Atomic Structure of the Nuclear Pore Complex

描述（由申请人提供）：进化的一个重要标志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条件，这是通过折叠蛋白质和蛋白质/核酸复合物在核膜双膜（NE）上的选择性运输来实现的。核孔复合体（NPC）是允许大分子通过NE的唯一通道，使这种转运细胞器成为真核生物必不可少的机器。npc被嵌入在穿透NE的圆形孔中，可以实现直径高达~40 nm的粒子的双向传输，速率为每秒数百次。电镜研究表明，鼻咽癌由一个中心核组成，其在核-胞质轴上具有8倍旋转对称，在NE平面上具有2倍旋转对称。这个对称的核与“细胞质丝”和“核篮”结构相连。鼻咽癌是由大约30种不同的蛋白质构成的，这些蛋白质被称为核孔蛋白（nups），它们被组织成6个不同的亚复合物。每个nup都以多个拷贝的形式存在于NPC中，使得整个组装在酵母中达到~60 MDa的非凡分子质量，在脊椎动物中甚至更高。NPC不仅作为转运通道发挥作用，还在其他基因调控模式中发挥全面作用，例如通过直接与转录和mRNA输出机制相互作用。因此，在多种人类疾病（如肿瘤或逆转录病毒疾病）中观察到鼻咽癌功能障碍就不足为奇了。这些关联以及NPC在真核细胞生物学中的存在作用激发了对其详细结构的研究。NPC的尺寸和灵活性，以及缺乏足够数量的合适材料，目前阻碍了对整个完整NPC结构的结晶学测定。本文提出的另一种方法旨在通过重组鼻咽癌亚复合物的重构和晶体学表征来阐明鼻咽癌的原子结构，这些亚复合物构成了完整鼻咽癌在体内的生理构建块。结合电镜重建、生化蛋白-蛋白相互作用图和细胞分析，该策略旨在建立整个NPC的复合伪原子模型，并为全面的结构-功能分析提供路线图。因此，这项研究的结果有望进一步加深我们对控制NPC参与核胞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“核孔蛋白疾病”创造机制基础。此外，本文开发的方法将挑战结构细胞生物学的当前边界，并作为其他具有重要细胞作用的大型大分子组件的范例，这些大分子组件的功能机制由于缺乏结构洞察力而仍然难以捉摸。

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.