High Resolution Assembly Structure of the Nuclear Pore Complex

 描述(申请人提供)：核是真核细胞的内部避难所，保存被核膜(NE)包围的遗传信息。NE由两个同心的膜双层组成，在特定的位置融合形成圆形开口，每个开口被一个核孔复合体(NPC)占据。作为进出细胞核的唯一通道，核祖细胞在细胞内稳态中发挥着关键作用，但它们不仅仅是运输复合体。我们开始认识到，NPC也是组织细胞核的参照点，在基因调控中发挥着关键作用。这项研究的重点是确定巨大的~40-80丙二醛鼻咽癌的分子结构。实现这一目标将为探讨其在运输、基因调控和核组织中的多种功能提供结构基础。重要的是，鼻咽癌及其组成部分的功能异常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、心肌病和各种病毒感染。NPC是一种模块化结构，由大约30种不同的蛋白质组成，称为核孔蛋白，它们围绕一个中心的8倍旋转轴排列成多个副本。由2-10个核孔素组成的亚复合体层次化地构成NPC。其中的两个亚复合体，异构体Y-和Nic96-复合体，组织了大部分的建筑核孔蛋白，并建立了NPC的稳定支架。不同实验室对这些亚络合物的结晶学分析结合冷冻电子断层扫描重建，最近导致了第一个模糊的组装模型。在这里，我们建议对NPC的支架组件进行全面的结构特征描述。在生物化学和细胞生物学方法的补充下，我们假设我们将产生数据来构建NPC的详细结构，NPC是真核细胞中最大的组装结构。该提案的核心是利用X射线结晶学和电子显微镜技术的组合，以近原子分辨率建立NPC的主要组成部分。这项研究的第二个重点是利用实验方法建立所有核孔蛋白的相互作用组。这些信息对于建立一个健全的全国人大模型至关重要。然后，这种NPC的3D再现可以用来在详细的分子水平上询问NPC的功能。

 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, each occupied by a nuclear pore complex (NPC). As the only gateway into and out of the nucleus, NPCs play a critical role in cellular homeostasis, but they are more than just transport complexes. We are beginning to appreciate that the NPC also serves as a reference point to organize the nucleus and plays a pivotal role in gene regulation. This research program focuses on determining the molecular architecture of the enormous ~40-80 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, known as 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. Crystallographic analysis of these subcomplexes by different labs combined with cryo-electron tomographic reconstitution has recently led to a first, vague assembly model. Here, we propose to fully structurally characterize the scaffold components of the NPC. Complemented by biochemical and cell biological methods, we hypothesize that we will generate the data to build a detailed structure of the NPC, the largest assembly structure in the eukaryotic cell. The proposal is centered on establishing the major building blocks of the NPC at near-atomic resolution, using a combination of X-ray crystallographic and electronmicroscopic techniques. A second focus of this study is to establish the interactome of all nucleoporins using experimental methods. This information will be critical in order to build a robust model of the NPC. Such a 3D rendition of the NPC can then be used to interrogate the NPC functions at a detailed, molecular level.