Atomic Structure of the Nuclear Pore Complex

核孔复合体的原子结构

• 批准号: 9398786
• 负责人: Andre Hoelz
• 金额: $ 5.62万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9398786
• 关键词: Affinity; Anatomy; Architecture; Biochemical; Caliber; Cell Nucleus; Cell physiology; Cells; Cellular Assay; Cellular biology; Characteristics; Communication; Complex; Crystallization; Cytoplasm; Cytoplasmic Filaments; Data; Development; Disease; Dissection; Electrons; Etiology; Eukaryotic Cell; Event; Evolution; Foundations; Functional disorder; Gene Expression Regulation; Generations; Genetic Transcription; Goals; Health; Human; In Vitro; Individual; Inherited; Interphase Cell; Investigation; Lead; Life; Link; Methodology; Microscopic; Mitosis; Modeling; Molecular; Molecular Conformation; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Structure; Nucleic Acids; Organelles; Outcome; Physiological; Protein-Protein Interaction Map; Proteins; Recombinant Proteins; Recombinants; Research; Ribosomes; Roentgen Rays; Role; Rotation; Site; Solid; Structure; Therapeutic; Time; Vertebrates; Yeasts; base; combinatorial; design; flexibility; genetic information; human disease; in vitro Assay; in vivo; insight; leukemia; mRNA Export; macromolecular assembly; macromolecule; molecular mass; neoplastic; novel; nucleocytoplasmic transport; particle; protein folding; public health relevance; reconstitution; reconstruction; stoichiometry; tool

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.

描述（由申请人提供）：进化的一个重要标志是遗传信息在细胞核中的封闭。这种空间分离产生了核与细胞质之间有效通讯的必要性，这是通过选择性运输折叠蛋白质和蛋白质/核酸复合物穿过核膜（NE）的双膜来实现的。核孔复合物（NPC）是允许大分子通过NE的唯一通道，使这种运输细胞器成为真核生命的重要机器。NPC被嵌入在渗透NE的圆形孔中，并且可以以每秒数百个事件的速率实现直径高达约40 nm的颗粒的双向传输。电子显微镜研究表明，NPC由一个中央核心组成，该核心在核质轴上具有8重旋转对称性，在NE平面上具有2重旋转对称性。这种对称的核心连接到“细胞质丝”和“核篮”结构。NPC由大约30种不同的蛋白质组成，称为核孔蛋白（nups），它们被组织成六种不同的亚复合物。每个nup以多个拷贝存在于NPC中，使得整个组装体在酵母中达到约60 MDa的非凡分子量，在脊椎动物中甚至更高。NPC不仅作为转运通道发挥作用，而且在其他基因调控模式中发挥综合作用，例如通过与转录和mRNA输出机制直接相互作用。因此，在多种人类疾病（如肿瘤或逆转录病毒疾病）中观察到NPC功能障碍并不令人惊讶。这些关联以及NPC在真核细胞生物学中的存在性作用激发了对其详细结构的研究。NPC的尺寸和柔韧性沿着，以及不能获得足够量的合适材料，目前排除了对整体完整NPC的结构进行晶体学测定。本文提出的另一种方法试图通过重组重建和NPC亚复合物的晶体学表征来阐明NPC的原子结构，所述亚复合物构成体内完整NPC的生理构建模块。结合电子显微镜重建，生化蛋白质-蛋白质相互作用图谱和细胞分析，该策略旨在为整个NPC提供复合伪原子模型，并为全面的结构-功能分析提供路线图。因此，拟议研究的结果预计将进一步加深我们对NPC参与核质转运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”奠定机制基础。“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分子组装体的范例，其功能机制由于缺乏结构洞察力而仍然难以捉摸。