title: Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis.
abstract: Granzyme B (GrB), acting similar to an apical caspase, efficiently activates a proteolytic cascade after intracellular delivery by perforin. Studies here were designed to learn whether the physiologic effector, GrB-serglycin, initiates apoptosis primarily through caspase-3 or through BH3-only proteins with subsequent mitochondrial permeabilization and apoptosis. Using four separate cell lines that were either genetically lacking the zymogen or rendered deficient in active caspase-3, we measured apoptotic indices within whole cells (active caspase-3, mitochondrial depolarization [DeltaPsim] and TUNEL). Adhering to these conditions, the following were observed in targets after GrB delivery: (a) procaspase-3-deficient cells fail to display a reduced DeltaPsim and DNA fragmentation; (b) Bax/Bak is required for optimal DeltaPsim reduction, caspase-3 activation, and DNA fragmentation, whereas BID cleavage is undetected by immunoblot; (c) Bcl-2 inhibits GrB-mediated apoptosis (reduced DeltaPsim and TUNEL reactivity) by blocking oligomerization of caspase-3; and (d) in procaspase-3-deficient cells a mitochondrial-independent pathway was identified which involved procaspase-7 activation, PARP cleavage, and nuclear condensation. The data therefore support the existence of a fully implemented apoptotic pathway initiated by GrB, propagated by caspase-3, and perpetuated by a mitochondrial amplification loop but also emphasize the presence of an ancillary caspase-dependent, mitochondria-independent pathway.
authors.affiliation: Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine, Northwestern University, Evanston, IL 60201, USA.
authors.forename: Sunil S
authors.initials: SS
authors.lastname: Metkar
authors.forename: Baikun
authors.initials: B
authors.lastname: Wang
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Ebbs
authors.forename: Jin H
authors.initials: JH
authors.lastname: Kim
authors.forename: Yong J
authors.initials: YJ
authors.lastname: Lee
authors.forename: Srikumar M
authors.initials: SM
authors.lastname: Raja
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Froelich
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D053938:DNA Fragmentation / Q000502:physiology; D004792:Enzyme Precursors / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts; D053804:Granzymes; D006801:Humans; D019169:Jurkat Cells; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000172:deficiency* / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000201:enzymology*; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C494827:Bax protein, mouse; C494815:Bid protein, mouse; D002352:Carrier Proteins; D004792:Enzyme Precursors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases