Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.
Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.
McStay
G P
GP
Department of Immunology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
Salvesen
G S
GS
Green
D R
DR
D020816:Amino Acid Motifs; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System; D045304:Cytochromes c / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D045304:Cytochromes c; D020169:Caspases