Mechanisms of Autophagy

自噬机制

• 批准号: 8557093
• 负责人: Richard James Youle
• 金额: $ 67.33万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557093
• 关键词: Amino Acids; Animal Model; Apoptosis; Autophagocytosis; Autophagosome; Bax protein; Binding; Cells; Complex; Docking; Encapsulated; Eukaryotic Cell; Generations; Heat-Shock Proteins 90; Knock-out; Lysosomes; Maintenance; Mammalian Cell; Mammals; Mediating; Membrane; Mitochondria; Molecular; Molecular Chaperones; Mus; Mutate; Nerve Degeneration; Neurons; Nutrient; Organelles; Parkinson Disease; Pathway interactions; Phosphorylation; Process; Proteins; Quality Control; Regulation; Role; Site; Starvation; Technology; Time; Up-Regulation; Yeasts; dopaminergic neuron; extracellular; membrane activity; mitochondrial membrane; novel; nuclease; protein aggregate

Autophagy is known to be required for eukaryotic cells to withstand nutrient starvation by mediating the degradation of cellular components to supply substrates for ATP generation thereby helping cells survive until extracellular nutrient availability returns. Autophagy also mediates forms of quality control within cells by engulfing and degrading protein aggregates and damaged mitochondria that mitigates neurodegeneration in mice. Autophagy substrates become encapsulated by a double membrane structure that then fuses with lysosomes to mediate degradation of the cargo. How autophagosomes form and how their substrates are recognized for engulfment remain poorly understood. We are exploring the regulation and mechanism of gene products including ATG6, ATG13, ATG14 that are known to be required for autophagy. ATG6 is known to form a complex with VPS34 and ATG14 and its recruitment to membranes is dependent on ATG14. We have found that a novel domain of ATG6 that is required for membrane binding and for autophagy induction. We have confirmed that the autophagy activity and membrane targeting function of this domain is conserved in yeast and mammals. Fusion of this domain to the apoptosis inducing protein Bax spontaneously activates Bax indicating that this domain can target mitochondrial membranes. We have also found that Atg6 is phosphorylated specifically upon autophagy induction and we have identified the amino acids phosphorylated allowing us to mutate these sites and determine the role of phosphorylation in autophagy regulation. To more conclusively understand the molecular mechanisms of autophagy we have knocked out ATG5, ATG6, ATG13, and ATG14 in mammalian cells using new Talen nuclease technology. These knock out cells reveal that ATG6 phosphorylation depends on expression of ATG14, a protein that specifically docks ATG6 to membranes and is crucial for autophagosome induction. We are using these knock out cells to understand how autophagosomes form and how they recognize and engulf specific cargo such as damaged mitochondria. In a collaborative project we found that the chaperone, HSP90 is required for the selective autophagy of mitochondria and accumulation of ATG13 on the mitochondria as a step in this process. We also plan to explore if the loss of dopaminergic neurons that can occur in animal models of Parkinson's disease can be rescued by up regulation of autophagy pathways.

已知真核细胞需要自噬来抵抗营养饥饿，通过介导细胞成分的降解来提供 ATP 生成的底物，从而帮助细胞生存，直到细胞外营养可用性恢复。自噬还通过吞噬和降解蛋白质聚集体和受损的线粒体来介导细胞内的质量控制，从而减轻小鼠的神经退行性变。 自噬底物被双层膜结构封装，然后与溶酶体融合以介导货物的降解。 自噬体是如何形成的以及它们的底物如何被识别并吞噬仍然知之甚少。 我们正在探索已知自噬所需的基因产物包括ATG6、ATG13、ATG14的调控和机制。已知 ATG6 与 VPS34 和 ATG14 形成复合物，并且其向膜的募集取决于 ATG14。 我们发现 ATG6 的一个新结构域是膜结合和自噬诱导所需的。 我们已经证实该结构域的自噬活性和膜靶向功能在酵母和哺乳动物中是保守的。 该结构域与细胞凋亡诱导蛋白 Bax 的融合会自发激活 Bax，表明该结构域可以靶向线粒体膜。 我们还发现 Atg6 在自噬诱导后特异性磷酸化，并且我们已经鉴定了磷酸化的氨基酸，使我们能够突变这些位点并确定磷酸化在自噬调节中的作用。为了更确切地了解自噬的分子机制，我们使用新的 Talen 核酸酶技术敲除哺乳动物细胞中的 ATG5、ATG6、ATG13 和 ATG14。这些敲除细胞表明，ATG6 磷酸化依赖于 ATG14 的表达，ATG14 是一种将 ATG6 特异性对接至细胞膜的蛋白质，对于自噬体诱导至关重要。 我们正在使用这些敲除细胞来了解自噬体如何形成以及它们如何识别和吞噬特定的货物，例如受损的线粒体。 在一个合作项目中，我们发现伴侣蛋白 HSP90 是线粒体选择性自噬和 ATG13 在线粒体上积累所必需的，作为该过程的一个步骤。 我们还计划探索是否可以通过上调自噬途径来挽救帕金森病动物模型中可能发生的多巴胺能神经元的丧失。