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

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

项目成果

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

用于亲和力分离的内源性蛋白质组件的天然质谱分析的强大工作流程。

• 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

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

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.

蛋白质组学技术内置的固有偏见的影响：比较接近性标记和亲和力捕获。

• 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