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（由申请人提供）：Torsin 是 AAA+（与多种细胞活动相关的 ATP 酶）超家族的重要成员，并且与蛋白质质量控​​制、膜形态调节和核包膜动力学有关。已知有四种不同的 Torsin 蛋白。多年来，Torsin 被认为缺乏 ATP 酶活性，相应地，Torsin ATP 酶的精确细胞功能和机制作用在很大程度上仍然难以捉摸。我们的知识阻碍了对 Torsin 系统突变引起的先天性疾病病因的全面理解，包括严重的运动障碍 DYT1 肌张力障碍。重要的是，我们最近的工作表明 Torsins 是 ATP 酶，其活性需要 LAP1 或 LULL1，而 LAP1 或 LULL1 是膜蛋白。与 Torsins 结合形成复合跨膜机器的跨越辅因子需要我们对 Torsin ATP 酶的功能和机制理解的进展。 (i) 膜结合 Torsin/辅因子组装的结构和动力学的定义，(ii) Torsin/辅因子机制的细胞靶标的识别，以及 (iii) Torsin/辅因子对底物作用的功能剖析。根据提议，我们将利用我们已建立的蛋白脂质体系统，通过冷冻电子显微镜研究有或没有其辅因子的全长 Torsin 组装的结构动力学（目标 1）。克服 Torsin 底物溶解度差的方法，允许通过消减蛋白质组学方法进行底物鉴定（目标 2）。底物及其对 Torsin/辅因子作用的依赖性将在细胞环境中进行分析，并使用遗传缺失。不同的成像技术以及重建系统（目标 3）的阐明 Torsin 功能和机制 - 以及其由疾病相关突变引起的功能障碍 - 将有助于开发靶向药物。用于治疗 Torsin 相关运动障碍的疗法，这是已知的最常见的遗传性运动障碍。