Molecular ordering of the caspase activation cascade initiated by the cytotoxic T lymphocyte/natural killer (CTL/NK) protease granzyme B.
Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.
Adrain
Colin
C
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Murphy
Brona M
BM
Martin
Seamus J
SJ
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053455:Caspase 10; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D002474:Cell-Free System; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D053804:Granzymes; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D040901:Proteomics; D012697:Serine Endopeptidases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*; D013997:Time Factors
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C494815:Bid protein, mouse; D002352:Carrier Proteins; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; C505526:Casp8 protein, mouse; D053455:Caspase 10; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D020169:Caspases; C506438:CASP10 protein, human