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.
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Research Article| March 02 2011
Molecular determinants involved in activation of caspase 7
Jean-Bernard Denault 1
*Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4
1To whom correspondence should be addressed (email Jean-Bernard.Denault@USherbrooke.ca).
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Dave Boucher, Véronique Blais, Marcin Drag, Jean-Bernard Denault; Molecular determinants involved in activation of caspase 7. Biosci Rep 1 August 2011; 31 (4): 283–294. doi: https://doi.org/10.1042/BSR20100111
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