In situ trapping of initiator caspases reveals intermediate surprises.
A novel method to identify initiator caspases is an in situ trapping approach using a cell-permeable biotinylated caspase inhibitor valine-alanine-aspartate-fluoromethyl ketone (bVAD) that binds covalently and irreversibly to the active cysteine site of caspases. This inhibits apoptosis and should allow precipitation of initiator caspases in their uncleaved forms. However, in our experiments TRAIL and FasL-induced apoptosis and bVAD labelling did not result in streptavidin precipitation of the procaspase-8 forms, but led to the pull-down of the intermediate and to a lesser extent fully cleaved forms (p41/43 and p18). These findings are contrary to other reports and are of relevance to apoptosis research as they challenge the general concept of the bVAD approach that procaspases are being trapped. We show that (partially) processed forms of initiator caspases rather than procaspases might be precipitated with this method.
Mohr
Andrea
A
National University of Ireland Galway, National Centre for Biomedical Engineering Science, Molecular Therapeutics Group, Galway, Ireland. andrea.mohr@nuigalway.ie
Zwacka
Ralf M
RM
D017209:Apoptosis / Q000502:physiology*; D019921:Biotinylation; D015153:Blotting, Western; D020169:Caspases / Q000145:classification / Q000378:metabolism*; D053471:Caspases, Initiator / Q000378:metabolism*; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics*; D004171:Diploidy; D006801:Humans; D007202:Indicators and Reagents; D019169:Jurkat Cells; D011994:Recombinant Proteins / Q000378:metabolism; D019809:Streptavidin / Q000494:pharmacology
D004273:DNA, Neoplasm; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D019809:Streptavidin; D020169:Caspases; D053471:Caspases, Initiator