导致 细胞器损伤和细胞退化。这将使用多方面的方法来实现，该方法 结合分子研究和功能细胞实验提供对Drp1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，低温电磁研究将 检查在胞浆状态下限制Drp1齐聚的自抑制相互作用。独特的构象 将进行研究，以确定和表征在招募和组装DRP1期间的中间结构 变成一个功能性的裂变复合体。我们认为，受调控的重排“开放”了分子的功能 在线粒体分裂的特定位置组装。对于特定目标2，重构实验提供了一个 评估驱动线粒体膜重塑的大分子相互作用的方法。特定的 将研究线粒体信号，包括脂质和伴侣蛋白，以评估每种信号的作用。 从成分到膜的重塑。将鼓励蛋白质-脂类小管收缩以评估 使用先进的结构方法进行收缩的程度。Liquid-EM将可视化动态缩窄 Drp1-脂质小管的实时检测和冷冻-ET将被用来解析各种drp1收缩的3D结构 事件是平行发生的。在具体目标3中，将在细胞水平上检查线粒体分裂的缺陷，以 确定DRp1的有害变化如何直接影响线粒体生物能量学。正直的 将研究ETC复合体，以揭示细胞器形态改变是如何影响代谢应激的。 同时，这种压力对ROS信号和有丝分裂吞噬的影响将被监测。总而言之， 从这项提案中获得的结构和功能洞察力将催化定向治疗策略， 在不同的疾病状态下抵消线粒体损伤。

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.