Granzyme B Proteolyzes Receptors Important to Proliferation and Survival, Tipping the Balance toward Apoptosis
2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês
10.1074/jbc.m604544200
ISSN1083-351X
AutoresCarly R.K. Loeb, Jennifer L. Harris, Charles S. Craik,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoGranzyme B is critical to the ability of natural killer cells and cytotoxic T lymphocytes to induce efficient cell death of virally infected or tumor cell targets. Although granzyme B can cleave and activate caspases to induce apoptosis, granzyme B can also cause caspase-independent cell death. Thirteen prospective granzyme B substrates were identified from a cDNA expression-cleavage screen, including Hsp70, Notch1, fibroblast growth factor receptor-1 (FGFR1), poly-A-binding protein, cAbl, heterogeneous nuclear ribonucleoprotein H′, Br140, and intersectin-1. Validation revealed that Notch1 is a substrate of both granzyme B and caspases, whereas FGFR1 is a caspase-independent substrate of granzyme B. Proteolysis of FGFR1 in prostate cancer cells has functionally relevant consequences that indicate its cleavage may be advantageous for granzyme B to kill prostate cancer cells. Therefore, granzyme B not only activates pro-death functions within a target, but also has a previously unidentified role in inactivating pro-growth signals to cause cell death. Granzyme B is critical to the ability of natural killer cells and cytotoxic T lymphocytes to induce efficient cell death of virally infected or tumor cell targets. Although granzyme B can cleave and activate caspases to induce apoptosis, granzyme B can also cause caspase-independent cell death. Thirteen prospective granzyme B substrates were identified from a cDNA expression-cleavage screen, including Hsp70, Notch1, fibroblast growth factor receptor-1 (FGFR1), poly-A-binding protein, cAbl, heterogeneous nuclear ribonucleoprotein H′, Br140, and intersectin-1. Validation revealed that Notch1 is a substrate of both granzyme B and caspases, whereas FGFR1 is a caspase-independent substrate of granzyme B. Proteolysis of FGFR1 in prostate cancer cells has functionally relevant consequences that indicate its cleavage may be advantageous for granzyme B to kill prostate cancer cells. Therefore, granzyme B not only activates pro-death functions within a target, but also has a previously unidentified role in inactivating pro-growth signals to cause cell death. Natural killer (NK) 4The abbreviations used are: NK, natural killer cell; CTL, cytotoxic T lymphocyte; GrB, granzyme B; PARP, poly(ADP-ribose) polymerase; z, benzyloxycarbonyl; FMK, fluoromethyl ketone; FGFR1, fibroblast growth factor receptor-1; FGFR1ic, FGFR-intracellular; ERK, extracellular signal-regulated kinase; CI, caspase inhibitors zVAD-FMK and zDEVD-FMK; PI, propidium iodide; GI, GrB inhibitor L-038597; BP, lipid protein-delivery reagent BioPORTER; N™, Notch1-TM; E:T, effector to target ratio; NICD, Notch intracellular domain.4The abbreviations used are: NK, natural killer cell; CTL, cytotoxic T lymphocyte; GrB, granzyme B; PARP, poly(ADP-ribose) polymerase; z, benzyloxycarbonyl; FMK, fluoromethyl ketone; FGFR1, fibroblast growth factor receptor-1; FGFR1ic, FGFR-intracellular; ERK, extracellular signal-regulated kinase; CI, caspase inhibitors zVAD-FMK and zDEVD-FMK; PI, propidium iodide; GI, GrB inhibitor L-038597; BP, lipid protein-delivery reagent BioPORTER; N™, Notch1-TM; E:T, effector to target ratio; NICD, Notch intracellular domain. cells and cytotoxic T lymphocytes (CTLs) are the primary defense of the immune system against viral infection and intracellular pathogens. These cytotoxic cells can also kill a variety of cancer cells, giving support to the immunosurveillance hypothesis (1Russell J.H. Ley T.J. Annu. Rev. Immunol. 2002; 20: 323-370Crossref PubMed Scopus (808) Google Scholar, 2Smyth M.J. Godfrey D.I. Trapani J.A. Nat. Immunol. 