Establishing a Mechanism for the Autophagic Degradation of Nuclear Components and its Relationship to Aging

核成分自噬降解机制的建立及其与衰老的关系

• 批准号: 10474326
• 负责人: Philip Mannino
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474326
• 关键词: Adaptor Signaling Protein; Aging; Animal Model; Autophagocytosis; Autophagosome; Binding Proteins; Biotin; Biotinylation; C-terminal; Catalogs; Cell Nucleus; Cell physiology; Cells; Chronology; Complement; Couples; Data; Disease; Electron Microscopy; Elements; Enzymes; Fluorescence; Fluorescence Microscopy; Gene Deletion; Genes; Genetic; Goals; Homeostasis; Knock-out; Label; Lamins; Light; Link; Longevity; Mammals; Mass Spectrum Analysis; Measures; Membrane; Molecular; N-terminal; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Outer Membrane; Nuclear Pore Complex; Organelles; Pathology; Process; Proteins; Reporting; Resolution; Role; Saccharomycetales; Specificity; Streptavidin; Structure; System; TimeLine; Work; Yeasts; age related; electron tomography; experimental study; innovation; insight; light microscopy; mutant; protein aggregation; protein function; recruit

Project Summary/Abstract Autophagy is a protective cellular mechanism with the capacity to maintain organelle homeostasis and thereby delay cellular and organismal aging. Interestingly, recent work suggests that degradation of components of the nucleus may be important for maintaining chronological lifespan (CLS). Consistent with this, there are many long-standing genetic links between the nucleus, and components of its bounding membranes, with aging. Thus, a priority for the field is to fully understand the nuclear autophagy (nucleophagy) mechanism to more fully define how clearance of nuclear components contributes to aging and age-related disease. However, there is no defined nuclear cargo adaptor in mammals making isolating the role of nucleophagy from autophagy more challenging. By contrast, a nucleophagy cargo adaptor, Atg39, has been identified in budding yeast providing an opportunity to investigate the nucleophagy mechanism and its role in maintaining CLS in this model organism. Key questions include which proteins are required for nucleophagy and how the nuclear envelope (NE) is remodeled to generate a subdomain of the nucleus competent for capture by autophagosomes. The goal of this proposal is to define key mechanistic steps in nucleophagy and provide insight into its potential role in slowing aging. I will achieve these goals by using proximity-labeling and Mass Spectrometry to identify proteins that cooperate with Atg39 to drive nucleophagy. The contribution of these proteins to nucleophagy will be determined by measuring nucleophagic flux in targeted gene deletions as well as colocalization experiments using live-cell fluorescence microscopy, which will inform the spatial and temporal dynamics of the function of each protein. The second aim will utilize an innovative combination of Bimolecular Fluorescence Complementation (BiFC) and Correlative Light Electron Microscopy (CLEM) to study the steps of nucleophagy at the resolution of the ultrastructure.

项目概要/摘要 自噬是一种保护性细胞机制，能够维持细胞器稳态，从而 延缓细胞和机体衰老。有趣的是，最近的工作表明， 细胞核对于维持时间寿命（CLS）可能很重要。与此相一致的是，还有很多 随着衰老，细胞核及其边界膜成分之间存在着长期的遗传联系。 因此，该领域的首要任务是充分了解核自噬（nucleophagy）机制，以更多地了解核自噬（nucleophagy）机制。 充分定义核成分的清除如何导致衰老和与年龄相关的疾病。然而， 哺乳动物中没有明确的核货物适配器，因此将核自噬的作用与 自噬更具挑战性。相比之下，一种核吞噬货物接头 Atg39 已在萌芽阶段被发现。 酵母提供了研究核吞噬机制及其在维持 CLS 中的作用的机会 这个模型生物。关键问题包括核吞噬需要哪些蛋白质以及核如何 包膜（NE）被重塑以生成能够捕获的细胞核子域 自噬体。该提案的目标是定义核吞噬的关键机制步骤并提供 深入了解其在延缓衰老方面的潜在作用。我将通过使用邻近标签和质量来实现这些目标 光谱分析法鉴定与 Atg39 协同驱动核吞噬的蛋白质。这些人的贡献 蛋白质对核吞噬的影响也将通过测量目标基因缺失中的核吞噬通量来确定 作为使用活细胞荧光显微镜的共定位实验，这将告知空间和 每种蛋白质功能的时间动态。第二个目标将利用创新组合 双分子荧光互补 (BiFC) 和相关光电子显微镜 (CLEM) 进行研究 超微结构分辨率中的核吞噬步骤。