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• W2063296970 abstract "The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates, and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro, and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown, its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore, it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner, and its cleavage is due solely to GrB and not other granule components. Furthermore, Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line, suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis, indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis. The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates, and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro, and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown, its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore, it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner, and its cleavage is due solely to GrB and not other granule components. Furthermore, Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line, suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis, indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis. Cytotoxic lymphocytes, including cytotoxic T lymphocytes and natural killer (NK) 6The abbreviations used are: NKnatural killerHipHsc70/Hsp70-interacting proteinGrBgranzyme BHspheat shock proteinHopHsp70/Hsp90-organizing proteinBag1Bcl-2-associated athano gene-1CIcaspase inhibitorsGIgranzyme B inhibitorzbenzyloxycarbonylFMKfluoromethyl ketonePARPpoly(ADP-ribose) polymeraseHPLChigh pressure liquid chromatographysiRNAsmall interfering RNAPIpropidium iodide. cells induce the death of virally infected or tumor cell targets through activation of tumor necrosis factor family death receptors or through granule exocytosis (1Russell J.H. Ley T.J. Annu. Rev. Immunol. 2002; 20: 323-370Crossref PubMed Scopus (829) Google Scholar). During granule exocytosis, the granzymes, a family of serine proteases, are released into the cytoplasm of the target cells with the assistance of perforin, where they induce target cell death. The human granzyme family includes five members that are termed A, B, H, K, and M. Granzymes A and B are the most well characterized and have been implicated as important contributors to target cell death (2Beresford P.J. Xia Z. Greenberg A.H. Lieberman J. Immunity. 1999; 10: 585-594Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 3Mahrus S. Craik C.S. Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 4Simon M.M. Hausmann M. Tran T. Ebnet K. Tschopp J. ThaHla R. Mullbacher A. J. Exp. Med. 1997; 186: 1781-1786Crossref PubMed Scopus (165) Google Scholar, 5Trapani J.A. Jans D.A. Jans P.J. Smyth M.J. Browne K.A. Sutton V.R. J. Biol. Chem. 1998; 273: 27934-27938Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). natural killer Hsc70/Hsp70-interacting protein granzyme B heat shock protein Hsp70/Hsp90-organizing protein Bcl-2-associated athano gene-1 caspase inhibitors granzyme B inhibitor benzyloxycarbonyl fluoromethyl ketone poly(ADP-ribose) polymerase high pressure liquid chromatography small interfering RNA propidium iodide. Previous substrate identification efforts have shown that granzyme B (GrB) induces target cell death by cleaving substrates that activate pro-apoptotic activities (i.e. caspase 3 (6Darmon A.J. Nicholson D.W. Bleackley R.C. Nature. 1995; 377: 446-448Crossref PubMed Scopus (647) Google Scholar)), dismantle the cytoskeleton (i.e. α-tubulin (7Adrain C. Duriez P.J. Brumatti G. Delivani P. Martin S.J. J. Biol. Chem. 2006; 281: 8118-8125Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar, 9Goping I.S. Sawchuk T. Underhill D.A. Bleackley R.C. J. Cell Sci. 2006; 119: 858-865Crossref PubMed Scopus (47) Google Scholar)), and inactivate proteins important for cellular homeostasis (i.e. DNA-dependent protein kinase catalytic subunit (10Andrade F. Roy S. Nicholson D. Thornberry N. Rosen A. Casciola-Rosen L. Immunity. 