2001; 2: 293-299Crossref PubMed Scopus (627) Google Scholar). Indeed, tumor immunotherapy approaches aim to treat cancers with NKs and CTLs that have potent anti-tumor recognition and killing activities. To that end, it is important to elucidate the mechanisms and pathways that are important to the ability of NKs and CTLs to kill a particular cancerous cell. NK cells and CTLs induce the death of their targets by either tumor necrosis factor family death receptor/death ligand interaction or by granule exocytosis. Granule exocytosis is critical for the efficient killing of virally infected or tumor cell targets (1Russell J.H. Ley T.J. Annu. Rev. Immunol. 2002; 20: 323-370Crossref PubMed Scopus (808) Google Scholar, 3Smyth M.J. Thia K.Y. Street S.E. MacGregor D. Godfrey D.I. Trapani J.A. J. Exp. Med. 2000; 192: 755-760Crossref PubMed Scopus (430) Google Scholar). Upon target recognition, killer cells release cytotoxic granules, the contents of which cross the immunological synapse and enter the target cell with the assistance of perforin, a putative pore-forming protein. Perforin may enable escape from endocytic vesicles or may allow entry directly through pores in the plasma membrane (1Russell J.H. Ley T.J. Annu. Rev. Immunol. 2002; 20: 323-370Crossref PubMed Scopus (808) Google Scholar). Granzymes are serine proteases that are released from the cytotoxic granules to induce target-cell death. There are five human granzymes, termed A, B, H, K, and M. Granzyme B (GrB) can initiate apoptosis by cleaving and activating several members of the caspases, the intracellular pro-apoptotic cysteine proteases (4Martin S.J. Amarante-Mendes G.P. Shi L. Chuang T.H. Casiano C.A. O'Brien G.A. Fitzgerald P. Tan E.M. Bokoch G.M. Greenberg A.H. Green D.R. EMBO J. 1996; 15: 2407-2416Crossref PubMed Scopus (273) Google Scholar, 5Darmon A.J. Nicholson D.W. Bleackley R.C. Nature. 1995; 377: 446-448Crossref PubMed Scopus (643) Google Scholar). GrB is required for efficient target cell lysis (6Mahrus S. Craik C.S. Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and DNA fragmentation (7Thomas D.A. Du C. Xu M. Wang X. Ley T.J. Immunity. 2000; 12: 621-632Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and treatment of target cells with GrB and a delivery agent such as perforin results in these and other common hallmarks of cell death, including nuclear condensation, phosphatidylserine exposure, mitochondrial depolarization, and cytochrome c release (1Russell J.H. Ley T.J. Annu. Rev. Immunol. 2002; 20: 323-370Crossref PubMed Scopus (808) Google Scholar). It has also become clear that GrB can induce cell death independently of caspases. GrB has a similar preference to caspases for cleaving proteins C-terminal to aspartic acid residues (8Harris J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 9Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar). GrB has been shown to cleave many caspase substrates such as poly(ADP-ribose) polymerase (PARP) (10Froelich C.J. Hanna W.L. Poirier G.G. Duriez P.J. D'Amours D. Salvesen G.S. Alnemri E.S. Earnshaw W.C. Shah G.M. Biochem. Biophys. Res. Commun. 1996; 227: 658-665Crossref PubMed Scopus (91) Google Scholar), inhibitor of caspase-activated DNase (7Thomas D.A. Du C. Xu M. Wang X. Ley T.J. Immunity. 2000; 12: 621-632Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), nuclear lamins (11Zhang D. Beresford P.J. Greenberg A.H. Lieberman J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5746-5751Crossref PubMed Scopus (130) Google Scholar), and Bid (12Barry M. Heibein J.A. Pinkoski M.J. Lee S.F. Moyer R.W. Green D.R. Bleackley R.C. Mol. Cell. Biol. 2000; 20: 3781-3794Crossref PubMed Scopus (284) Google Scholar). Although the morphologies associated with caspase-independent cell death caused by GrB are chiefly characterized by mitochondrial disruption (13Pinkoski M.J. Waterhouse N.J. Heibein J.A. Wolf B.B. Kuwana T. Goldstein J.C. Newmeyer D.D. Bleackley R.C. Green D.R. J. Biol. Chem. 2001; 276: 12060-12067Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and lysis (6Mahrus S. Craik C.S. Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), the exact cleavage events leading to these hallmarks have not all been completely elucidated. This is especially true in light of the fact that GrB can cause apoptosis in the absence of Bid, Bax, and Bak (14Thomas D.A. Scorrano L. Putcha G.V. Korsmeyer S.J. