title: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation.
abstract: The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.
authors.affiliation: Laboratoire d'Immunologie, Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal, H3C 3J7.
authors.forename: E
authors.initials: E
authors.lastname: Sharif-Askari
authors.forename: A
authors.initials: A
authors.lastname: Alam
authors.forename: E
authors.initials: E
authors.lastname: Rhéaume
authors.forename: P J
authors.initials: PJ
authors.lastname: Beresford
authors.forename: C
authors.initials: C
authors.lastname: Scotto
authors.forename: K
authors.initials: K
authors.lastname: Sharma
authors.forename: D
authors.initials: D
authors.lastname: Lee
authors.forename: W E
authors.initials: WE
authors.lastname: DeWolf
authors.forename: M E
authors.initials: ME
authors.lastname: Nuttall
authors.forename: J
authors.initials: J
authors.lastname: Lieberman
authors.forename: R P
authors.initials: RP
authors.lastname: Sékaly
mesh_terms: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D053804:Granzymes; D006801:Humans; D019169:Jurkat Cells; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D009687:Nuclear Proteins; D011506:Proteins; C109664:caspase-activated DNase inhibitor; D003851:Deoxyribonucleases; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases