Structure-Function Mapping of the Nuclear Pore Complex-Renewal

核孔复合体更新的结构-功能图谱

• 批准号: 10658474
• 负责人: JOHN D. AITCHISON
• 金额: $ 70.35万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658474
• 关键词: Address; Architecture; Binding; Cell Nucleus; Cell physiology; Cells; Chromatin; Communicable Diseases; Complex; Cytoplasm; DNA; Data; Defect; Development; Disease; Drug Targeting; Elements; Epigenetic Process; Freezing; Functional disorder; Gene Expression; Gene Silencing; Genes; Hand; Human; Knowledge; Lead; Light; Malignant Neoplasms; Maps; Measures; Mechanics; Mediating; Membrane; Messenger RNA; Modeling; Molecular; Mutation; Nature; Nuclear; Nuclear Pore Complex; Nucleoplasm; Oncogenic; Phenotype; Positioning Attribute; Process; Protein Isoforms; Proteins; Side; Site-Directed Mutagenesis; Structure; Testing; Therapeutic; Time; Viral; Work; Yeasts; chromatin remodeling; design; developmental disease; human disease; macromolecular assembly; messenger ribonucleoprotein; mutant; nervous system disorder; nucleocytoplasmic transport; protein function

ABSTRACT Nuclear pore complexes (NPCs) are huge macromolecular assemblies that serve as the only conduit for bidirectional transport between the nucleus and cytoplasm. We have determined the constituents, architecture, and detailed high precision structure of the archetypal yeast NPC. However, despite our increasing structural information on NPCs, we still lack a fundamental understanding of the mechanics of numerous of its functions. With our detailed maps in hand, we are, for the first time, in a unique position to map and reveal the structural changes associated with functional states that throw light on mechanisms underlying critical aspects of NPC function. Our hypothesis is that, despite some overlap, discrete and distinct structural stages and states are associated with NPCs’ varied functions. We have therefore established a powerful pipeline for analyzing NPCs and their vicinal associated complexes both structurally and functionally in defined functional states onto which we map quantitative phenotypic information. This information will allow us to move from static models of NPCs to working models of the machine in action, breathing life into our NPC maps and dissecting out how particular functionalities are mechanistically supported at the structural level. We focus on two such functionalities that are central to nuclear function at two related levels: first, as a regulator of transport, NPCs control mRNA packaging and export to the cytoplasm to both mediate and regulate gene expression; and second, NPCs directly control genes by binding chromatin and its regulators to alter expression states epigenetically. Both processes are incompletely understood at the molecular level, and have profound effects on cellular function as evidenced by the fact that disruptions of NPC-associated proteins associated with these functions lead to many human diseases. For Aim 1, we will determine the molecular machinery of NPC-mediated mRNP export by studying NPCs effectively “frozen” in defined intermediate stages of mRNP export. For Aim 2, we will determine the molecular machinery of NPC-mediated chromatin organization, specifically focusing on subtelomeric gene silencing. Using our established pipeline, we will identify and structurally characterize these NPC stages and states and their vicinal interactomes. Realizing these Aims will generate NPC structure-function maps in unprecedented detail, which will be of great use to the field to understanding how the mRNP export and chromatin remodeling machineries act in concert with different parts of the NPC to enable their functionalities and will shed light on the nature of numerous disorders associated with dysfunction in these processes. The resulting structure-function NPC maps promise to set the stage for tapping the NPC’s tremendous potential as a drug target for many human conditions ranging from cancers to infectious diseases to developmental and neurological disorders. 1