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14985-14990Crossref PubMed Scopus (102) Google Scholar). Moreover, GrB may cleave other substrates to ensure target cell death but that do not result in a currently recognizable physiological trait. Elucidation of all GrB substrates will unveil a more detailed mechanism of cytotoxic cell death. To identify substrates of GrB, a cDNA expression and cleavage screen was employed. Several new prospective substrates of granzyme B were identified, and three, Hsp70, FGFR1 and Notch1, were validated. We show here these proteolytic events as well as how the cleavage of FGFR1 by GrB could be important in targeting prostate cancer. Reagents—The caspase inhibitor zDEVD-FMK was purchased from Alexis Biochemicals. The caspase inhibitor zVAD-FMK was purchased from EMD Biosciences and Alexis Biochemicals. When present, both caspase inhibitors are used in equimolar amounts and are designated as CI. The Notch1 antibody Nic927 was a gift from Anthony Capobianco (University of Cincinnati). Antibodies to Lamin B (C-20), Myc (9E10), PARP (H-250), Notch1 (C-20), and Flg (H-76) were purchased from Santa Cruz Biotechnology. Another FGFR1 antibody (Ab-2) was from EMD Biosciences. Purchased from Cell Signaling Technologies were antibodies to PARP, Notch1 (6A5), FGFR1, phospho-FGFR1, phospho-ERK, and ERK. Production of rat granzyme B (GrB) was previously described (8Harris J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Human GrB was a generous gift from either Dr. Nancy Thornberry (Merck) or Dr. Sandra Waugh Ruggles (Catalyst Biosciences.) The small molecule GrB inhibitor L-038597 (GI) was developed and described in a previous study (15Willoughby C.A. Bull H.G. Garcia-Calvo M. Jiang J. Chapman K.T. Thornberry N.A. Bioorg Med. Chem. Lett. 2002; 12: 2197-2200Crossref PubMed Scopus (52) Google Scholar) and was a generous gift from Dr. Nancy Thornberry (Merck). Plasmid Construction—All inserts were ligated into the Invitrogen mammalian expression vector pcDNA 3.1 Myc/Hisx6. Each overexpressed protein therefore was C-terminally Myc/Hisx6-tagged. The Notch1-ic clone was generated from a Notch1-ic vector that was a generous gift from Anthony Capobianco (University of Cincinnati). The insert coding for Notch1-ic residues 1759-2556 was liberated by BamHI and XbaI digestion and ligated into the expression vector. The plasmids for FGFR1-ic, FGFR1-VSAD, and FGFR1-VSAN were generated from the clone for mouse FGFR1 (IIIc isoform), a generous gift from Dr. David Ornitz (Washington University at St. Louis). The primers used to generate FGFR1-ic were 5′-CAC AAG CTT AAG ATG AAG AGC GGC ACC AAG-3′ and 5′-GCC GAA TTC GCG CCG TTT GAG TCC ACT GTT GGC-3′. The latter primer was also used along with the forward primer 5′-CAC AAG CTT ATG TGG AGC TGG AAG TGC CTC CTC TTC TG-3′ to generate the full-length insert for cloning both FGFR1-VSAD and FGFR1-VSAN. The FGFR1-VSAN clone was made from FGFR1-VSAD using the QuikChange site-directed mutagenesis kit (Stratagene) and the mutagenesis primers 5′-CAG ACA GGT AAC AGT GTC AGC TAA CTC CAG TGC ATC CAT GAA CTC-3′ and its reverse complement. Human FGFR1-gBEC was cloned by reverse transcription-PCR on mRNA prepared from LNCaP cells using the RNeasy Mini Kit and Omniscript reverse transcriptase (both from Qiagen) and the primers 5′-CAC AAG CTT ATG TGG AGC TGG AAG TGC CTC CTC TTC TG-3′ and 5′-GCC GAT ATC GTC AGC AGA CAC TGT TAC CTG TCT GCG-3′. Cell Lines and Culture—All cell lines were available from the American Type Culture Collection (ATCC.) K562 chronic myelogenous leukemia, Jurkat T-cell leukemia, LNCaP, and PC3 prostate cancer cell lines were all propagated in RPMI 1640 media containing 10% fetal bovine serum and penicillin/streptomycin. NK-92 human natural killer cells were cultured in RPMI 1640 containing 10% fetal bovine serum, 10% fetal calf serum, glutamine, non-essential amino acids, sodium pyruvate, β-mercaptoethanol, penicillin and streptomycin, and 100 units/ml interleukin-2. All cells and transfectants were maintained in a humidified 37 °C, 5% CO2 incubator. Small Pool cDNA Screening—A cDNA library was constructed from RNA isolated from a 14-day post coitus mouse embryo as previously described (16Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1033) Google Scholar). Briefly, the cDNA library was cloned into the high copy number plasmid pCS2+ to yield 200,000 independent clones. The library was divided into pools of ∼100 unique plasmids. [35S]Methionine-labeled protein pools were prepared directly from the cDNA pools using the coupled transcription-translation rabbit reticulocyte lysate system (Promega) (17Lustig K.D. Stukenberg P.T. McGarry T.J. King R.W. Cryns V.L. Mead P.E. Zon L.I. Yuan J. Kirschner M.W. Methods Enzymol. 