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  • Atg cleaves Atg at the peptide

    2020-11-13

    Atg4 cleaves Atg8 at the peptide bond on the glycine residue at the C-terminus, thus allowing the conjugation of Atg8 to phosphatidylethanolamine (PE) with the participation of other autophagy molecules. Atg4 can also serve as a deconjugating enzyme, which cleaves the amide bond of the conjugated Atg8 and releases it from the autophagosomal membrane. The latter step is important for reusing Atg8 upon autophagy completion. Moreover, this deconjugation process may also occur on isolation membrane, which would positively or negatively affect membrane formation (Nair et al., 2012, Scherz-Shouval et al., 2007, Yu et al., 2012). Therefore, Atg4-mediated deconjugation of Atg8 from the membrane may regulate the autophagy process. Autophagic activity is diminished in both Atg4-deficient yeast and Atg4B-deficient mice (Marino et al., 2010, Nakatogawa et al., 2012, Yu et al., 2012), while deletion of Atg4C in mice displayed minimal impact on autophagy (Marino et al., 2007), indicating that Atg4B is likely the principal mammal Atg4 homolog. Inhibition of Atg4A and Atg4B activity by reactive oxygen species has been shown in one study to block starvation-induced autophagy (Scherz-Shouval et al., 2007), suggesting that the cysteine protease nature of Atg4 can be regulated by redox events. A more recent study found that the yeast Atg4 could be regulated by thioredoxin on the reduction of the disulfide bond formed by Cys338 and Cys394 (Perez-Perez, Zaffagnini, Marchand, Crespo, & Lemaire, 2014). Mutation of the two cysteines resulted in an increased recruitment of Atg8 to the phagophore assembly site. In addition, Akin et al. have shown that Atg4B knockdown in osteosarcoma cell line Saos-2 and breast cancer cell line MDA-MB468 reduced starvation-induced autophagy and that Saos-2 ubiquitin activating enzyme lacking Atg4B failed to survive in amino acid-starvation conditions and failed to grow as xenografted tumors in mice (Akin et al., 2014). Atg4 proteins may thus have a role in cancer development. Finally, some studies have suggested a potential link between Atg4D and apoptosis, indicating a putative role for Atg4D at the interface between autophagy and apoptosis (Kaminskyy and Zhivotovsky, 2014, Norman et al., 2010).
    The Structure and Regulatory Machinery of Atg4 Atg4 proteases range from 393 to 474 amino acids in size and possess several structural features of cysteine proteases. The crystal structure of Atg4A (PDB ID: 2P82) has been resolved (Fig. 2A). Atg4A shares a similar catalytic triad (Cys77/Asp279/His281) with Atg4B (Marino et al., 2003). Structure studies have shown that all the residues of Atg4B that interact with LC3B are conserved in Atg4A except Leu232. Atg4A possesses Ile233 instead of Leu232 at the corresponding position. When Ile233 is changed to Leu, the mutated Atg4A acquires a notable ability to cleave LC3B (Satoo et al., 2009). The crystal structure of human Atg4B (PDB ID: 2CY7) has also been resolved (Fig. 2B). The structure of Atg4B contains a papain-like fold and a small α/β-fold domain, which is thought to be the binding sites for Atg8 homologues. The active site of Atg4B is composed of Cys74, Asp278, and His280. Mutation of these sites is associated with the complete loss of its catalytic activity (Sugawara et al., 2005). The active site of free Atg4B is masked by a regulatory loop (residues 259–262) (Satoo et al., 2009). A large conformational change of Atg4B is induced in the regulatory loop and the N-terminal tail (residues 1–24) when Atg4B interacts with LC3 (Fig. 2C and D). In this process, the regulatory loop masking the entrance of the active site of Atg4B is lifted by LC3 Phe119, resulting in the formation of a groove, into which the LC3 tail can enter, gaining the access to the active site. Besides, the N-terminal tail is originally positioned at the back of the active sites and undergoes a large conformational change as well upon interaction with LC3, which could affect the exit of the cleaved substrates. Thus, deletion of this N-terminal region has been found to increase Atg4B activity (Satoo et al., 2009).