Establishing a blueprint for nuclear pore complex assembly

建立核孔复合体组装蓝图

• 批准号: 9250184
• 负责人: Charles Patrick Lusk
• 金额: $ 31.54万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9250184
• 关键词: Alzheimer' s Disease; Animal Model; Automobile Driving; Biogenesis; Cell Nucleus; Cell membrane; Cell physiology; Coupled; Cytoplasm; Data; Data Set; Development; Dimerization; Disease; Drug Targeting; Eukaryota; Event; Fluorescent Probes; Future; Generations; Genes; Genetic; Goals; Health; Heart Diseases; Home environment; Human; In Vitro; Individual; Infection; Interphase; Light; Liposomes; Malignant Neoplasms; Membrane; Membrane Fusion; Membrane Proteins; Mitotic; Molecular; Mutation; Neurodegenerative Disorders; Nuclear Envelope; Nuclear Outer Membrane; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoplasm; Parkinson Disease; Pathology; Pathway interactions; Permeability; Process; Proteins; Recruitment Activity; Saccharomyces cerevisiae; Saccharomycetales; Series; Sirolimus; Stream; System; Tertiary Protein Structure; Testing; Therapeutic; Time; TimeLine; Tissues; Translational Research; Triple A Syndrome; Virus; Virus Diseases; Work; Yeasts; base; combinatorial; design; dimer; experimental study; genetic resource; in vivo; insight; macromolecule; nucleocytoplasmic transport; overexpression; programs; public health relevance; reconstitution; success

DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) provide the sole gateways that control the bidirectional exchange of molecules across the nuclear envelope (NE) in all eukaryotes. From the perspective of human health, compromised function of several of the components of the NPC (nucleoporins/nups) is associated with diverse diseases including cancers, heart disease, triple A syndrome, and neurodegenerative diseases like Alzheimer's and Parkinson's. Further, to propagate and promote infection, viruses often modulate nup function. The wide spectrum of these pathologies suggests that the NPC impacts a broad array of essential cellular processes, although the mechanisms are poorly defined. In addition, several nup genes are up- or down- regulated, and NPC number is altered, in developmental and disease contexts. Thus, a better understanding of mechanisms that contribute to NPC function will likely reveal translational drug targets in the future. While, as a field, we have a good understanding of the underlying mechanisms governing nuclear transport, a major remaining challenge is determining the mechanism of de novo NPC assembly. Specifically, it is not understood how the ~30 individual nups are coordinately assembled in space and time to form the ~50 MD NPC. Further, the assembly of individual nups coincides with the generation of membrane curvature that leads to the close apposition and eventual fusion of the inner and outer nuclear membranes to generate a nuclear pore. It is not known how nup assembly and membrane fusion are coordinated, nor has the fusion machinery been clearly identified. In this proposal, we aim to tackle two key challenges in understanding the assembly of the NPC. First, we propose an experimental strategy designed to elucidate the order by which nups are assembled at the NE during interphase. We will achieve this by exploiting the genetic toolkit of the yeast, S. cerevisiae, to generate a system where we rapidly and specifically inactivate newly synthesized nups one at a time, leaving mature NPCs unaffected. After inactivation of each target nup, we will comprehensively examine the distribution of other nups to assign an up- or down-stream relationship in the order of assembly. Integrating this data set, we will systematically define the steps in the assembly process. Second, we will use both the order analysis and a candidate approach to identify proteins that generate membrane curvature to support pore formation in the NE during NPC assembly. Using a series of in vivo and in vitro approaches, we will test whether these candidate curvature generators are capable of directly driving membrane curvature and home in on their curvature-generating domains. In this way, we will shed significant new light onto the molecular mechanisms of pore formation and directly test how membrane curvature impacts NPC assembly. The experiments outlined in this proposal will provide much needed mechanistic insight into the essential process of NPC biogenesis, universal to all eukaryotes.

描述（由申请人提供）：核孔复合物（npc）在所有真核生物中提供控制分子在核包膜（NE）上双向交换的唯一通道。从人类健康的角度来看，NPC（核孔蛋白/核孔蛋白）的几个组成部分的功能受损与多种疾病有关，包括癌症、心脏病、aaa综合征和阿尔茨海默病和帕金森病等神经退行性疾病。此外，为了繁殖和促进感染，病毒经常调节nup功能。这些病理的广泛性表明鼻咽癌影响了广泛的基本细胞过程，尽管其机制尚不明确。此外，在发育和疾病背景下，一些nup基因被上调或下调，NPC数量被改变。因此，更好地了解NPC功能的机制可能会在未来揭示转化药物靶点。虽然，作为一个领域，我们已经很好地理解了控制核传输的潜在机制，但一个主要的挑战是确定从头开始的NPC组装机制。具体来说，目前还不清楚这30个独立的星云是如何在空间和时间上协调地组装成50个MD的NPC的。此外，单个核结的组装与膜曲率的产生相一致，从而导致内外核膜的紧密靠近和最终融合，从而产生核孔。目前尚不清楚nup组装和膜融合是如何协调的，也没有清楚地确定融合机制。在这个提案中，我们的目标是解决了解全国人大会议的两个主要挑战。首先，我们提出了一种实验策略，旨在阐明在间期期间nup在NE组装的顺序。我们将通过利用酵母的遗传工具包来实现这一目标，酿酒酵母，产生一个系统，在这个系统中，我们可以一次快速地特异性地灭活新合成的核苷酸，而不影响成熟的npc。在每个目标nup失活后，我们将全面检查其他nup的分布，以按照组装顺序分配上游或下游关系。整合此数据集，我们将系统地定义装配过程中的步骤。其次，我们将使用顺序分析和候选方法来识别在NPC组装过程中产生膜曲率以支持NE孔形成的蛋白质。使用一系列体内和体外方法，我们将测试这些候选曲率发生器是否能够直接驱动膜曲率并返回其曲率产生域。通过这种方式，我们将为孔形成的分子机制提供重要的新见解，并直接测试膜曲率如何影响NPC组装。本提案中概述的实验将为所有真核生物普遍存在的NPC生物发生的基本过程提供急需的机制见解。