1997; 283: 83-99Crossref PubMed Scopus (66) Google Scholar). Briefly, 5 μl of transcription-translation mix was incubated at 30 °C with 0.1 μg of pooled DNA. After 2 h, half of the labeled protein reaction mix was incubated with 1nm recombinant rat granzyme B, 50 μm CI, and 50 μm Ac-IETD-FMK in a buffer containing 100 mm Hepes, pH 7.4, and 100 mm NaCl. After 1 h, loading dye was added to each reaction mix, and the mixture was heated at 65 °C for 10 min. The samples were loaded side-by-side onto 10-20% Tris-glycine gels and separated by SDS-PAGE. Separated proteins were visualized by autoradiography and scored for cleavage by the disappearance or appearance of bands in the granzyme B-incubated samples that were not found in the control samples. Proteolysis of Hsp70 in Vitro—Purified Hsp70 protein was purchased from Stressgen. Hsp70 protein at 0.64 μm was incubated at 37 °C with 50 nm human GrB in GrB activity buffer containing 50 mm Na HEPES, pH 8, 100 mm NaCl, and 0.01% Tween 20. Aliquots of 15 μl containing 670 ng of Hsp70 were taken at specific intervals and mixed with sample loading buffer. Samples were loaded onto 4-20% Tris-glycine gels and separated by SDS-PAGE. Separated proteins were visualized by Coomassie staining. In Vitro Transcription-Translation—Plasmids encoding FGFR1-ic and Notch1-ic were subject to T7 Quick Coupled in vitro transcription-translation (Promega) in the presence of [35S]methionine. Samples were then diluted into GrB activity buffer and incubated with human GrB at 37 °C for 1-3 h. Aliquots were separated by SDS-PAGE; gels were imaged by autoradiography. Lysis and Immunoblotting—Cytoplasmic lysates were generated by resuspending cells at 1 × 107 cells/ml in 50 mm Tris, pH 8.0, 150 mm NaCl, and 1% Nonidet P-40 (Lysis Buffer). Aliquots from assays described below were lysed in Lysis Buffer including the Complete Protease Inhibitor Mixture tablets from Roche Applied Science (Lysis Buffer plus PI) to stop proteolysis. After incubating on ice for 30 min, lysates were spun at 10,000 rpm for 10 min at 4 °C to remove the insoluble fraction. Protein concentrations were determined with the BCA Protein Assay Reagent (Pierce), and equal amounts of total protein from each sample were separated by SDS-PAGE. Proteins were then transferred to nitrocellulose or polyvinylidene difluoride membranes and blocked in TBST containing 5% milk or 5% bovine serum albumin, as per the antibody manufacturer's instructions. Membranes were then incubated with substrate-specific antibodies, washed, and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were developed on film with the ECL detection system. The locations of pre-stained protein markers were traced by overlaying the film onto the membrane. Immunoblots presented are representative of at least three independent experiments. Confirming Proteolysis in Cell Lysates—K562 cytoplasmic extracts were generated in the absence or presence of 50 μm CI in Lysis Buffer. The lysate was incubated with 50 nm human GrB in the absence or presence of 20 μm GI, and aliquots were removed from 1 to 6 h. Samples were then subjected to SDS-PAGE and immunoblotting. NK-mediated Cytotoxicity—NK-92 effectors (E) and K562 targets (T) were collected, counted, and mixed at varying E:T ratios as in Ref. 6Mahrus S. Craik C.S. Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, with 2 × 105 K562s in 24-well plates. Killing was stopped at intervals from 1 to6hby collecting and lysing in Lysis Buffer plus PI. Activation of Caspase-mediated Apoptosis in Jurkat T-cell Leukemia Cells—Jurkat cells were plated in a 96-well plate at 4 × 105 cells/well in 200 μl of RPMI 1640 media containing 2% serum. Cells were stimulated with 200 ng/ml CH-11 anti-Fas antibody (Upstate) and incubated from 2 to 6 h. At the indicated time intervals, aliquots were lysed in Lysis Buffer plus PI. GrB Delivery to LNCaPs—Human GrB was delivered to LNCaP cells with the lipid protein-delivery reagent Bio-PORTER (BP, Gene Therapy Systems). BioPORTER has been used for intracellular delivery of a variety of proteins (18Zelphati O. Wang Y. Kitada S. Reed J.C. Felgner P.L. Corbeil J. J. Biol. Chem. 2001; 276: 35103-35110Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). This approach has been used to deliver GrB and is an excellent method for analyzing components of cytotoxic granules (19Thiery J. Dorothee G. Haddada H. Echchakir H. Richon C. Stancou R. Vergnon I. Benard J. Mami-Chouaib F. Chouaib S. J. Immunol. 2003; 170: 5919-5926Crossref PubMed Scopus (28) Google Scholar). GrB·BP complexes were established in QuikEase Single-Use tubes according to the manufacturer's instructions and delivered to pre-seeded LNCaPs at 75 nm GrB. Cells were incubated and time points were collected for lysis in Lysis Buffer plus PI. For flow cytometry analysis, serum was added to 30% after 3-4 h of GrB delivery, and samples were incubated for another 2-3 h. The cells were incubated overnight in media containing 10% serum. The following morning cells were harvested, stained with Annexin V-FITC (BioVision), and analyzed on a FACS-Calibur according to the manufacturer's instructions. Dead cells were counted as the Annexin V-positive (FL1) population. Transient Transfections—Mammalian expression plasmids with FGFR1-VSAD, FGFR1-VSAN, or FGFR1gBEC were transiently transfected into LNCaPs or PC3s with Lipofectamine 2000 (LF) reagent (Invitrogen) according to the manufacturer's instructions. Briefly, LNCaPs were seeded in 500 μl of media without antibiotics so that ∼0.5-2 × 105 cells were in one well of a 24-well plate the day of the transfection. A DNA·LF complex was formed in OPTI-MEM by combining 0.8 μg of plasmid DNA with 2 μl of LF; the complex was then added to one well of a 24-well plate. PC3s were transfected in 12-well (trypan blue) or 96-well (proliferation) plates and scaled accordingly. After 6 h all cells were changed into fresh media and incubated for 24-48 h, depending on the experiment. FGF2 Stimulation—LNCaPs were transiently transfected with FGFR1-VSAD as outlined above to monitor FGFR1 cleavage. At 36 h post transfection, cells were incubated with or without GrB·BP complexes in serum-free media as above. After 6 h, transfectants were stimulated for 10 min in the presence or absence 50 ng/ml purified recombinant FGF2 (R&D Systems) and 50 μg/ml heparin. Samples were then lysed in Lysis Buffer plus PI containing the phosphatase inhibitors 1 mm sodium orthovanadate and 25 mm β-glycerophosphate, pH 7.3. To monitor overexpressed FGFR1-VSAD or FGFR1-VSAN directly after FGF2 stimulation, the receptors were immunoprecipitated from the lysate with anti-Myc antibodies and protein G-Sepharose. The pulled-down receptors were separated by SDS-PAGE and subjected to immunoblotting for both their Myc tags and for phospho-FGFR1. Trypan Blue Staining and Proliferation Assay—Viability of PC3s transiently overexpressing FGFR1-VSAD or FGFR1-gBEC for 48 h was determined by trypan blue staining (Invitrogen). Cells that were still adherent were collected by mild trypsinization, washed, resuspended in complete media, and counted on a hemocytometer. The data are presented as the average percent dead and alive cells, and error bars represent one standard deviation above and below the mean of at least three independent transfection experiments. Proliferation of PC3s 48 h post transfection was assessed with the CellTiter 96 AQueous One Solution cell proliferation assay (Promega) according to the manufacturer's instructions. Briefly, 20 μl of AQueous One Solution reagent was added to the cells in 100 μl of media. The cells were incubated for 2 h, and then the absorbance at 490 nm was recorded. Readings are presented as the mean A490 with error bars representing one standard deviation above and below