ATG4B is a cysteine protease that serves as a critical regulator of autophagy through dual enzymatic functions. Primarily, ATG4B proteolytically activates ATG8 family proteins (MAP1LC3A/B/C, GABARAPL1/2, GABARAP) by cleaving their C-terminal amino acids to expose a glycine residue 123. This exposed glycine is essential for ATG8 conjugation to phosphatidylethanolamine (PE) and membrane insertion, enabling autophagosome formation 12. Beyond proteolytic activation, ATG4B catalyzes delipidation of PE-conjugated ATG8 proteins during macroautophagy and mediates deconjugation of ATG8 from other proteins, preventing pathological high-molecular weight conjugates 45. ATG4B also participates in non-canonical autophagy by catalyzing delipidation of ATG8 proteins conjugated to phosphatidylserine at endolysosomal compartments 6. Additionally, ATG4B regulates phagophore growth during mitophagy independently of its protease activity by modulating ATG9A trafficking 5. Dysregulation of ATG4B is implicated in disease pathogenesis: elevated lipocalin 2 binding inhibits ATG4B activity in dry age-related macular degeneration 7, while impaired ATG4B expression due to TDP-43 dysfunction contributes to amyotrophic lateral sclerosis pathology 8. ATG4B also participates in ferroptotic cell death 9.