High Resolution Assembly Structure of The Nuclear Pore Complex

描述（申请人提供）：细胞核是真核细胞的内部避难所，将遗传信息保存在核被膜（NE）中。NE由两个同心的膜双层在特定位点融合形成由核孔复合物（NPC）占据的圆形开口组成。作为进出细胞核的唯一通道，NPC在细胞内稳态中起着关键作用，但它不仅仅是一个运输细胞器。我们开始意识到，NPC也可以作为组织细胞核的参考点，并可能在基因调控中发挥关键作用。该研究计划的重点是确定巨大的~50 MDa NPC的分子结构。实现这一目标将提供一个结构基础，询问其在运输，基因调控和核组织的无数功能。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、心肌病和各种病毒感染。NPC是一种模块化结构，由约30种不同的蛋白质组成，这些蛋白质被称为核孔蛋白，它们围绕一个中心八重旋转轴排列成多个拷贝。2-10个核孔蛋白的亚复合物分级构建NPC。这些亚复合物中的两个，异聚Y-和Nic 96-复合物，组织了大多数结构核孔蛋白，并建立了NPC的稳定支架。因此，这两个子综合体是本提案的主要重点。一旦两者都被完全结构表征，NPC的支架结构就可能以高置信度生成。该提案围绕四个目标展开。目的1 -普遍保守的七聚体Y-复合物核心的结构表征。575 kDa的Y-复合物是关键的NPC结构支架单位。基于最近发表的关于Y复合体片段的工作，现在可以从结构上表征整个复合体。将采用晶体学和电子显微镜方法相结合的方法。目的2 -特定物种的Y-复形扩张的结构特征。人类Y-复合物含有三种额外的蛋白质，其功能作用包括NPC结构和组装。这些蛋白质将在结构上与Y复合物的核心成分复合研究。目的3 -Nic 96复合物的结构表征。Nic 96复合体只有部分特征，但它与Y复合体有共同的祖先。人们普遍认为，阐明Nic 96复合物结构是关键的，因为它是除了Y-复合物之外的NPC支架结构的第二大构件。目标4 -NPC和相互作用组的组装结构。NPC亚复合体必须相互作用以形成NPC的高阶组装体，但关于这些亚复合体间接触的信息仍然不完整。为了解决这个问题，将产生基于酵母双杂交的相互作用组，对所有核孔蛋白之间的二元相互作用进行采样。 公共卫生相关性：这项研究将导致对大规模核孔复合物（NPC）的结构理解，NPC是进出细胞核的唯一运输通道，也是许多致病病毒（包括HIV）的通道。这一知识将打开中期的可能性，影响运输性能，可能阻止病毒进入细胞核。 NPC还涉及无数其他人类疾病，包括癌症和心肌病，因此对这些机制的分子理解可能提供新的长期治疗策略。

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. PUBLIC HEALTH RELEVANCE: This study will lead to a structural understanding of the massive nuclear pore complex (NPC), the sole transport channel to and from the cell's nucleus and the passage route for many pathogenic virus, including HIV. This knowledge will open mid-term possibilities to influence transport properties, possibly to block viral entry to the nucleus. The NPC is also involved in a myriad of other human diseases, including cancer and cardiomyopathies, thus a molecular understanding of these mechanisms may provide new long-term therapeutic strategies.