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.

项目摘要/摘要 自噬是一种保护性蜂窝机制，具有维持细胞器稳态的能力，从而 延迟细胞和生物衰老。有趣的是，最近的工作表明 细胞核对于维持时间顺序寿命（CLS）可能很重要。与此一致，有很多 核之间长期存在的遗传联系，其边界膜的成分随老化。 因此，该领域的优先级是充分理解核自噬（接核）机制 充分定义了核成分的清除如何有助于衰老和与年龄有关的疾病。然而， 在哺乳动物中没有确定的核货物适配器，可以使亲核的作用从 自噬更具挑战性。相比之下，已经确定 酵母提供了研究亲核机制及其在维持CLS中的作用的机会 这个模型有机体。关键问题包括核含量需要哪种蛋白质以及核如何 重塑包络（NE）以生成一个拟合捕获的细胞核的子域 自噬体。该提案的目的是定义亲核的关键机械步骤，并提供 深入了解其在减慢衰老中的潜在作用。我将通过使用邻近标记和质量来实现这些目标 光谱法鉴定与ATG39合作以驱动亲核的蛋白质。这些的贡献 蛋白质至亲核能也将通过测量靶基因缺失中的亲核通量来确定 作为使用活细胞荧光显微镜进行共定位实验，这将告知空间和 每种蛋白质功能的时间动力学。第二个目标将利用创新的组合 双分子荧光互补（BIFC）和相关光显微镜（CLEM）研究 在分辨率的分辨率下的亲核步骤。