Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.
Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.
Wolf
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BB
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA. 102251.1444@compuserve.com
Schuler
M
M
Echeverri
F
F
Green
D R
DR
D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism*; D002474:Cell-Free System; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D053804:Granzymes; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D007700:Kinetics; D008954:Models, Biological; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; C113696:DFFB protein, human; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases