Granzyme B Mimics Apical Caspases

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7. Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7. granzyme B replication-deficient adenovirus type 2 polyacrylamide gel electrophoresis. A family of cytosolic cysteine proteases, the caspases, stored in most cells as zymogens play an essential role in the execution of apoptosis. The caspases involved in cell death are divided into apical (-2, -8, -9, and -10) and executioner subsets (-3, -6, and -7) (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar, 2Froelich C.J. Dixit V.M. Yang X. Immunol. Today. 1998; 19: 30-36Abstract Full Text PDF PubMed Scopus (116) Google Scholar). The proteolytic signal initiated by the apical caspases is transmitted to the executioners (caspases-3, -6, and -7) (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar, 2Froelich C.J. Dixit V.M. Yang X. Immunol. Today. 1998; 19: 30-36Abstract Full Text PDF PubMed Scopus (116) Google Scholar) whose action on cellular proteins defines apoptosis. The caspases are processed to form active heterodimeric enzymes by cleavage at specific Asp residues. Activation is not thought to require removal of the propeptide; cleavage between the large and small subunits is the activating event. The zymogens of apical caspases are recruited by specific adapter molecules, either at the cytosolic face of death receptors (in the case of caspase-8 and -10) (3Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar), or via a post-mitochondrial route (caspase-9) (4Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Crossref PubMed Scopus (590) Google Scholar, 5Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X.D. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). Unlike the apical caspases where the propeptide plays a crucial role in signal transmission, the function of this segment in executioner caspases has not been clarified. The third pathway that activates the caspase cascade is initiated by cytotoxic cells. The immune system utilizes lymphocyte granule-mediated apoptosis to protect the host from intracellular pathogens and tumor cells through perforin/granzyme induced apoptosis where granzyme B (GrB)1 plays a vital role. Our laboratory has proposed that perforin and GrB are internalized to endosomes of the target cell during granule-mediated apoptosis. Perforin then permeabilizes the vesicles delivering the granzyme to the cytosol where GrB rapidly induces cell death by activating a preferred caspase substrate (6Froelich C.J. Orth K. Turbov J. Seth P. Babior B.M. Gottlieb R.A. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W.L. J. Biol. Chem. 1996; 271: 29073-29081Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 7Stoorvogel W. Strous G.J. Geuze H.J. Oorschot V. Schwartz A.L. Cell. 1991; 65: 417-427Abstract Full Text PDF PubMed Scopus (231) Google Scholar). GrB has been an important tool in analyzing the maturation of caspase-3 (8Darmon A.J. Nicholson D.W. Bleackley R.C. 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A. 1996; 93: 11002-11007Crossref PubMed Scopus (77) Google Scholar), the apparent polyspecificity of GrB for the caspases has made it difficult to establish which member is directly activated by granzyme to initiate the death pathway in vivo. Given this dilemma, it was argued that the caspase that is processed most rapidly by the granzyme in vitro would be a prime target for initial activation in vivo. Testing the human caspases, GrB was found to have the greatest preference for caspase-7 (21Talanian R.V. Yang X. Turbov J. Seth P. Ghayur T. Casiano C.A. Froelich C.J. J. Exp. Med. 1997; 186: 1323-1331Crossref PubMed Scopus (165) Google Scholar, 22Zhou Q. Salvesen G.S. Biochem. J. 1997; 324: 361-364Crossref PubMed Scopus (123) Google Scholar). During GrB-mediated apoptosis, however, the propeptide of caspase-7 is removed first. This observation indicated that GrB is unable to interact with procaspase-7 and that an upstream activator, possibly the less preferred caspase-3 or -10, removes the propeptide. We hypothesized that the caspase (-3 or -10) that removes the propeptide of caspase-7 would also be the member activated by GrB to initiate the death pathway (6Froelich C.J. Orth K. Turbov J. Seth P. Babior B.M. Gottlieb R.A. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W.L. J. Biol. Chem. 1996; 271: 29073-29081Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 21Talanian R.V. Yang X. Turbov J. Seth P. Ghayur T. Casiano C.A. Froelich C.J. J. Exp. Med. 1997; 186: 1323-1331Crossref PubMed Scopus (165) Google Scholar). Using caspase-3/10a-deficient MCF-7 cells (21Talanian R.V. Yang X. Turbov J. Seth P. Ghayur T. Casiano C.A. Froelich C.J. J. Exp. Med. 1997; 186: 1323-1331Crossref PubMed Scopus (165) Google Scholar, 23Jänicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar, 24Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar) and stable transfectants expressing the deficient proteases, we have decisively proven that GrB triggers the caspase pathway by activating caspase-3, which then cleaves the propeptide of caspase-7. Remarkably, the removal of propeptide is crucial for the release of caspase-7 from a sequester site so it may then interact with the granzyme. MCF-7 cells were maintained RPMI 1640, 10% heat-inactivated fetal calf serum supplemented with 2 mml-glutamine, 100 units/ml penicillin and 50 μg/ml streptomycin. The caspase-3/-10a-deficient MCF-7 line was kindly provided by J. Boothman (University of Wisconsin). The absence of detectable caspase-3 and -10 was verified by immunoblotting with anti-caspase-3 and -10a/b rabbit polyclonal antibodies. The absence of caspase-3 is because of a 47-base pair deletion within exon 3 of the Casp-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing thereby abrogating translation of the caspase-3 mRNA (23Jänicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar). The caspase-deficient MCF-7 cells were transfected with the following constructs to generate stable lines: caspase-3 in a retroviral vector (pBabe-puro) (provided by Dr. T. Sladek) and caspase-10a in pCl-neo vector. After the selection with puromycin and geneticin, respectively, stable expression of the particular caspase was verified by immunoblotting. MCF-7 control cell lines were generated by transfecting empty pBabe-puro and pCl-neo vectors. Human GrB was isolated to homogeneity from a human natural killer cell line (YT) (25Hanna W.L. Zhang X. Turbov J. Winkler U. Hudig D. Froelich C.J. Protein Purif. Exp. 1993; 4: 398-402Crossref PubMed Scopus (66) Google Scholar) and a nonreplicating strain of adenovirus type 2 was cultured and isolated as described (26Seth P. Biochem. Biophys. Res. Commun. 1994; 203: 582-587Crossref PubMed Scopus (16) Google Scholar). Active caspase-3, -7, and -10a were expressed in Escherichia coli and isolated as described previously (27Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar). The expression constructs for caspase-3 and -7 contained a His6 tag at the C terminus of the full-length protein. Full-length procaspase 7 was obtained by reducing the previously described expression times in the presence of 0.2 mm isopropyl-1-thio-β-d-galactopyranoside to 30 min. This protocol has the advantage because it yields the full-length zymogens of both caspases and not the truncated form of procaspase 7 previously described by Zhou and Salvesen (22Zhou Q. Salvesen G.S. Biochem. J. 1997; 324: 361-364Crossref PubMed Scopus (123) Google Scholar). The concentrations of the purified enzymes were determined from the absorbance at 280 nm based on the molar absorption coefficients for the caspases calculated from the Edelhoch relationship: Caspase-3 (e280 = 26,000 m−1 cm−1), caspase-7 (e280 = 24,510 m−1cm−1) and caspase-10 (e280 = 29,140m−1 cm−1). The processing pattern of procaspase 7 by caspases 3 and 7 were determined by incubating 2.65 mm procaspase 7 with caspase 3 at concentration ranging from ∼0.2 to 400 nm or with caspase 7 at concentration ranging from ∼0.2 to 450 nm. The reactions were incubated for 30 min at 37 °C, stopped by boiling in SDS sample buffer for 5 min, and the reaction products separated by SDS-PAGE on an 8–18% gradient gel and visualized by Coomassie staining. Target cells were treated with isolated GrB and AD as described previously (6Froelich C.J. Orth K. Turbov J. Seth P. Babior B.M. Gottlieb R.A. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W.L. J. Biol. Chem. 1996; 271: 29073-29081Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar); unless indicated cells (1 × 106/ml) were mixed with GrB (1 μg/ml–30 nm) and AD (100 pfu) in 1-ml microcentrifuge tubes containing RPMI 1640 supplemented with 0.5% bovine serum albumin. To minimize in vitro processing of caspases in cell lysates before immunoblotting, targets were washed with phosphate-buffered saline 5 min before the end of the assay to remove excess GrB, transferred to a fresh tube, and lysed in buffer containing the GrB-specific anti-protease, anti-GraB (6Froelich C.J. Orth K. Turbov J. Seth P. Babior B.M. Gottlieb R.A. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W.L. J. Biol. Chem. 1996; 271: 29073-29081Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Detection of processed caspase -3, -7, -8, -9, and -10 was performed as described previously (6Froelich C.J. Orth K. Turbov J. Seth P. Babior B.M. Gottlieb R.A. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W.L. J. Biol. Chem. 1996; 271: 29073-29081Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Anti-caspase-3 and -7 were supplied by V. Dixit. Anti-caspase-8, -9, and -10 were supplied by Pharmingen, Inc., D. Green, and A. Srinivasan, respectively. Treated cells (106/ml) were lysed, resolved by SDS-PAGE (10–15%), and transferred to nitrocellulose. Rabbit antisera were used at a dilution of 1:1,000 followed by incubation with anti-rabbit Ig-horseradish peroxidase (Amersham Pharmacia Biotech) at a dilution of 1:10,000. Signal was visualized with the ECL During in caspase-7 is processed by removal of the propeptide then maturation occurs by a between the large and small subunits (6Froelich C.J. Orth K. Turbov J. Seth P. Babior B.M. Gottlieb R.A. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W.L. J. Biol. Chem. 1996; 271: 29073-29081Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, K. O'Rourke K. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). in MCF-7 cells caspase-3 and processing of caspase-7 not this we predicted that either or both these caspases are to caspase-7 in vivo. caspase-3 and -10a were to either removed the propeptide of caspase-7. Caspase-3 was found to the propeptide of but it was unable to procaspase-7 to active form The of cleavage by caspase-3 was verified by the was between the Asp and in the not removal of the propeptide has been to caspases with we also examined the pattern of processing that caspase-7 Active caspase-7 was found to proteolytic on procaspase-7 in with that caspase 7 not activate the truncated form of procaspase-7 not undergo activation (22Zhou Q. Salvesen G.S. Biochem. J. 1997; 324: 361-364Crossref PubMed Scopus (123) Google Scholar) This that removal of the not caspase-7 with and that caspase-3 is required to caspase-7. is to be upstream of caspase-3 and -7 (21Talanian R.V. Yang X. Turbov J. Seth P. Ghayur T. Casiano C.A. Froelich C.J. J. Exp. Med. 1997; 186: 1323-1331Crossref PubMed Scopus (165) Google Scholar). We then examined the and pattern of cleavage that caspase-10a generated the family Unlike caspase-3, caspase-10 preferred to the zymogen of caspase-7 between the large and small subunits and not release the propeptide verified that the made by caspase-10a was between the Asp and in the to the large and small subunits not with a preference for cleavage the T. M. V.M. S. Nicholson D.W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), caspase-10a also processed caspase-3 at 2 caspase-10a to caspase-3 caspase-7 at this not with that GrB activates a pattern in vitro where an apical caspase for active caspase-3 removes the propeptide of caspase-7 but then caspase-10a is required to the maturation of caspase-7. the of this we the processing of caspase-7 in specific MCF-7 cell Using cleavage of the propeptide of caspase-7 as an of caspase-3 the pattern and of processing of caspase-7 was in a MCF-7 line and in cells with stable expression of cells were induced to undergo GrB-mediated apoptosis, cleavage of procaspase-7 was the granzyme was unable to this caspase. To that caspase-7 was expressed and undergo processing in the caspase-3 was examined in a cytosolic of the The predicted cleavage between the large and small subunits was that the caspase was 3 to the apoptosis of the MCF-7 cells was with removal of the caspase-7 propeptide and followed by cleavage between the large and small subunits. The of processing of caspase-7 with the of caspase-3 in the transfected line 3 and in the cytosolic