Abstract
During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7's IDC for its activation and proteolytic activity.
| Original language | English |
|---|---|
| Pages (from-to) | 283-94 |
| Number of pages | 12 |
| Journal | Bioscience Reports |
| Volume | 31 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Aug 2011 |
Keywords
- Amino Acid Sequence
- Binding Sites
- Caspase 3/chemistry
- Caspase 7/chemistry
- Caspase 8/chemistry
- Caspases/chemistry
- Caspases, Initiator/chemistry
- Cells, Cultured
- Dimerization
- Humans
- Kinetics
- Protein Structure, Tertiary