the mean of six independent transfections. A Student's t test with a two-tailed distribution was calculated to determine the statistical significance of FGFR1gBEC from FGFR1-VSAD transfectants. An in Vitro cDNA Expression Cleavage Screen Identified Several Substrates of Granzyme B—To identify new GrB substrates, a cDNA library containing ∼200,000 unique cDNAs was screened. The library was divided into pools each containing ∼100 unique plasmids. The cDNA pools were in vitro transcribed and translated, and the resulting [35S]methionine-labeled protein pools were incubated in the presence or absence of GrB and the caspase inhibitors zVAD-FMK and zDEVD-FMK (CI). Proteins were separated by SDS-PAGE, visualized by autoradiography, and scored for caspase-independent proteolysis. Cleavage-positive pools were split into smaller sub-pools, which were then re-transformed and subjected to a subsequent round of DNA purification and in vitro transcribed and translated and screening. Fig. 1A is an autoradiograph of seven subpools from one initial positive pool. In sub-pool #5, a band at ∼47 kDa decreased in intensity and a smaller band at 40 kDa was enriched when GrB was added. Positive sub-pools like #5 continued to be split into smaller sub-pools and screened until individual cDNAs were isolated. The cDNAs were sequenced and subjected to a BLAST search. The cDNA isolated from positive sub-pool #5 was identified as the fibroblast growth factor receptor-1 (FGFR1), and other prospective GrB substrates identified were: Notch1, cAbl, Hsp70, heterogeneous nuclear ribonucleoprotein H′, Br140, intersectin-1, and poly-A-binding protein. Five additional clones matched cDNAs that have undefined protein identities. Lamin B, a known GrB substrate (11Zhang D. Beresford P.J. Greenberg A.H. Lieberman J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5746-5751Crossref PubMed Scopus (130) Google Scholar), was also isolated, demonstrating the efficacy of the screen. Each Identified Substrate Has a Prospective Granzyme B Cleavage Site—GrB has a strict requirement for cleaving C-terminal to Asp residues due to Arg-226 in its P1 pocket (20Waugh S.M. Harris J.L. Fletterick R. Craik C.S. Nat. Struct. Biol. 2000; 7: 762-765Crossref PubMed Scopus (86) Google Scholar, 21Rotonda J. Garcia-Calvo M. Bull H.G. Geissler W.M. McKeever B.M. Willoughby C.A. Thornberry N.A. Becker J.W. Chem. Biol. 2001; 8: 357-368Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Armed with this fact as well as the well characterized extended substrate specificity of the enzyme (8Harris J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 9Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar), protein sequences were searched for possible cleavage sites. Table 1 presents the prospective GrB cleavage sites within the identified proteins. According to protease substrate nomenclature, the (N-terminal to C-terminal) P4 thru P2′ residues are listed, with the scissile bond located between P1 and P1′. The optimal substrate of GrB is the sequence (I/V)EPD∼SG. However, specificity profiling data as well as research into the structural determinants of the specificity of GrB (22Ruggles S.W. Fletterick R.J. Craik C.S. J. Biol. Chem. 2004; 279: 30751-30759Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) has revealed that the P1 and P4 sites are of critical importance, whereas GrB can accommodate a broader range of residues at the other positions. Depicted in Table 1, the importance of the P4 thru P2′ sites is proportional to the size of the letter. Other known substrates of GrB such as caspase 3, PARP, and Bid are cleaved at sites that are a close but not exact match to IEPD SG (Table 1). This understanding created an appropriate sieve for eliminating poly-A-binding protein, which has a less than optimal P4 Leu, from further investigation.TABLE 1Prospective granzyme B cleavage sites within each identified substrate Hsp70, FGFR1, and Notch1 Are Substrates of Granzyme B in Vitro—The ability of GrB to proteolyze Hsp70 was assessed in vitro by incubating purified recombinant Hsp70 with 50 nm GrB (Fig. 1B). The cleavage products that appear as early as 1 h (h) are 40 and 