in targets expressing caspase-3 but GrB to have the capacity to directly activate preferred by is and be processed directly by the granzyme unless the propeptide is by After removal of the caspase-7 to activation by GrB that a may to this executioner caspase. The data suggest that GrB directly activates caspase-3 into cells deficient in based on of in GrB initiate the caspase cascade by activating which then induces caspase-3 As for however, caspase-10a also to be to the granzyme into the target cell Using MCF-7 cells transfected with we examined the of processing during apoptosis and in cytosolic GrB was to the caspase-10a processing was cleavage of caspase-10a be in cytosolic Although caspase-10a is a preferred substrate for because of the of GrB to this zymogen in the granzyme initiate the cascade by activating this has been based on performed with and purified GrB, that the granzyme directly activate caspase-8 to trigger the death pathway Scaffidi C. R.A. Peter M.E. R. Krammer P.H. J. Immunol. 1997; PubMed Scopus Google Scholar). Although caspase-8 is not a preferred substrate processed by GrB in the zymogen be accessible to GrB in vivo. This was examined by of caspase-8 in and caspase cells. was not processed in cells during the cleavage of caspase-8 was detectable at 2 in the line the of processing of caspase-8 in cytosolic of was apparent at by a in the of the zymogen 5 results that caspase-8 is processed in cells GrB-mediated apoptosis, but occurs in the presence of activated We also the that GrB activate the cascade through caspase-9 by a action on which to be an important in apoptosis for by J.C. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). Using the described processing of caspase-9 in the presence and absence of caspase-3 was examined intracellular of GrB. Although processing was in cells during the for cells expressing caspase-3, the zymogen of caspase-9 was to a form at 30 min with the large at The is with cleavage at caspase-9 S.M. T. J. N. Armstrong R.C. Wang L. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-9 is a substrate for GrB, the caspase-9 zymogen was not in cytosolic from cells to caspase-9 is processed in but is on the presence of active GrB a for Asp with caspases, the initiate the caspase pathway by activation of an apical by an apical caspase or by as an We on caspase-7 as a of in the because most family the propeptide of the zymogen is removed in vivo. This is in cells as as and tumor apoptosis. Using procaspase-7 to the role of in the processing of this vitro and described cell lines to the pattern of cleavage in we have been to caspase-3 caspase-10 were of the cascade initiated by the granzyme and to the of procaspase-7 to processing by GrB. The granzyme activates caspase-3 this substrate is not the most preferred member of the death Caspase-3 then cleaves the of procaspase-7 it accessible to activation by the granzyme. GrB caspases in the granzyme has been as a of the caspase cascade in vivo. The of the granzyme to directly -8, -9, and -10, as as caspase-6 not in the absence of caspase-3, was an that a pathway may for cytotoxic activation of this death We have that GrB an apical caspase in an where to to the in which the two executioner caspases are by the granzyme. Our observation an is caspase-7 to activation by GrB but is activated by procaspase-7 is processed by GrB of the cells 3 the zymogen to be in an have based on cell that procaspase-7 is to the and G.M. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) a of apical and executioner Although it is the of caspase-7 is the of the executioner caspase-7 a previously of in this As a of this two are required for maturation of caspase-7. Using the MCF-7 treated with tumor caspase-8 to function to the granzyme H.R. Yang X. Zhou Q. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (647) Google Scholar). results may a by the granzyme and apical caspases (-2, -8, -9, and -10) to the executioners apical caspases may be activated in a that to the of the cascade that at caspase-3 the role of this in cell of the proteolytic signal from the apical to executioner caspases into the and intracellular of the family in the cell as as the that a particular caspase We Dr. Dixit for kindly the anti-caspase-3 and -7 the for We also of the MCF-7 line from Dr.