Altered Communication between the nucleus and the mitochondria under oncogenic states

致癌状态下细胞核与线粒体之间通讯的改变

• 批准号: 9764927
• 负责人: MICHAEL P ROUT
• 金额: $ 38.77万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764927
• 关键词: Address; Affect; Affinity; Antineoplastic Agents; Apoptosis; Apoptotic; Architecture; Behavior; Biological Assay; Biological Models; Cell Fractionation; Cell Line; Cell Nucleus; Cell Shape; Cell Size; Cell physiology; Cells; Cellular Morphology; Chimeric Proteins; Chromosomal translocation; Communication; Complex; Defect; Disease; Drug Targeting; Elements; FGFR1 gene; Failure; Gene Expression; Genetic Materials; Goals; Health; Homeostasis; Human; Immunofluorescence Microscopy; Impairment; Interphase Cell; Knowledge; Lead; Link; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Methodology; Mitochondria; Modification; Monitor; Morphology; Multiprotein Complexes; Mutation; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Structure; Oncogenic; Organelles; Pathologic; Pathway interactions; Pharmaceutical Preparations; Process; Protein Export Pathway; Protein Import; Protocols documentation; Reagent; Research; Role; Signal Pathway; Signal Transduction; Stress; Structure; Techniques; Testing; cell behavior; cell growth; experimental study; improved; leukemia; macromolecule; mutant; nanobodies; novel; nucleocytoplasmic transport; outcome prediction; overexpression; receptor; sarcoma; tissue culture; tool; trafficking; tumorigenesis; virtual

Project summary Nuclear pore complexes (NPCs) are multi-protein assemblies embedded in the nuclear envelope, forming channels that mediate the regulated bidirectional nucleocytoplasmic transport of macromolecules. This transport allows for communication between the central organelle, the nucleus, and the rest of the cell, providing a crucial means to control gene expression, signaling networks, and cell homeostasis. Due to its central role, even modest disruption of the NPC has profound effects on cellular function, leading to many cancers such as leukemia and sarcomas. Crucially, a major causative connection in cancer and the NPC is the communication between the nucleus and the mitochondria in the apoptotic signaling pathway, triggered by intracellular damage or by oncogenic stress. The examples of Selinexor and Verdinexor have now established nucleocytoplasmic trafficking as a valid and powerful anti-cancer drug target; and there is strong evidence that at least in part these drugs are effective because they regulate this nuclear-mitochondrial communication pathway. We hypothesize that cancer- associated NPC alterations change the NPCs’ structure and interactome that in turn impact their overall capability to serve in transport and intracellular communication. The proposed study is divided into two independent but synergistic aims that will decipher the structural and functional defects caused by these oncogenic alterations, focusing on downstream effects on the nuclear-mitochondrial apoptotic signaling pathway. The first Aim (1.1) is to characterize the changes in NPC interactome caused by oncogenic Nup alterations; specifically the overexpression of Nup62 or Nup88 or presence of TPR-FGFR1 or Nup214-Abl1 fusion mutations. We will establish a tissue culture model system expressing each of these cancer-associated Nup alterations and then compare their interactomes to the normal state using affinity-capture and mass spectrometry methodologies already established in our lab. As an additional tool, we suggest (Aim 1.2) to produce novel research tools in the form of nanobodies against cancer-associated Nups. Importantly, Aim 1.1 is not dependent on Aim 1.2. Aim 2 will focus on the functional impact of the oncogenic alterations. We will first (Aim 2.1) examine how these alterations affect nuclear and mitochondrial morphology and behavior. First we will assess changes in cellular morphology and behavior in cell lines bearing oncogenic Nup alterations. Next, we will assess how the Nup oncogenic alterations affect the mitochondria. Finally (Aim 2.2), we will look for changes in nucleocytoplasmic trafficking under oncogenic conditions and how it affects nuclear – mitochondrial communication. We will monitor for changes in protein import and export, localization of transport factors and we will look for changes in specific mediators of nuclear-mitochondrial signaling. Our techniques and tools will (i) allow us to identify disease-causing alterations in NPC architecture, (ii) detect altered amounts or localizations of Nups due to these alterations, and correlate (i) with (ii) to determine the underlying mechanism of the Nup-induced oncogenic changes.

项目摘要 核孔复合物（NPC）是嵌入核包膜中的多蛋白组件，形成 介导大分子的受调节双向核核核质胞质转运的通道。这辆运输 可以在中央细胞器，核和其他细胞的其余部分之间进行通信，从而提供至关重要的 控制基因表达，信号网络和细胞稳态的手段。由于其核心角色，甚至谦虚 NPC的破坏对细胞功能具有深远的影响，导致许多癌症，例如白血病和 肉瘤。 conc依的是，癌症和NPC中的主要符号联系是 细胞核和凋亡信号通路中的线粒体，由细胞内损伤或由 致癌应力。 Selinexor和Verdinexor的例子现已建立了核细胞贩运 作为有效而强大的抗癌药物目标；有强有力的证据表明，至少部分这些药物是 有效，因为它们调节了这种核核素通信途径。我们假设癌症 - 相关的NPC更改会改变NPC的结构和互动群，从而影响其整体 在运输和细胞内通信中服务的能力。拟议的研究分为两个 独立但协同的目的将破坏由这些造成的结构和功能缺陷 致癌的改变，重点是对核用凋亡信号通路的下游影响。 第一个目的（1.1）是表征由致癌NUP改变引起的NPC相互作用组的变化。 特别是NUP62或NUP88或TPR-FGFR1或NUP214-ABL1融合突变的过表达。 我们将建立一个表达与癌症相关的NUP改变和的组织培养模型系统 然后使用亲和力捕获和质谱法比较其相互作用与正常状态 已经在我们的实验室中建立。作为额外的工具，我们建议（AIM 1.2）在 针对癌症相关的NUPS的纳米体形式。重要的是，目标1.1不取决于目标1.2。 AIM 2将集中于致癌改变的功能影响。我们将首先（AIM 2.1）检查如何 这些改变会影响核和线粒体形态和行为。首先，我们将评估更改 细胞的形态和行为，带有致癌性NUP改变的细胞系。接下来，我们将评估如何 NUP致癌改变会影响线粒体。最后（AIM 2.2），我们将寻找变化 在肿瘤条件下进行核细胞质运输及其如何影响核 - 线粒体 沟通。我们将监控蛋白质进出口的变化，运输因素的定位以及 我们将寻找核用光信号传导的特定介体的变化。 我们的技术和工具将（i）允许我们确定NPC架构中引起疾病​​的改变，（ii）检测 由于这些变化而改变的NUP的数量或本地化，并将（i）与（i）相关联（i）以确定 NUP诱导的致癌变化的基本机制。