Mechanisms of Autophagy

已知自噬是真核细胞抵御营养饥饿所必需的，它通过介导细胞成分的降解来提供ATP生成的底物，从而帮助细胞存活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线粒体介导细胞内的质量控制，从而减轻小鼠的神经退行性变。自噬底物被双膜结构包裹，然后与溶酶体融合，介导货物的降解。自噬体是如何形成的，以及它们的底物是如何被识别并吞噬的，人们对其了解甚少。我们正在探索包括ATG6、ATG13、ATG14在内的已知自噬所需基因产物的调控及其机制。已知ATG6与VPS34和ATG14形成复合物，其在膜上的募集依赖于ATG14。我们发现了ATG6的一个新的结构域，它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在自噬诱导时被特异性磷酸化，我们已经确定了磷酸化的氨基酸，使我们能够使这些位点突变，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，我们利用新的Talen核酸酶技术敲除了哺乳动物细胞中的ATG5、ATG6、ATG13和ATG14。这些敲除的细胞表明，ATG6磷酸化依赖于ATG14的表达，ATG14是一种特异性地将ATG6连接到膜上的蛋白质，对自噬体的诱导至关重要。我们正在利用这些被敲除的细胞来了解自噬体是如何形成的，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，我们发现伴侣蛋白HSP90是线粒体选择性自噬和线粒体上ATG13积累所必需的，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多巴胺能神经元的丢失是否可以通过上调自噬通路来挽救。

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.