抽象的 核孔复合物（NPC）是巨大的大分子组件，作为唯一的渠道 核与细胞质之间的双向转运。我们已经确定了构造，体系结构， 以及原型酵母NPC的详细高精度结构。但是，尽管我们的结构增加了 关于NPC的信息，我们仍然对其许多功能的机制缺乏基本理解。 有了我们的详细地图，我们首次处于独特的位置，可以映射并揭示结构 与功能状态相关的变化，这些状态阐明了NPC关键方面的基础机制 功能。我们的假设是，目的地某些重叠，离散和独特的结构阶段和状态是 与NPC的各种功能相关联。因此，我们已经建立了一个强大的管道来分析NPC 在结构和功能上，它们在定义的功能状态下在其上都在此上 我们绘制定量表型信息。这些信息将使我们从NPC的静态模型转变 到机器的工作模型，将生命呼吸到我们的NPC地图，并剖析特定的 功能在结构层面得到机械支持。我们专注于两个这样的功能 NPC控制mRNA包装，首先是两个相关级别的核功能的中心：首先，作为运输的调节剂 并导出到细胞质中介导和调节基因表达；其次，NPC直接控制 通过结合染色质及其调节剂以表观遗传态度改变表达状态的基因。两个过程都是 在分子水平上不完全理解，对细胞功能具有深远影响 与这些功能相关的NPC相关蛋白的破坏导致了许多人的事实 疾病。对于AIM 1，我们将通过研究NPC介导的MRNP导出的分子机制 NPC在MRNP导出的定义中间阶段有效地“冷冻”。对于AIM 2，我们将确定 NPC介导的染色质组织的分子机械，特别关注亚端粒基因 沉默。使用我们已建立的管道，我们将在结构上识别并在结构上表征这些NPC阶段，并且 国家及其典型相互作用。意识到这些目标将在 前所未有的细节，这对该领域非常有用，以了解MRNP出口和 染色质重塑机与NPC的不同部分共同起作用以实现其功能 并将阐明这些过程中与功能障碍相关的众多疾病的性质。 由此产生的结构功能NPC地图有望为挖掘NPC的巨大潜力设定舞台 从癌症到感染性疾病再到发育和发育和 神经系统疾病。 1

项目成果

Heh2/Man1 may be an evolutionarily conserved sensor of NPC assembly state.

• DOI: 10.1091/mbc.e20-09-0584
• 发表时间: 2021-07-15
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Borah S;Thaller DJ;Hakhverdyan Z;Rodriguez EC;Isenhour AW;Rout MP;King MC;Lusk CP
• 通讯作者: Lusk CP

A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies.

• DOI: 10.1021/acs.analchem.5b04477
• 发表时间: 2016-03-01
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Olinares PD;Dunn AD;Padovan JC;Fernandez-Martinez J;Rout MP;Chait BT
• 通讯作者: Chait BT

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

GBF1 的病毒蛋白参与通过合成致死作用诱导宿主细胞脆弱性。

• DOI: 10.1101/2020.10.12.336487
• 发表时间: 2020
• 期刊: bioRxiv : the preprint server for biology
• 作者: Navare,ArtiT;Mast,FredD;Olivier,JeanPaul;Bertomeu,Thierry;Neal,Maxwell;Carpp,LindsayN;Kaushansky,Alexis;Coulombe-Huntington,Jasmin;Tyers,Mike;Aitchison,JohnD
• 通讯作者: Aitchison,JohnD

The Evolution of Organellar Coat Complexes and Organization of the Eukaryotic Cell.

细胞器外壳复合物的进化和真核细胞的组织。

• DOI: 10.1146/annurev-biochem-061516-044643
• 发表时间: 2017
• 期刊: Annual review of biochemistry
• 影响因子: 16.6
• 作者: Rout,MichaelP;Field,MarkC
• 通讯作者: Field,MarkC

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.

• DOI: 10.1016/j.jbc.2022.102726
• 发表时间: 2023-01
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Moreira, Claudia Maria do Nascimento;Kelemen, Cristina D.;Obado, Samson O.;Zahedifard, Farnaz;Zhang, Ning;Holetz, Fabiola B.;Gauglitz, Laura;Dallagiovanna, Bruno;Field, Mark C.;Kramer, Susanne;Zoltner, Martin
• 通讯作者: Zoltner, Martin