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: 10248415
• 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/10248415
• 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（Nuclear pore complex，NPC）是一种嵌入核膜的多蛋白质组装体， 介导大分子调节性双向核质转运的通道。此传输 允许中央细胞器，细胞核和细胞其余部分之间的通信，提供了一个关键的 控制基因表达、信号网络和细胞稳态的手段。由于其核心作用，即使是温和的 NPC的破坏对细胞功能有深远的影响，导致许多癌症，如白血病和 肉瘤至关重要的是，癌症和NPC之间的一个主要致病联系是肿瘤细胞之间的沟通。 细胞核和线粒体在凋亡信号通路中的作用，由细胞内损伤或 致癌应激Selinexor和Verdinexor的例子现在已经建立了核质运输 作为有效和强大的抗癌药物靶点;有强有力的证据表明，至少部分这些药物是 有效，因为它们调节这种核-线粒体通讯途径。我们假设癌症- 相关的NPC改变改变了NPC的结构和相互作用组，反过来又影响了他们的整体 在运输和细胞内通讯中起作用的能力。拟议的研究分为两部分 独立但协同的目标，将破译这些造成的结构和功能缺陷， 致癌改变，重点是对核-线粒体凋亡信号通路的下游影响。 第一个目的（1.1）是描述由致癌Nup改变引起的NPC相互作用物组的变化; 特别是Nup 62或Nup 88的过表达或TPR-FGFR 1或Nup 214-FGFR 1融合突变的存在。 我们将建立一个组织培养模型系统，表达这些癌症相关的Nup改变， 然后使用亲和捕获和质谱方法将它们的相互作用组与正常状态进行比较， 已经在我们的实验室里建立起来了。作为一个额外的工具，我们建议（目标1.2）在全球范围内开发新的研究工具。 针对癌症相关Nups的纳米抗体形式。重要的是，目标1.1并不依赖于目标1.2。 目标2将集中于致癌改变的功能影响。我们将首先（目标2.1）研究如何 这些改变影响核和线粒体的形态和行为。首先，我们将评估 具有致癌Nup改变的细胞系中的细胞形态和行为。接下来，我们将评估 Nup致癌改变影响线粒体。最后（目标2.2），我们将寻找 致癌条件下的核质运输及其如何影响核-线粒体 通信我们将监测蛋白质进出口的变化，运输因子的定位， 我们将寻找核-线粒体信号传导特定介质的变化。 我们的技术和工具将（i）使我们能够识别NPC结构中的致病性改变，（ii）检测 由于这些改变而改变的Nups的量或定位，并将（i）与（ii）相关联以确定 NUP诱导的致癌变化的潜在机制。