Mechanisms of Autophagy

National Institute Of Neurological Disorders And Stroke

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Abstract

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.