High Resolution Assembly Structure of The Nuclear Pore Complex

核孔复合体的高分辨率组装结构

• 批准号: 8708103
• 负责人: Thomas Schwartz
• 金额: $ 34.84万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708103
• 关键词: Architecture; Autoimmune Diseases; Biochemical; Biological; Biological Assay; Cardiomyopathies; Cell Nucleus; Cells; Collaborations; Complement; Complex; Cryoelectron Microscopy; Crystallization; Data; Electrons; Eukaryotic Cell; Funding; Gene Expression Regulation; Genetic; Goals; HIV; Homeostasis; Human; In Vitro; Knowledge; Lead; Length; Malignant Neoplasms; Maps; Membrane; Methods; Microscopic; Modeling; Molecular; N-terminal; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Organelles; Phase; Play; Problem Solving; Property; Proteins; Publishing; Research; Resolution; Role; Route; Sampling; Site; Solutions; Structure; Techniques; Tertiary Protein Structure; Therapeutic; Universities; Viral; Virus; Virus Diseases; Work; Yeasts; base; human disease; in vivo; leukemia; milligram; novel; particle; programs; reconstitution; scaffold; stoichiometry; yeast two hybrid system

DESCRIPTION (provided by applicant): The nucleus is the inner sanctuary of the eukaryotic cell, keeping the genetic information enclosed by the nuclear envelope (NE). The NE consists of two concentric membrane bilayers fused at specific sites to form circular openings occupied by the nuclear pore complex (NPC). As the only gateway into and out of the nucleus, the NPC plays a critical role in cellular homeostasis, but it is more than a transport organelle. We are beginning to appreciate that the NPC also serves as a reference point to organize the nucleus and is likely to play a pivotal role in gene regulation. This research program focuses on determining the molecular architecture of the enormous ~50 MDa NPC. Attaining this goal will provide a structural basis to interrogate its myriad functions in transport, gene regulation, and nuclear organization. Importantly, aberrant function of the NPC and its constituents is a primary cause of many human diseases, including leukemia, other cancers, autoimmune diseases, cardiomyopathies, and a variety of viral infections. The NPC is a modular structure, composed of ~30 different proteins, so-called nucleoporins that arrange in multiple copies around a central eightfold rotational axis. Subcomplexes of 2-10 nucleoporins hierarchically build up the NPC. Two of these subcomplexes, the heteromeric Y- and Nic96-complexes, organize the majority of architectural nucleoporins and establish the stable scaffold of the NPC. Consequently, these two subcomplexes are the main focus of this proposal. Once both are fully structurally characterized, the scaffold structure of the NPC can likely be generated with high confidence. This proposal is organized around four aims. Aim 1 - Structural characterization of the universally conserved, heptameric Y-complex core. The 575 kDa Y-complex is the pivotal NPC architectural scaffold unit. Building on recently published work on fragments of the Y-complex it is now feasible to structurally characterize the entire complex. A combined approach using crystallographic and electron microscopic methods will be employed. Aim 2 - Structural characterization of the species-specific Y-complex extensions. The human Y-complex contains three additional proteins, whose functional roles include NPC architecture and assembly. These proteins will be studied structurally in complex with the core components of the Y-complex. Aim 3 - Structural characterization of the Nic96 complex. The Nic96 complex is only partially characterized, but it shares common ancestry with the Y-complex. It is widely accepted that it is pivotal to elucidate the Nic96 complex structure as it is, beside the Y-complex, the second large building block of the NPC scaffold structure. Aim 4 - Assembly structure of the NPC and interactome. NPC subcomplexes have to interact to form the higher order assembly of the NPC, yet information on these inter-subcomplex contacts is still incomplete. To solve this problem, a yeast-two-hybrid based interactome will be generated sampling binary interactions between all nucleoporins.

描述（由申请人提供）：核是真核细胞的内部保护区，保留核包膜（NE）包含的遗传信息。 NE由两个融合在特定位点融合的同心膜双层组成，形成由核孔复合物（NPC）占据的圆形开口。作为唯一进入核的门户，NPC在细胞稳态中起着至关重要的作用，但它不仅仅是运输细胞器。我们开始理解NPC也是组织核的参考点，并且很可能在基因调节中起关键作用。该研究计划着重于确定〜50 MDA NPC的分子体系结构。实现这一目标将提供一个结构性的基础，以询问其在运输，基因调节和核组织中的无数功能。重要的是，NPC及其成分的异常功能是许多人类疾病的主要原因，包括白血病，其他癌症，自身免疫性疾病，心肌病和多种病毒感染。 NPC是一个模块化结构，由〜30种不同的蛋白质组成，即所谓的核孔蛋白，在中央八重旋转轴周围以多个副本排列。 2-10个核孔蛋白分层的子复合物构建NPC。这些子复合物中的两个，即杂体Y-和NIC96-复合物，组织了大多数建筑核孔蛋白，并建立了NPC的稳定支架。因此，这两个子复合是该提案的主要重点。一旦两者都在结构上表征，NPC的脚手架结构可能会高度置信。该提议围绕四个目标组织。目标1-普遍保守的七聚体Y复合核的结构表征。 575 kDa y-complex是关键的NPC建筑支架单元。在最近发表的有关Y复合物碎片的工作的基础上，现在可以在结构上表征整个综合体。将采用晶体学和电子显微镜方法的组合方法。 AIM 2-物种特异性Y复合物扩展的结构表征。人类Y-复合物包含另外三种蛋白质，其功能作用包括NPC结构和组装。这些蛋白质将与Y复合物的核心成分结构研究。目标3- NIC96复合物的结构表征。 NIC96复合体仅部分表征，但它与Y-复合物共享共同的血统。人们普遍认为，阐明NIC96复合结构的关键是，在Y-Complex旁边是NPC脚手架结构的第二个大构件。 AIM 4- NPC和Interactome的组装结构。 NPC子复合物必须相互作用才能形成NPC的高阶组件，但是有关这些补充串联触点的信息仍然不完整。为了解决这个问题，将生成所有基于酵母的基于杂交的相互作用组，从而在所有核孔蛋白之间生成采样二进制相互作用。