Autophagy is an essential cellular process whereby damaged proteins and organelles are degraded and recycled. Autophagy, while happening constantly at a basal level, is tightly regulated and can be further induced under cellular stress. One of the regulators of the early steps of autophagy is ATG4. The ATG4 family of cysteine proteases consists of 4 homologs: ATG4A, ATG4B, ATG4C, and ATG4D. This family of proteins regulates the initiation of autophagosome assembly by cleaving ATG8-like proteins, including LC3, at a conserved glycine residue and thereby activating it. Cleaved LC3 can then be lipidated by the ATG12-ATG5-ATG16 protein complex. LC3 conjugated to the lipid PE is inserted into the assembling autophagosome membrane where it recruits the core autophagy machinery. Without LC3 activation by ATG4 autophagosome assembly is blocked. ATG4 is also responsible for cleaving and removing LC3 from the autophagosome. This serves to mediate fusion of the autophagosome with the lysosome and to recycle LC3. Like the other ATG4 homologs, ATG4D is widely expressed. A study in mice identified expression in the heart, lung, kidney, and testis through western blotting with an ATG4D antibody (1). A study out of the University of Bristol has found ATG4D specifically cleaves the ATG8 paralog GABARAP-L1 (2). This same study identified an important mechanism to regulate ATG4D levels and activity. The authors identified a caspase cleavage sequence in the N-terminus of ATG4D and showed cleavage of ATG4D in apoptotic cells. This finding indicates a dual role for ATG4D in induction of both autophagy and apoptosis. A follow-up to this study showed ATG4D is imported into mitochondria in response to cellular stress and sensitizes cells to cell death (3). While both of these studies relied on overexpression of GFP-ATG4D, further investigations using ATG4D antibodies may allow the examination of endogenous ATG4D. Recently missense mutation in ATG4D were found to be associated with neurodegenerative disease indicating a previously unknown role in neuronal homeostasis (4). Further investigations of ATG4D’s neuronal function will benefit from ATG4D antibodies to monitor protein localization to elucidate the mechanism of neurodegeneration. ATG4D antibodies have also been used to identify a autophagy fingerprint for ischemia and reperfusion injuries (5). This study found ATG4A, ATG4C, and ATG4D were increased in cardiac tissue and may inform treatment of these types of cardiac injuries. While the functions of ATG4D remain unclear, these recent studies have helped to understand its role in various contexts ranging from neurodegenerative disease to apoptosis. Further experiments with ATG4D antibodies will allow examination of endogenous ATG4D protein and help characterize its function in various cellular contexts.
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