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。许多类型的选择性自噬已经被研究过，在确定货物选择性的基础上已经取得了一些进展。已知自噬受体蛋白OPTN、NDP52、TAX1BP1和p62在先天免疫的细菌自噬中起重要作用，而OPTN、NDP52以及TAX1BP1在与帕金森病相关的PINK1/帕金森介导的有丝分裂中起重要作用。然而，蛋白质聚集体如何被自噬体识别尚不清楚。

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. Numerous types of selective autophagy have been examined, and some progress has been made in determining the basis of cargo selectivity. The autophagy receptor proteins OPTN, NDP52, TAX1BP1 and p62 are known to be important in autophagy of bacteria for innate immunity, whereas OPTN, NDP52, and to a lesser extent, TAX1BP1 are essential for PINK1/Parkin-mediated mitophagy linked to Parkinsons disease. How protein aggregates are recognized by autophagosomes, however, is unclear. 1) Protein aggregates disrupt cellular homeostasis, causing toxicity linked to neurodegeneration and ways to eliminate them are under intense investigation, including autophagic engulfment and degradation of them. We compared the requirements for autophagy receptor proteins: OPTN, NBR1, p62, NDP52, and TAX1BP1 in clearance of proteotoxic aggregates. Endogenous TAX1BP1 is recruited to and required for the clearance of stress-induced aggregates, whereas ectopic expression of TAX1BP1 increases clearance through autophagy, promoting viability of human induced pluripotent stem cell-derived neurons. In contrast, TAX1BP1 depletion sensitizes cells to several forms of aggregate-induced proteotoxicity. Furthermore, TAX1BP1 is more specifically expressed in the brain compared to other autophagy receptor proteins. In vivo, loss of TAX1BP1 results in accumulation of high molecular weight ubiquitin conjugates and premature lipofuscin accumulation in brains of young TAX1BP1 knockout mice. TAX1BP1 mediates clearance of a broad range of cytotoxic proteins indicating therapeutic potential in neurodegenerative diseases. 2) Autophagy of mitochondria and protein aggregates is initiated by the ULK complex, which consists of the kinase ULK1, FIP200, ATG13, and ATG101. In collaboration with a group at UC Berkeley, we mapped the structural basis and biological consequences of their mutual interactions. The N-terminal domain of FIP200 interact with the C-terminal domain of ATG13. Mutations in these regions abolish their interaction. FIP200 forms a dimer, while only a single molecule each of the other subunits is present in the ULK complex. The FIP200 N-terminal domain is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C 20 nm across. The ULK1 EAT domain interacts loosely with the N-terminal domain dimer, while the ATG13:ATG101 HORMA dimer does not contact the N-terminal domain. Cryo-EM of the N-terminal domain dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex. Interestingly, TBK1 functions in PINK1/Parkin mediated mitophagy and in STING activation of innate immunity and is under our investigation in both those pathways. The claw domain on Fib200 appears to be a potential drug pocket that we are screening for high affinity ligands. Identification of such may allow AutoTACs development to eliminate protein aggregates involved in neurodegeneration.