Abstract
Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. Lipidated LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. Endogenous ATG4‐mediated deconjugation of LC3‐PE is required for LC3 recycling. However, the Legionella effector RavZ irreversibly deconjugates LC3‐PE to inhibit autophagy. It is not clear how ATG4 and RavZ process LC3‐PE with distinct modes. Herein, a series of semisynthetic LC3‐PE proteins containing C‐terminal mutations or insertions were used to investigate the relationship of the C‐terminal structure of LC3‐PE with ATG4/RavZ‐mediated deconjugation. Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3‐PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ. The molecular ruler mechanism exists in the active site of ATG4B, but not in RavZ. Met63 and Gln64 at the active site of RavZ are involved in accommodating LC3 C‐terminal motif. Our findings show that the distinct binding modes of the LC3 C‐terminal motif (116–120) with ATG4 and RavZ might determine the specificity of cleavage site.
Highlights
RavZ irreversibly deconjugates light chain 3 (LC3)-PE to inhibit autophagy
Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3-PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ
The ATG4B-LC3 complex revealed that the C-terminal motif
Summary
Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. The Legionella effector RavZ is injected into the cell and functions as a cysteine protease that irreversibly deconjugates LC3-PE to inhibit autophagosome formation.[7] Unlike endogenous ATG4 that cleaves the amide bond between terminal glycine and PE, RavZ cleaves the amide bond before glycine. These semisynthetic LC3-PE proteins were used to investigate the relationship of the Cterminal structure of LC3-PE with the ATG4/RavZ-mediated deconjugation. Our results revealed that the C-terminal motif of Autophagy is an evolutionarily conserved “self-eating” process to eliminate damaged organelles and proteins in eukaryotes. It plays an essential role in cellular homeostasis in response to various environmental and cellular stresses.
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