1998; 8: 451-460Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar)). Recent substrate identification efforts have expanded the third class of GrB substrates to include receptors that transmit pro-proliferative signals from the extracellular environment (11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and members of the heat shock/stress response family (8Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar, 11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Bredemeyer A.J. Carrigan P.E. Fehniger T.A. Smith D.F. Ley T.J. J. Biol. Chem. 2006; 281: 37130-37141Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 13Caruso J.A. Reiners J.J. Apoptosis. 2006; 11: 1877-1885Crossref PubMed Scopus (12) Google Scholar). These efforts and others (reviewed in Refs. 14Andrade F. Casciola-Rosen L.A. Rosen A. Acta Haematol. 2004; 111: 28-41Crossref PubMed Scopus (36) Google Scholar and 15Waterhouse N.J. Sedelies K.A. Trapani J.A. Immunol. Cell Biol. 2006; 84: 72-78Crossref PubMed Scopus (42) Google Scholar) emphasize the critical role that substrate identification plays in understanding GrB-induced death. We undertook a candidate-based approach to identify new GrB substrates. The extended substrate specificity of GrB has been comprehensively identified by the use of positional scanning substrate combinatorial libraries (16Thornberry 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 (1852) Google Scholar, 17Harris 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 (166) Google Scholar). These data sets provide a four-amino acid “zip code” to identify potential cleavage sites within a protein. Algorithms such as PoPS (18Boyd S.E. Pike R.N. Rudy G.B. Whisstock J.C. J. Bioinform Comput Biol. 2005; 3: 551-585Crossref PubMed Scopus (81) Google Scholar, 19Boyd S.E. Proc. IEEE. Comput Syst. Bioinform Conf. 2004; : 372-381PubMed Google Scholar) and GraBCas (20Backes C. Kuentzer J. Lenhof H.P. Comtesse N. Meese E. Nucleic Acids Res. 2005; 33: W208Crossref PubMed Scopus (91) Google Scholar) leverage the comprehensive nature of the positional scanning substrate combinatorial library-derived zip code to go beyond sequence gazing. However, searching the entire proteome for substrates with only the zip code yields too many hits to practically screen. Additional information is required to filter the hits to a reasonable number. Proteomic screens (8Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar, 11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) have proven to be useful filters by identifying proteins that are likely cut by GrB. Here, we restricted our search for GrB substrates to the heat shock/stress response family because of the well documented interplay between stress response and cell death (21Parcellier A. Gurbuxani S. Schmitt E. Solary E. Garrido C. Biochem. Biophys. Res. Commun. 2003; 304: 505-512Crossref PubMed Scopus (319) Google Scholar, 22Beere H.M. J. Cell Sci. 2004; 117: 2641-2651Crossref PubMed Scopus (519) Google Scholar, 23Beere H.M. J. Clin. Investig. 2005; 115: 2633-2639Crossref PubMed Scopus (355) Google Scholar) and the identification of several heat shock proteins as GrB substrates (8Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar, 11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Caruso J.A. Reiners J.J. Apoptosis. 2006; 11: 1877-1885Crossref PubMed Scopus (12) Google Scholar). Heat shock proteins (Hsps) are a family of ubiquitous and highly conserved chaperone proteins that serve a maintenance function in the cell, helping to fold newly synthesized peptides. They are also expressed in response to environmental, physical, and chemical stress, including heat shock, oxidative free radicals, chemotherapeutic agents, nutrient withdrawal, and irradiation (21Parcellier A. Gurbuxani S. Schmitt E. Solary E. Garrido C. Biochem. Biophys. Res. Commun. 2003; 304: 505-512Crossref PubMed Scopus (319) Google Scholar, 22Beere H.M. J. Cell Sci. 2004; 117: 2641-2651Crossref PubMed Scopus (519) Google Scholar, 23Beere H.M. J. Clin. Investig. 