Mechanisms of Autophagy

已知自噬是真核细胞抵御营养饥饿所必需的，它通过调节细胞成分的降解来为ATP的生成提供底物，从而帮助细胞存活，直到细胞外营养恢复。自噬还通过吞噬和降解蛋白质聚集体和受损的线粒体来调节细胞内的质量控制形式，从而减轻小鼠的神经退化。自噬底物被双层膜结构包裹，然后与溶酶体融合，以调节货物的降解。自噬小体是如何形成的，它们的底物是如何被吞噬的，目前还知之甚少。我们正在探索包括ATG6、ATG13、ATG14在内的基因产物的调节和机制，这些基因产物是自噬所必需的。已知ATG6与Vps34和ATG14形成复合体，其在膜上的募集依赖于ATG14。我们发现ATG6的一个新结构域是膜结合和自噬诱导所必需的。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。该结构域与凋亡诱导蛋白Bax的融合可自发激活Bax，表明该结构域可靶向线粒体膜。我们还发现，ATG6在自噬诱导时被特异性地磷酸化，我们已经确定了被磷酸化的氨基酸，这使得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核酸酶技术敲除了哺乳动物细胞中的ATG5、ATG6、ATG13和ATG14。这些敲除细胞揭示，ATG6的磷酸化依赖于ATG14的表达，ATG14是一种专门将ATG6对接到膜上的蛋白质，对自噬诱导至关重要。我们正在使用这些敲除细胞来了解自噬小体是如何形成的，以及它们如何识别和吞噬特定的货物，如受损的线粒体。在一个合作项目中，我们发现作为伴侣的HSP90是选择性自噬线粒体和在线粒体上积累ATG13所必需的，这是这一过程的一个步骤。我们还计划探索帕金森病动物模型中可能发生的多巴胺能神经元的丢失是否可以通过上调自噬途径来挽救。

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.