30 kDa, as expected given the predicted cleavage site at Asp-366. Proteolysis by GrB of FGFR1 and Notch1 (Fig. 1, C and D, respectively) was first confirmed in vitro by incubating GrB with in vitro transcribed and translated peptides corresponding to the intracellular portions of each transmembrane protein (Fig. 1, C (panel ii) and D (panel ii)). The mobility shift of intracellular FGFR1 (FGFR1ic, Fig. 1C), after incubation with GrB, confirms that it is a substrate of GrB in vitro, and the size change is consistent with cleavage at Asp-432. Intracellular Notch1 (Notch1-ic, Fig. 1D), which runs at ∼85 kDa, appears to be cleaved at one major site yielding a product of ∼75 kDa (arrowhead) but also appears to be proteolyzed further (asterisks) upon longer incubation with increasing concentrations of GrB. The banding pattern of the cleavage products is consistent with proteolysis occurring in the region of Notch1-ic that aligns to the location of the predicted cleavage sites (Fig. 1D and Table 1). FGFR1 and Notch1 Are Caspase-independent Substrates of Granzyme B—Proteolysis was next confirmed by treating human K562 chronic myelogenous leukemia lysates with GrB. Using cell extracts is a valid approach, because GrB enters the cytoplasm of target cells with the assistance of perforin to induce apoptosis. Aliquots were removed over time, separated by SDS-PAGE, and immunoblotted for Notch1, FGFR1, or PARP (Fig. 2). Notch1 exists on the cell surface as a heterodimer (Fig. 1D, panel i); the peptide that contains the transmembrane and intracellular portions of the receptor is ∼120 kDa and is known as Notch1-TM (N™). Within only 1 h, both FGFR1 and N™ have been proteolyzed upon incubation with GrB (Fig. 2, left panels). Including CI did not prevent cleavage, indicating that proteolysis is independent of caspase activation (Fig. 2, right panels). However, N™ cleavage appears to be less efficient in the presence of CI when comparing the 2-h time points, indicating that N™ may also be a caspase substrate. PARP, a substrate of both GrB and caspases, was also monitored in this system (bottom panels). The addition of a small molecule-specific granzyme B inhibitor (GI, right panels) largely blocked the proteolysis of the full-length proteins. Notch1 and FGFR1 were also confirmed to be proteolyzed during NK cell-mediated killing of K562 targets (Fig. 3). NK cells kill K562s, which do not express a death receptor, exclusively by granule exocytosis. Potent NK-92 effectors (E) were co-incubated with K562 targets (T), and proteolysis was monitored by immunoblotting. Notch1 cleavage during NK-mediated cell killing of K562s is presented in Fig. 3 (A and B). N™ is proteolyzed within2hand almost completely by4hatanE:T of 2:1 (Fig. 3A, left). At 4:1, N™ is totally absent at 4h (Fig. 3A, right). The proteolysis is not due to NK cells killing themselves, because the 2:0 and 4:0 controls show no destruction of the N™ band. Furthermore, the addition of GI (Fig. 3, A and B) blocks efficient cleavage, and therefore proteolysis of these substrates is due to GrB and cannot be rescued by other granzymes. The addition of CI (Fig. 3B, left) shows that N™ is cleaved during cell killing in a caspase-independent manner. The antibody used to detect N™ in these experiments recognizes a cross-reactive species at 100 kDa. FGFR1 is also proteolyzed during NK-mediated cytotoxicity to a significant extent by 4 h and completely by 6 h under an E:T of 4:1 (Fig. 3C). FGFR1 is also a caspase-independent substrate during cell killing (Fig. 3C, right panel). In fact, proteolysis of FGFR1 seems to occur more efficiently by2hinthe presence of CI. This may indicate that, without apoptotic amplification by caspases, GrB can cleave other substrates more robustly. The cytotoxic cell would therefore still induce the death of a target whose caspase cascade is blocked. Proteolysis of PARP was also assessed during the cell killing experiments (Fig. 3D). At either 2:1 or 4:1 PARP is cleaved, and the 89-kDa caspase product and 55-kDa GrB products appear (8Harris J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Bi
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