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The in vitro cleavage of the hAtg proteins by cell death proteases
Joanna M Norman1, Gerald M Cohen, Edward T W Bampton
1MRC Toxicology Unit, The University of Leicester, Leicester, England, UK.
Abstract:
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.
Insights
Crosstalk between apoptosis and autophagy involves caspases cleaving autophagy proteins. This study identifies specific caspase and calpain targets within human Atg proteins, impacting autophagosome formation and p62 degradation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Autophagy and apoptosis pathways exhibit crosstalk, with autophagy potentially regulating programmed cell death.
- Autophagy-related (Atg) proteins are emerging as potential targets for caspases, suggesting novel regulatory mechanisms and functions for protein fragments.
- A detailed understanding of which Atg proteins are cleaved by specific death proteases is lacking.
Purpose of the Study:
- To investigate the cleavage of human Atg (hAtg) proteins by death proteases, specifically caspases and calpain 1.
- To determine the specific Atg proteins targeted by different caspases (caspase-3, -6, -8) and calpain 1.
- To assess the functional consequences of caspase-mediated cleavage of Atg proteins on the autophagic process.
Main Methods:
- In vitro cleavage assays using purified human Atg proteins and recombinant caspases/calpain 1.
- Analysis of protein cleavage products using techniques like Western blotting.
- Cell-based assays in HeLa cells treated with apoptosis inducers (staurosporine, TRAIL) and caspase inhibitors.
- Monitoring of autophagy markers such as GFP-LC3 lipidation, GFP-LC3 puncta formation, and p62 degradation.
Main Results:
- The majority of hAtg proteins were found to be substrates for calpain 1.
- hAtg3 was cleaved by caspases-3, -6, and -8.
- hAtg6 (Beclin 1) was cleaved by caspases-3 and -6.
- hAtg9, hAtg7, and hAtg4 homologues were cleaved by caspase-3.
- Cleavage of Beclin 1, Atg3, and Atg4C was observed in staurosporine- and TRAIL-induced apoptosis of HeLa cells.
- Caspase inhibition showed subtle effects on GFP-LC3 lipidation but significant effects on GFP-LC3 puncta formation and p62 degradation.
- p62 was identified as a direct target for caspase-6 and -8 cleavage.
Conclusions:
- Specific caspases and calpain 1 cleave various human Atg proteins, indicating a significant level of protease-mediated regulation of autophagy.
- Caspase cleavage of key autophagy proteins, including Beclin 1 and Atg3, directly impacts autophagosome formation and cargo degradation (p62).
- These findings reveal novel interactions between apoptotic and autophagic pathways, highlighting the role of proteolysis in modulating cellular death and survival processes.
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