Atomic Structure of the Nuclear Pore Complex

描述(申请人提供)：进化的最大标志之一是遗传信息被包围在细胞核中。这种空间分离为细胞核和细胞质之间的有效通信创造了必要性，这是通过选择性地跨核膜(NE)的双膜运输折叠的蛋白质和蛋白质/核酸复合体来实现的。核孔复合体(NPC)是允许大分子通过NE的唯一通道，使这个运输细胞器成为真核生物生命所必需的机器。核祖细胞被嵌入在贯穿NE的圆形孔隙中，可以完成直径达~40 nm的粒子的双向传输，速度为每秒数百次。电子显微镜研究表明，鼻咽癌由一个中心核组成，在核质轴上具有8次旋转对称性，在NE平面上具有2次旋转对称性。这个对称的核心连接到“细胞质细丝”和“核篮子”结构。NPC由大约30种不同的蛋白质组成，这些蛋白质被称为核孔蛋白(NUP)，它们被组织成六个不同的亚复合体。每个NUP都以多个副本存在于NPC中，因此整个组装在酵母中达到了~60MDA的非凡分子质量，在脊椎动物中甚至更多。NPC不仅作为运输通道发挥作用，而且在其他基因调控模式中也发挥着广泛的作用，例如通过与转录和mRNA输出机制的直接相互作用。因此，在多种人类疾病中观察到鼻咽癌功能障碍也就不足为奇了，例如肿瘤或逆转录病毒疾病。这些联系以及鼻咽癌在真核细胞生物学中的存在作用促使人们对其详细结构进行研究。鼻咽癌的大小和灵活性，以及目前缺乏足够数量的合适材料，使得结晶学无法确定完整的鼻咽癌的结构。本文提出的另一种方法试图通过重组NPC亚复合体和结晶学特征来阐明NPC的原子结构，这些亚复合体构成了完整的NPC在体内的生理构件。结合电子显微镜重建、生化蛋白质-蛋白质相互作用图和细胞分析，这一策略旨在为整个鼻咽癌建立一个复合伪原子模型，并为全面的结构功能分析提供路线图。因此，这项拟议研究的结果有望进一步加深我们对NPC参与核质运输和其他细胞过程的分子机制的理解，同时为目前无法治疗的“核孔素疾病”创造一个机制基础。此外，本文开发的方法学将挑战当前结构细胞生物学的边界，并为其他具有重要细胞作用的大分子组件提供范例，由于缺乏结构洞察力，其作用机制仍然难以捉摸。

DESCRIPTION (provided by applicant): One of the great hallmarks of evolution is the enclosure of genetic information in the nucleus. This spatial separation creates the necessity for efficient communication between the nucleus and the cytoplasm, which is achieved through the selective transport of folded proteins and of protein/nucleic acid complexes across the double membrane of the nuclear envelope (NE). The nuclear pore complex (NPC) is the sole gateway that allows passage of macromolecules through the NE, making this transport organelle an essential machine for eukaryotic life. NPCs are embedded in circular pores permeating the NE and can accomplish the bidirectional transport of particles of up to ~40 nm in diameter and at a rate of several hundred events per second. Electron microscopic studies have revealed that the NPC consists of a central core with an 8-fold rotational symmetry across a nucleo-cytoplasmic axis and a two-fold rotational symmetry across the plane of the NE. This symmetric core links to "cytoplasmic filaments" and a "nuclear basket" structure. The NPC is built from approximately 30 distinct proteins, termed nucleoporins (nups) that are organized into six distinct subcomplexes. Each nup is present in the NPC in multiple copies such that the entire assembly reaches the extraordinary molecular mass of ~60 MDa in yeast and even more in vertebrates. The NPC functions not just as a transport channel, but has a comprehensive role in other modes of gene regulation, for example through direct interaction with the transcription and mRNA export machineries. As such, it is less surprising that NPC dysfunction has been observed in a diverse set of human illnesses, such as neoplastic or retroviral disease. These associations as well as the NPC's existential role in eukaryotic cell biology have motivated investigations into its detailed architecture. The NPC's size and flexibility along with the unavailability of sufficient quantities of suitable material presently preclude the crystallographi determination of the structure of the entire intact NPC in one piece. An alternative approach proposed herein seeks to elucidate the atomic architecture of the NPC through recombinant reconstitution and crystallographic characterization of NPC subcomplexes, which constitute the physiological building blocks of the intact NPC in vivo. Combined with electron microscopic reconstruction, biochemical protein-protein interaction maps and cellular assays, this strategy is designed to lead to a composite pseudo-atomic model for the entire NPC and provide a roadmap for comprehensive structure-function analyses. As such, the outcome of the proposed research is expected to further our understanding of the molecular mechanisms that govern the involvement of the NPC in nucleocytoplasmic transport and other cellular processes, while at the same time creating a mechanistic basis for currently untreatable "nucleoporin diseases." Furthermore, the methodologies developed herein will challenge the current boundaries of structural cell biology and serve as a paradigm for other large macromolecular assemblies with essential cellular roles whose functional mechanism has remained elusive due to lack of structural insight.