2005; 115: 2633-2639Crossref PubMed Scopus (355) Google Scholar). Hsps are able to prevent protein aggregation and stimulate the refolding and renaturation of proteins that have misfolded under stress. These functions limit the extent of stress-induced damage and facilitate cellular recovery. Conversely, apoptosis removes damaged or unwanted cells. Both of these opposing mechanisms are so well conserved that it is likely the two evolved together. It is not surprising, then, that components of these two pathways are increasingly found to oppose each other at the molecular level. Indeed, many Hsps have been shown to directly interfere with components of the apoptotic machinery, and this inhibition is sometimes, but not always, dependent on chaperone activity (21Parcellier A. Gurbuxani S. Schmitt E. Solary E. Garrido C. Biochem. Biophys. Res. Commun. 2003; 304: 505-512Crossref PubMed Scopus (319) Google Scholar, 23Beere H.M. J. Clin. Investig. 2005; 115: 2633-2639Crossref PubMed Scopus (355) Google Scholar, 24Beere H.M. Wolf B.B. Cain K. Mosser D.D. Mahboubi A. Kuwana T. Tailor P. Morimoto R.I. Cohen G.M. Green D.R. Nat. Cell Biol. 2000; 2: 469-475Crossref PubMed Scopus (1295) Google Scholar). Therefore, Hsps respond to stress by preventing the misfolding and aggregation of proteins while directly antagonizing the apoptotic machinery to tip the balance toward cell survival. Given the antagonistic interactions between the stress response/heat shock pathway and apoptosis, GrB may also target Hsps to ensure the death of the target cell. This hypothesis is supported by the identification of Hsp70, Hsp70/Hsp90-organizing protein (Hop), and Hsc70/Hsp70-interacting protein (Hip) as GrB substrates (8Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar, 11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Caruso J.A. Reiners J.J. Apoptosis. 2006; 11: 1877-1885Crossref PubMed Scopus (12) Google Scholar). To identify additional stress response substrates of GrB, members of the chaperone family of proteins were chosen as candidates for a sequence-based search for GrB cleavage sites. Three putative GrB substrates were identified. Hsp90, the Bcl-2-associated athano gene-1 (Bag1), and Hip were found to be substrates of GrB in vitro, and an additional cleavage site was identified and validated in Hip. Prior to this work, Hsp90 and Bag1 were known chaperone family members with documented anti-apoptotic roles, but Hip was not known to play an anti-apoptotic role and was therefore selected for further investigation. Proteolysis of Hip at both cleavage sites was caspase-independent and GrB-dependent in cell based assays. GrB cleavage is predicted to result in a loss of function by compromising the oligomerization domain of Hip and its Hsc70/Hsp70-interacting domain. To determine whether Hip loss of function made a contribution to natural killer cell-mediated cytotoxicity, RNA interference was used to reduce cellular levels of Hip within the K562 chronic myelogenous leukemia cell line. Hip deficiency rendered the cells slightly more susceptible to natural killer cell-mediated lysis, indicating that the proteolysis of Hip by GrB contributes to death induction. The modest impact of Hip deficiency is consistent with previous work showing that no single substrate or mechanism is required for GrB-mediated death and demonstrates the highly redundant nature of natural killer cell-mediated cytotoxicity. Reagents-The caspase inhibitor z-DEVD-FMK was purchased from Alexis Biochemicals (catalog number ALX-260-072-M001) and Calbiochem (catalog number 264155). The caspase inhibitor z-VAD-FMK was purchased from EMD Biosciences (catalog number 627610) and Bachem (catalog number N-1560). When present, both caspase inhibitors were used in equimolar amounts (50 μm each) and are designated as CI. Antibodies to PARP were from Santa Cruz Biotechnology (catalog number sc-7150) or from Cell Signaling Technologies (catalog number 9542). The antibody to the p48 Hsc/Hsp70-Interacting Protein, Hip, was from Affinity BioReagents (catalog number MA3–413). The anti-Bag1 antibody was from Stressgen (catalog number AAM-400). The anti-Hsp90 antibody (recognizing both α and β isoforms) was from Cell Signaling (catalog number 4874). Both the α-specific (catalog number SPS-771) and β-specific (catalog number SPA-843) anti-hsp90 antibodies were from Stressgen. The antibody to c-Myc was from Santa Cruz Biotechnology (catalog number sc-40). 