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: 9502300
• 负责人: Christian Dirk Schlieker
• 金额: $ 32万
• 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/9502300
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Ablation; Alleles; Area; Biochemical; Biological; 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; 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; disease-causing mutation; early onset; effective therapy; genetic manipulation; imaging approach; 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（由适用提供）：Torsins是AAA+（与各种细胞活动相关的ATPases）的重要成员，并且已在蛋白质质量控​​制，膜形态的调节和核隐膜动力学中隐含。在人类中已知四种不同的扭曲蛋白。多年来，Torsins被认为缺乏ATPase活性。相应地，Torsin Atpases的精确细胞功能和机械作用在很大程度上仍然是弹性。我们知识中的这一差距阻碍了人们对由Torsin系统突变引起的先天性疾病的病因，包括严重的运动障碍Dyt1 dystonia。重要的是，我们最近的工作表明Torsins是ATPases，其活性需要LAP1或LULL1，它们是跨膜的辅助因子，它们与Torsins相关联，形成复合膜的跨膜机器。我们对Torsin ATPases的功能和机械理解的进步将需要（i）（i）定义膜结合的Torsin/cofactor组装的结构和动力学，（II）鉴定Torsin/cofactor机械的细胞靶标，以及（III）对Torsin/cofactor对底物的功能解剖。在此提案中，我们将利用我们已建立的蛋白质体系统，以通过低温电子显微镜研究带有和没有辅助因子的全长Torsin组装的结构动力学（AIM 1）。我们将利用一种新颖的方法来克服Torsin底物的溶解度差，从而通过减法蛋白质组学方法可以鉴定底物（AIM 2）。底物及其对Torsin/cofactor作用的依赖将在细胞环境中分析，使用与不同的成像技术以及重构系统的遗传缺失（AIM 3）。阐明Torsin功能和机制 - 以及与疾病相关的突变引起的功能障碍 - 将使靶向疗法的发展用于治疗Torsin相关运动障碍，这是最常见的遗传运动障碍。