Chaperone Mechanisms in Clathrin Mediated Neuronal Vesicle Trafficking

网格蛋白介导的神经元囊泡贩运中的伴侣机制

• 批准号: 9090391
• 负责人: EILEEN M. LAFER
• 金额: $ 42.74万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9090391
• 关键词: Acceleration; Address; Affect; Alternative Splicing; Alzheimer' s Disease; Bacteria; Binding; Binding Sites; Biological; Biological Process; Cell membrane; Characteristics; Clathrin; Clathrin-Coated Vesicles; Clinical; Complement; Complex; Cytosol; Data; Disease; Dissociation; Dynamin; Endocytic Vesicle; Endocytosis; Eukaryota; Family member; Generations; Guanine Nucleotide Exchange Factors; Heat shock proteins; Huntington Disease; In Vitro; Intervention; Lampreys; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Mitochondria; Modeling; Molecular; Molecular Chaperones; Monitor; Motor; Mutation; Nerve; Neurodegenerative Disorders; Neurons; Nucleotides; Parkinson Disease; Phosphorylation; Power stroke; Process; Property; Protein Binding Domain; Protein translocation; Proteins; Reaction; Recycling; Regulation; Role; Speed; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Testing; Therapeutic; Time; Variant; Vesicle; Work; base; coated pit; design; fight against; flexibility; genetic regulatory protein; in vivo; intermolecular interaction; mutant; nervous system disorder; polymerization; pressure; protein aggregate; protein complex; protein folding; protocol development; public health relevance; research study; trafficking; transmission process

 DESCRIPTION (provided by applicant): Protein and membrane trafficking depends on choreographed reactions which deform membranes and sequentially assemble and disassemble protein complexes. Hsp70s--the ubiquitous chaperones involved in protein folding and many cellular protein processing reactions--function as motors in trafficking processes such as translocation of proteins into ER and mitochondria, and disassembly of the clathrin coats that form around nascent endocytic vesicles. The ubiquitous role of Hsp70s suggests a common mechanism is being harnessed in these seemingly disparate reactions, but while we understand how Hsp70s bind and release protein substrates, our understanding of how Hsp70s generate force to move proteins or take apart protein complexes is limited. Clathrin coat disassembly provides an exceptional system to study these force generation mechanisms: unlike Hsp70 mediated processes such as protein translocation or dissociation of heterogeneous aggregates, coat disassembly can be precisely monitored in real time, structures of all players and reaction snapshots are known, coat stability can be easily controlled, and the single Hsc70 binding site in clathrin required for disassembly is known. By exploiting these features and our development of protocols for producing functional clathrin in bacteria, we have obtained evidence that neither previously proposed power- stroke nor Brownian ratchet/steric wedge models can explain how Hsc70 disassembles coats. Instead, our data indicate that coats are disassembled through pressure generated by collisions between coat walls and Hsc70s bound to flexible tethers in close apposition to these walls. We also discovered that self-association, characteristic of all Hsp70s, amplifies this force, thus providing a biological function for a ubiquitous phenomenon that has never had one. In our proposed work we will test and refine this collision pressure mechanism of Hsp70 force generation (Aim 1). Hsc70 cooperates with other chaperones, among them Hsp110, which acts as an Hsp70 nucleotide exchange factor (NEF). We determined the structure of the Hsc70:Hsp110 complex, and used the structural information to design experiments which revealed that Hsp110 regulates Hsc70 chaperoning of clathrin during synaptic vesicle recycling. The latter experiments used the giant lamprey reticulospinal synapse, which represents the in vivo experimental complement to our in vitro clathrin/Hsc70 system. Because neuronal synapses are compartments devoted primarily to membrane trafficking, they provide an exceptional system for addressing fundamental biological questions in trafficking and the role of chaperones in these processes. In our proposed studies we will exploit both the in vitro clathrin/Hsc70 system and the in vivo, lamprey RS synapse to further define the nucleotide exchange mechanism of Hsp110 (Aim 2), and the mechanism by which it regulates Hsc70 and clathrin availability during membrane trafficking (Aim 3). These studies will advance our understanding of how Hsp70s generate the forces by which they translocate proteins and remodel protein complexes, and of how their activities are regulated. They will also elucidate fundamental mechanisms underlying synaptic transmission and clathrin mediated vesicular trafficking.

 描述（由申请人提供）：蛋白质和膜运输依赖于使膜变形并依次组装和分解蛋白质复合物的编排反应。Hsp 70--参与蛋白质折叠和许多细胞蛋白质加工反应的普遍存在的分子伴侣--在运输过程中起马达的作用，例如蛋白质易位到ER和线粒体中，以及在新生内吞囊泡周围形成的网格蛋白外壳的分解。Hsp 70的普遍作用表明，在这些看似不同的反应中，一种共同的机制正在被利用，但是尽管我们了解Hsp 70如何结合和释放蛋白质底物，但我们对Hsp 70如何产生力来移动蛋白质或分解蛋白质复合物的理解是有限的。网格蛋白外壳拆卸提供了一个特殊的系统来研究这些力的产生机制：不像热休克蛋白70介导的过程，如蛋白质易位或解离的异质性聚集体，外壳拆卸可以精确地监测在真实的时间，结构的所有球员和反应快照是已知的，外壳稳定性可以很容易地控制，和单一的Hsc 70结合位点网格蛋白所需的拆卸是已知的。通过利用这些特征和我们开发的用于在细菌中产生功能性网格蛋白的方案，我们已经获得了证据，即先前提出的动力冲程模型和布朗棘轮/空间楔模型都不能解释Hsc 70如何分解外壳。相反，我们的数据表明，大衣拆卸通过之间的碰撞产生的压力大衣壁和HSC 70绑定到灵活的系绳在这些墙壁紧密并列。我们还发现，所有热休克蛋白70的特征--自我缔合--放大了这种力量，从而为一种从未有过的普遍现象提供了生物学功能。在我们提出的工作中，我们将测试和完善这种Hsp 70力产生的碰撞压力机制（目标1）。Hsc 70与其他分子伴侣合作，其中包括Hsp 110，其充当Hsp 70核苷酸交换因子（NEF）。我们确定了Hsc 70：Hsp 110复合物的结构，并使用结构信息设计实验，揭示了Hsp 110调节Hsc 70在突触囊泡回收过程中的网格蛋白伴侣作用。后者的实验使用了巨大的七鳃鳗网状脊髓突触，这代表了我们在体外网格蛋白/Hsc 70系统的体内实验补充。由于神经元突触是主要用于膜运输的隔室，因此它们为解决运输中的基本生物学问题以及分子伴侣在这些过程中的作用提供了一个特殊的系统。在我们提出的研究中，我们将利用在体外网格蛋白/Hsc 70系统和在体内，七鳃鳗RS突触，以进一步确定Hsp 110（目标2）的核苷酸交换机制，以及它调节Hsc 70和网格蛋白膜运输过程中的可用性（目标3）的机制。这些研究将促进我们对Hsp 70如何产生使其转运蛋白和重塑蛋白质复合物的力以及如何调节其活性的理解。他们还将阐明突触传递和网格蛋白介导的囊泡运输的基本机制。