Structure-function analysis of Torsin ATPases in the context of the membrane

膜背景下 Torsin ATP 酶的结构功能分析

• 批准号: 9105864
• 负责人: Christian Dirk Schlieker
• 金额: $ 33.19万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9105864
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Ablation; Alleles; Area; Binding; Biochemical; Biological; Bulla; Bypass; Cell Line; Cell physiology; Cells; Cellular biology; Complex; Congenital Disorders; Cryoelectron Microscopy; Dependence; Development; Disease; Dissection; Dystonia; Embryo; Etiology; Functional disorder; Genetic; Genome engineering; Goals; Human; Image; Imaging Techniques; In Vitro; Inherited; Knowledge; Lead; Length; Medical; Membrane; Methodology; Molecular Motors; Morphology; Movement Disorders; Mus; Mutation; Nature; Nuclear Envelope; Nucleotides; Pathway interactions; Patients; Primary Dystonias; Proteins; Proteome; Proteomics; Quality Control; Role; Solubility; Structure; System; TorsinA; TorsinB; Variant; Work; base; cellular targeting; cofactor; early onset; effective therapy; genetic manipulation; in vitro Assay; mammalian genome; member; novel; novel strategies; novel therapeutics; postnatal; proteoliposomes; public health relevance; reconstitution; stem; stoichiometry; targeted treatment; treatment strategy

 DESCRIPTION (provided by applicant): Torsins are essential members of the AAA+ (ATPases associated with a variety of cellular activities) superfamily and have been implicated in protein quality control, modulation of membrane morphology, and nuclear envelope dynamics. Four different Torsin proteins are known in humans. For years Torsins were thought to lack ATPase activity. Correspondingly, the precise cellular functions and the mechanistic roles of Torsin ATPases remained largely elusive. This gap in our knowledge hinders a comprehensive understanding of the etiology of congenital disorders caused by mutations in the Torsin system, including the severe movement disorder DYT1 dystonia. Importantly, our recent work has revealed that Torsins are ATPases whose activity requires LAP1 or LULL1, which are membrane-spanning cofactors that associate with Torsins to form a composite, membrane-spanning machine. Advances in our functional and mechanistic understanding of Torsin ATPases will require (i) definition of the structure and dynamics of the membrane-bound Torsin/cofactor assembly, (ii) identification of the cellular targets of the Torsin/cofactor machinery, and (iii) functional dissection of Torsin/cofactor action on substrates. In this proposal, we will capitalize on our established proteoliposome system to investigate the structural dynamics of full-length Torsin assembly with and without its cofactors via cryo-electron microscopy (Aim 1). We will utilize a novel methodology to overcome the poor solubility of Torsin substrates, allowing for substrate identification via a subtractive proteomic approach (Aim 2). The substrates, and their dependence on Torsin/cofactor action, will be analyzed in a cellular context, using genetic deletions in concert with different imaging techniques, as well as in reconstituted systems (Aim 3). The elucidation of Torsin function and mechanism -as well as its dysfunction resulting from disease-associated mutations- will enable the development of targeted therapies for the treatment of Torsin-related movement disorders, which are the most common inherited movement disorders known.

 描述（由申请方提供）：扭转蛋白是AAA+（与多种细胞活性相关的ATP酶）超家族的重要成员，并参与蛋白质质量控制、膜形态调节和核膜动力学。人类已知有四种不同的扭转蛋白。多年来，扭转蛋白被认为缺乏ATP酶活性。相应地，扭转蛋白ATP酶的确切细胞功能和机制作用在很大程度上仍然难以捉摸。我们知识上的这一空白阻碍了对扭转蛋白系统突变引起的先天性疾病病因学的全面理解，包括严重的运动障碍DYT 1肌张力障碍。重要的是，我们最近的工作揭示了扭转蛋白是ATP酶，其活性需要LAP 1或LULL 1，这是与扭转蛋白结合形成复合跨膜机器的跨膜辅因子。在我们的Torsin ATP酶的功能和机制的理解的进展将需要（i）的结构和动力学的膜结合的Torsin/辅因子组件的定义，（ii）识别的Torsin/辅因子机制的细胞目标，和（iii）功能解剖的Torsin/辅因子作用的基板。在这个提议中，我们将利用我们建立的蛋白脂质体系统，通过冷冻电子显微镜（目的1）研究全长扭转蛋白组装及其辅因子的结构动力学。我们将利用一种新的方法来克服扭转蛋白底物的溶解性差，允许通过减法蛋白质组学方法（目的2）的底物识别。底物及其对扭转蛋白/辅因子作用的依赖性将在细胞环境中进行分析，使用遗传缺失与不同的成像技术以及重建系统（目的3）。扭转蛋白功能和机制的阐明-以及由疾病相关突变引起的功能障碍-将使开发用于治疗扭转蛋白相关运动障碍的靶向疗法成为可能，扭转蛋白相关运动障碍是已知的最常见的遗传性运动障碍。