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 as described in Ref. 25Willoughby 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 (53) Google Scholar and was a generous gift from Dr. Nancy Thornberry (Merck). Purified recombinant Hip (catalog number SPP-767), Hsp90 (catalog number SPP-770), and Hsp27 (catalog number SPP-715) proteins were from Stressgen. Cell Lines and Culture-All of the cell lines are available from the American Type Culture Collection. K562 chronic myelogenous leukemia and Jurkat T-cell leukemia cell lines were all propagated in RPMI 1640 medium containing 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate. NK-92 human natural killer cells were cultured in RPMI 1640 containing 10% fetal bovine serum, 10% bovine calf serum, 2 mm glutamax-1 (Invitrogen; catalog number 35050-81), nonessential amino acids (l-Ala, l-Asn, l-Asp, l-Glu, Gly, l-Pro, and l-Ser), 110 μg/ml sodium pyruvate, 0.1 mm β-mercaptoethanol, 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate, and 100 units/ml interleukin-2. All of the cells and transfectants were maintained in a humidified 37 °C, 5% CO2 incubator. Plasmid Construction-All of the inserts were ligated into the Invitrogen mammalian expression vector pcDNA 3.1 Myc/His6 that contains a neomycin resistance gene. Each overexpressed protein, therefore, was C-terminally Myc and His6-tagged. Human Hip was cloned by reverse transcription-PCR from mRNA prepared from K562 cells by using the RNeasy Mini Kit and Omniscript reverse transcriptase (both from Qiagen), Pfx DNA polymerase, and the primers 5′-GCC GAA TTC CGC TTG ACC TCC-3′ (reverse) and 5′-CAC AAG CTT ATG GAC CCC CGC AAA GTG-3′ (forward). The reverse transcription-PCR product was cloned into a Invitrogen TOPO TA cloning kit as an intermediate step for blue/white screening and then sequenced. Positive clones were then excised by HindIII and EcoRI digestion and ligated into the pcDNA 3.1 Myc/His6 vector for mammalian expression. The uncleavable mutant Hip-DDNN was cloned using a Stratagene QuikChange mutagenesis kit first by synthesizing and confirming the D92N single mutant and then subjecting this clone to a second round to make the D180N mutation. The mutagenesis primers used were the following: 5′-GAA GGT GTG ATT GAA CCA AAC ACT GAT GCT CCT CAA GA-3′ (D92N), the complement of primer D92N, 5′-GCC ATT GAA ATA AAT CCG AAT TCA GCT CAG CCT TAC AAG TGG-3′ (D180N), and the complement of primer D180N. Proteolysis of Chaperones in Vitro-Purified Hip, Hsp90, or Hsp27 protein at 45 μg/ml was incubated at 37 °C with 50 nm human GrB in GrB activity buffer containing 50 mm Na-HEPES, pH 8.0, 100 mm NaCl, and 0.01% Tween 20. Aliquots of 15 μl containing 670 ng of either Hip or Hsp90 were taken at specific intervals and were mixed with sample loading buffer. The samples were loaded onto 4–20% Tris-glycine gels and separated by SDS-PAGE. Separated proteins were visualized by Coomassie Blue staining. On-line Capillary Liquid Chromatography-Mass Spectrometry Analysis-A 1-μl aliquot of the digestion mixture was injected manually into an Eldex capillary liquid chromatography system and separated by a silica-based monolithic reverse phase capillary column (Onyx column from Phenomenex, Torrance, CA) at a flow rate of ∼1 μl/min. The HPLC eluent was connected directly to the micro-ion electrospray source of a QSTAR Pulsar QqTOF mass spectrometer (Applied Biosystem/MDS Sciex, Foster City, CA). Typical performance characteristics were >8000 resolution with 30 ppm mass measurement accuracy in mass spectrometry mode. Lysis and Immunoblotting-Cytoplasmic lysates to be treated with GrB 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 cell killing or Fas stimulation experiments described below were lysed in lysis buffer including the Complete Protease Inhibitor mixture tablet from Roche Applied Science (product number 11 697 498 001) (Lysis Buffer + PI) to stop proteolysis. After incubating on ice for 30 min, the lysates were spun at 16,000 × g for 10 min at 4 °C to remove the insoluble fraction. The 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. The proteins were then transferred to nitrocellulose or polyvinylidene difluoride membranes and blocked in Tris-buffered saline Triton X-100 containing 5% milk or 5% bovine serum albumin, as per the antibody manufacturer's instructions. The 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 (Amersham Biosciences Bioscience) or ECL Plus detection systems (GE Healthcare). The locations of prestained 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. The samples were then subjected to SDS-PAGE and immunoblotting. NK Cell-mediated Cytotoxicity-NK-92 effectors (E) and K562 targets (T) were collected, counted, and mixed at varying E:T ratios as in Refs. 3Mahrus S. Craik C.S. Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar and 11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, with 2 × 105 K562 cells in 24-well plates. For analysis of proteolysis by immunoblotting, killing was stopped at intervals from 1 to 6 h by collecting and lysing the cells in Lysis Buffer + PI. For analysis by flow cytometry, the cells were mixed as described in Ref. 3Mahrus S. Craik C.S. Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar. Briefly, 20,000 K562 cells were mixed with 20,000–80,000 NK-92 effectors in a total volume of 50 μl of medium in 5 ml, 12 × 75 mm polystyrene round bottom test tubes (BD Biosciences, catalog number 352054). Prior to mixing, the cells were preincubated for at least 1 h in 50 μm CI; CI concentration was kept constant throughout the experiment. The cells were incubated for 1–6 h at 37 °C, and phosphate-buffered saline was added to 475 μl to stop the experiment. The cells were stained with 20 μl of an anti-CD56 allophycocyanin-conjugated antibody (Becton Dickinson; catalog number 555518) and 5 μl of propidium iodide (BioVision, catalog number K101) and incubated in the dark at room temperature for 30 min. The samples were then transferred to ice, kept in the dark, and analyzed on a FACSCalibur (Becton Dickinson) according to the manufacturer's instructions. Dead target cells were counted as the population of PI+, CD56 cells, excluding cellular debris. Analysis was conducted with FlowJo v.6.4.1 software. 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 medium containing 2% fetal bovine serum. The cells were stimulated with 200 ng/ml anti-Fas antibody CH-11 (Upstate Biotechnology, Inc.; product number 05-201) and incubated from 2 to 6 h. At the indicated time intervals, the aliquots were lysed in Lysis Buffer + PI. Stable Transfectant Generation-All of the transfections were executed using an Amaxa electroporator system and the Nucleofector Solution V, program T-16, according to the manufacturer's instructions. For generation of stable cell lines expressing Myc-tagged cleavable Hip and uncleavable Hip-DDNN proteins, 1 million K562 cells were transfected with 2 μg of plasmid DNA. At 48 h post-transfection, the cells were plated in ClonaCell-TCS (Stem Cell Technologies; product number 03814) methylcellulose-based medium containing 1.2–1.7 mg/ml G418 according to the manufacturer's instructions. Colonies were allowed to form for up to 2 weeks and were then selected and expanded in culture medium containing 1.5 mg/ml G418. Clones expressing the constructs were maintained, expanded, weaned off of G418, and periodically checked for expression of the desired Hip protein. siRNA Knock-down of Hip-All of the transfections were generated by using the Amaxa electroporation instrument and solution kits as described above. Of the four siRNA duplexes available from Ambion, siRNA 13835 resulted in optimal Hip knock-down. Hip-deficient cells were compared with either K562 cells transfected with nuclease-free water or the nontargeting siRNA negative control number 4 (Ambion; catalog number 4641). The ON-TARGETplus SMARTpool from Dharmacon (catalog number L-017380-00-0005) was also used to silence Hip expression. Hip-deficient cells were compared with K562 cells transfected with ON-TARGETplus siCONTROL Nontargeting pool (Dharmacon; catalog number D-001810-10-05). On the day of the transfection, 1 million K562 cells were transfected with ∼3 μg of Ambion siRNA or Dharmacon siRNA according to the instructions from Amaxa. Transfected cells were maintained in 12-well plates at a maximum concentration of 500,000 cells/ml for up to 6 days post-transfection. The transfectants were then either used for lysis and immunoblotting or for cell killing experiments, both described above. Statistical Analysis-All of the analyses were performed using the freely available R language (26Team R.D.C. R: A Language and Environment for Statistical Computing. 