The in vitro cleavage of the hAtg proteins by cell death proteases.
It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.
MRC Toxicology Unit, The University of Leicester, Leicester, England, UK.
Joanna M
JM
Norman
Gerald M
GM
Cohen
Edward T W
ET
Bampton
D000595:Amino Acid Sequence; D002154:Calpain / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D002154:Calpain; D020169:Caspases; D019311:Staurosporine