Caspase-7 is a member of a family of cysteine proteases that includes apoptotic initiators (caspases-8, -9 and -10) and executors (caspases-3, -6 and -7). During apoptosis, executioner caspases are cleaved by initiator caspases either by the extrinsic (death receptors) or by the intrinsic (mitochondrial) pathway of caspase activation. Caspase-7 is an obligate dimer in the cell and cleavage of the interdomain connector (IDC), which split the catalytic domain in two subunits, at either site 1 or site 2 allows the conversion of the enzyme from the zymogen (inactive) state to the active state through a conformation switch that leads to the creation of a substrate binding pocket and the catalytic site. During caspase-7 activation, a 23-residue N-terminal peptide is also cleaved. Consequently, caspase-7 displays different N-terminal residues from those of its zymogen. This can change the stability of caspase-7 according to the N-end rule, which relates the half-life of a protein with the residue presented at its N-terminus. This degradation pathway controls the ubiquitination of the protein based on the N-terminus. To replicate the different forms of caspase-7 produced during its activation process in a controlled manner, a TEV protease cleavage site [ENLYFQ[arrow down](S/A)] was strategically inserted to mimic the different possibilities of IDC cleavage: 1) cleavage at site 1 only, 2) cleavage at site 2 only, or 3) a double cleavage. This was done in order to obtain the N-terminal residues normally presented during the cleavage of caspase-7. These constructions have been also optimized to preserve the proteolytic activity of the enzyme with as little change as possible to the length of the IDC. These constructs were cleaved by TEV protease in vitro and in cellulo and allowed the activation of apoptosis. Furthermore, the cellular half-life of caspase-7 seems to be changed by its cleavage. In conclusion, we have developed an interesting tool for the study of caspase-7.
| Identifer | oai:union.ndltd.org:usherbrooke.ca/oai:savoirs.usherbrooke.ca:11143/4081 |
| Date | January 2011 |
| Creators | Tremblay, Alexandre |
| Contributors | Denault, Jean-Bernard |
| Publisher | Université de Sherbrooke |
| Source Sets | Université de Sherbrooke |
| Language | French |
| Detected Language | English |
| Type | Mémoire |
| Rights | © Alexandre Tremblay |
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