2005; Google Scholar). To analyze the association of dead cell ratio in K562 cells with siRNA transfection, a multivariate linear model was fit with dead cell ratio as response and siRNA transfection indicator as the independent variable. To control for length of incubation and K562/natural cell ratio, time and K562/natural cell ratio covariates were added. Time was considered as a continuous variable because it had a strong linear association with the response, whereas K562/natural cell ratio was considered as categorical because there was no obvious trend. To take into account the blocking effect of the experiments being performed in pairs for each time point and K562/natural cell ratio, a random blocking effect for each pair was also included. The model was fit using the nlme package in R (27Lindstrom M.L. Bates D.M. Biometrics. 1990; 46: 673-687Crossref PubMed Scopus (1336) Google Scholar). Several Members of the Chaperone Family Are Proteolyzed by Granzyme B in Vitro-Recently, three chaperone family members have been shown to be GrB substrates: Hsp70 (11Loeb C.R. Harris J.L. Craik C.S. J. Biol. Chem. 2006; 281: 28326-28335Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar); the Hsp70-organizing Protein, Hop (8Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar, 12Bredemeyer A.J. Carrigan P.E. Fehniger T.A. Smith D.F. Ley T.J. J. Biol. Chem. 2006; 281: 37130-37141Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar); and the Hsc/Hsp70-interacting Protein, Hip (13Caruso J.A. Reiners J.J. Apoptosis. 2006; 11: 1877-1885Crossref PubMed Scopus (12) Google Scholar). Other chaperone proteins were therefore investigated for their ability to be proteolyzed by GrB. As a first pass approach to determining whether a particular chaperone family member could be a prospective GrB substrate, the amino acid sequence was scanned for putative GrB cleavage sites. GrB has an absolute requirement to cleave C-terminally to aspartic acid residues because of Arg226 in its S1 pocket (28Rotonda 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 (67) Google Scholar, 29Waugh S.M. Harris J.L. Fletterick R. Craik C.S. Nat. Struct Biol. 2000; 7: 762-765Crossref PubMed Scopus (89) Google Scholar). Moreover, because the extended substrate specificity of GrB has been defined (16Thornberry 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 (1852) Google Scholar, 17Harris 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 (166) Google Scholar), a substrate amino acid sequence can be scanned to predict cleavage sites. The GrB optimal substrate has the sequence (I/V)EPD (supplemental Table S1). Specificity profiling data indicated that the preference of GrB for Ile or Val at P4 is of critical importance, but that its preference at P3 and P2 is much more broad. The likelihood of a site being cleaved can also be evaluated on the basis of the preference of all proteases to cleave specifically at extended β strands (30Tyndall J.D. Nall T. Fairlie D.P. Chem. Rev. 2005; 105: 973-999Crossref PubMed Scopus (349) Google Scholar). The chaperones selected for cleavage site analysis included those with known and unknown anti-apoptotic roles. Cleavage sites were identified by both sequence gazing and by GraBCas (20Backes C. Kuentzer J. Lenhof H.P. Comtesse N. Meese E. Nucleic Acids Res. 2005; 33: W208Crossref PubMed" @default.
• W2063296970 created "2016-06-24" @default.
• W2063296970 creator A5019878226 @default.
• W2063296970 creator A5034563487 @default.
• W2063296970 creator A5084136590 @default.
• W2063296970 creator A5087001996 @default.
• W2063296970 date "2007-09-01" @default.
• W2063296970 modified "2023-10-11" @default.
• W2063296970 title "Hip Is a Pro-survival Substrate of Granzyme B" @default.
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