Conformational Dynamics of the Dynamin PH domain in Synaptic Vesicle Endocytosis

突触小泡胞吞作用中 Dynamin PH 结构域的构象动力学

• 批准号: 10057144
• 负责人: Rajesh Ramachandran
• 金额: $ 44.28万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057144
• 关键词: Address; Alzheimer' s Disease; Behavior; Binding; Biomimetics; Cell membrane; Centronuclear myopathy; Charcot-Marie-Tooth Disease; Consensus; Coupled; Coupling; Cryoelectron Microscopy; Data; Defect; Detection; Dictyostelium discoideum dynamin A; Diglycerides; Dimerization; Disease; Down Syndrome; Drug Design; Dynamin; Electrostatics; Endocytic Vesicle; Endocytosis; Epilepsy; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Foundations; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Huntington Disease; Hydrolysis; Hydrophobicity; Kinetics; Label; Length; Lipids; Location; Maps; Measurement; Mediating; Membrane; Micelles; Molecular; Molecular Conformation; Motion; Mutation; NMR Spectroscopy; Nanotubes; Nature; Neck; Nucleotides; Outcome; Outcomes Research; PH Domain; Parkinson Disease; Phosphatidylinositol 4,5-Diphosphate; Play; Polymers; Recycling; Regulation; Research; Resolution; Retrieval; Role; SERPINA4 gene; Site; Structure; Surface; Synaptic Transmission; Synaptic Vesicles; System; Techniques; Therapeutic; Thinness; Time; Torque; Vesicle; Water; base; constriction; design; disease-causing mutation; fluorophore; improved; in vivo; membrane model; mutant; nanodisk; nervous system disorder; neurotransmission; novel therapeutics; polymerization; premature; presynaptic; public health relevance; self assembly; uptake

PROJECT SUMMARY Synaptic transmission relies critically on the rapid uptake of emptied exocytic vesicle membrane remnants from the presynaptic plasma membrane via the coupled and compensatory mechanisms of endocytosis catalyzed by the large GTPase dynamin. Defects in synaptic vesicle recycling have been implicated in various neurological disorders including Epilepsy, Down’s syndrome, Alzheimer’s, Parkinson’s and Huntington’s diseases. Emerging evidence indicates that in addition to dynamin’s better-characterized helical polymerization and mechanoenzymatic membrane constriction activities, a third distinct activity involving the alternative tilting or orientation of its pleckstrin homology (PH) domain at the membrane surface governs synaptic vesicle scission. The mechanisms remain largely uncharacterized. Disease-causing mutations in dynamin, which precipitate centronuclear myopathy (CNM) and Charcot-Marie-Tooth (CMT) disease, map largely to the PH domain or to its various intermolecular interfaces. Although this underscores the importance of the PH domain in dynamin function, it is unclear how these mutations specifically influence PH domain interactions or conformational behavior at the membrane surface. It is our long-term goal to understand the various molecular mechanisms at play in dynamin-mediated endocytic vesicle scission. In this proposal, we seek to address several unknown or unresolved fundamental issues concerning the role of the PH domain in dynamin function, both in solution and on membranes. These include: 1) the regulatory mechanisms and conformational rearrangements that underlie the transition of dynamin from stable, self-limited, cytosolic tetramers to dynamic, self-assembled, membrane- bound helical polymers, 2) the conformational coupling of dynamin PH domain-membrane insertion and alternate orientations to helical self-assembly and the coordination of assembly-dependent GTPase activity, and 3) the molecular nature and structural basis of alternate PH domain orientations on the membrane surface. To address these, we will use a powerful combination of multiple independent fluorescence spectroscopic techniques including Förster resonance energy transfer (FRET), fluorescence lifetime analysis, quenching and stopped-flow kinetic measurements, coupled to sophisticated NMR spectroscopic measurements of the dynamin PH domain on various biomimetic lipid templates. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the mechanisms of dynamin function that underlie rapid synaptic vesicle scission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate synaptic vesicle endocytosis under various disease states.

项目总结 突触传递关键依赖于排空的胞外囊泡膜残留物的快速摄取。 突触前质膜通过偶联和代偿机制的内吞作用催化 大的GTP酶动力蛋白。突触小泡再循环缺陷与多种神经疾病有关 疾病包括癫痫、唐氏综合症、阿尔茨海默氏症、帕金森氏症和亨廷顿氏症。新兴 有证据表明，除了动力素更好地表征螺旋聚合和 机械酶促膜收缩活性，第三种不同的活性涉及交替倾斜或 膜表面Peckstrin同源(PH)结构域的定位决定了突触小泡的断裂。 这些机制在很大程度上仍然没有定论。动力素的致病突变，它使 中心核肌病(CNM)和夏科-玛丽-图思(CMT)病，主要映射到PH域或其 各种分子间界面。尽管这强调了PH域在动力方面的重要性 功能，目前还不清楚这些突变是如何具体影响PH结构域相互作用或构象的 膜表面的行为。我们的长期目标是了解不同的分子机制 在动力蛋白介导的胞内囊泡断裂中发挥作用。在这项提案中，我们寻求解决几个未知或 关于PH结构域在动力蛋白功能中的作用的未解决的基本问题，无论是在溶液中还是在 在膜上。这些包括：1)调控机制和构象重排 动力蛋白从稳定的、自限的胞质四聚体向动态的、自组装的膜四聚体的转变. 结合的螺旋聚合物，2)Dynamin PH结构域的构象偶联-膜插入和交替 螺旋自组装的定向和依赖于组装的GTP酶活性的协调，以及3) 膜表面交替的PH结构域取向的分子性质和结构基础。致信地址 这些，我们将使用多种独立荧光光谱技术的强大组合 包括Förster共振能量转移(FRET)、荧光寿命分析、猝灭和停流 动力蛋白PH结构域的动力学测量和复杂的核磁共振波谱测量 在各种仿生类脂模板上。这项研究的成功结果将提供(I)从根本上 提高了对突触小泡快速断裂背后的动力蛋白功能机制的理解， 以及(Ii)可以有益地调节突触的药物和疗法设计的分子基础 各种疾病状态下的囊泡内吞作用。