Artigo Acesso aberto Revisado por pares

Caspase-8 Cleaves Histone Deacetylase 7 and Abolishes Its Transcription Repressor Function

2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês

10.1074/jbc.m800331200

ISSN

1083-351X

Autores

Fiona L. Scott, Greg J. Fuchs, Sarah Boyd, Jean‐Bernard Denault, Christine J. Hawkins, Samuel Dequiedt, Guy S. Salvesen,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

Caspase-8 is the initiator caspase of the extrinsic apoptosis pathway and also has a role in non-apoptotic physiologies. Identifying endogenous substrates for caspase-8 by using integrated bioinformatics and biological approaches is required to delineate the diverse roles of this caspase. We describe a number of novel putative caspase-8 substrates using the Prediction of Protease Specificity (PoPS) program, one of which is histone deacetylase 7 (HDAC7). HDAC7 is cleaved faster than any other caspase-8 substrate described to date. It is also cleaved in primary CD4+CD8+ thymocytes undergoing extrinsic apoptosis. By using naturally occurring caspase inhibitors that have evolved exquisite specificity at concentrations found within the cell, we could unequivocally assign the cleavage activity to caspase-8. Importantly, cleavage of HDAC7 alters its subcellular localization and abrogates its Nur77 repressor function. Thus we demonstrate a direct role for initiator caspase-mediated proteolysis in promoting gene transcription. Caspase-8 is the initiator caspase of the extrinsic apoptosis pathway and also has a role in non-apoptotic physiologies. Identifying endogenous substrates for caspase-8 by using integrated bioinformatics and biological approaches is required to delineate the diverse roles of this caspase. We describe a number of novel putative caspase-8 substrates using the Prediction of Protease Specificity (PoPS) program, one of which is histone deacetylase 7 (HDAC7). HDAC7 is cleaved faster than any other caspase-8 substrate described to date. It is also cleaved in primary CD4+CD8+ thymocytes undergoing extrinsic apoptosis. By using naturally occurring caspase inhibitors that have evolved exquisite specificity at concentrations found within the cell, we could unequivocally assign the cleavage activity to caspase-8. Importantly, cleavage of HDAC7 alters its subcellular localization and abrogates its Nur77 repressor function. Thus we demonstrate a direct role for initiator caspase-mediated proteolysis in promoting gene transcription. During apoptosis cells are systematically dismantled and packaged into small membrane-bound particles ready for removal by professional phagocytes, by a process that is driven by members of the caspase family of proteases. Members of the caspase family have generally been separated into two groups as follows: those involved in apoptosis (caspase-2, -3, and -6–10) and those involved in non-apoptotic processes such as inflammation and differentiation (caspase-1, -4, -5, and -14) (1Fuentes-Prior P. Salvesen G.S. Biochem. J. 2004; 384: 201-232Crossref PubMed Scopus (710) Google Scholar). However, this simple demarcation is complicated by evidence suggesting that some apoptotic caspases may have functions in non-apoptotic physiologies, including, but not limited to, cell differentiation, migration, proliferation, T and B cell activation, and nuclear factor-κB (NF-κB) 3The abbreviations used are: NF-κB, nuclear factor-κB; Ac, acetyl; afc, amino-4-trifluoromethylcoumarin; AICD, activation-induced cell death; CrmA, cowpox response modifier A; HDAC, histone deacetylase; NES, nuclear export signal; NLS, nuclear localization signal; PoPS, prediction of protease specificity; RT, reverse transcriptase; TNF, tumor necrosis factor; XIAP, X-linked inhibitor of apoptosis; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. activation (2Lamkanfi M. Festjens N. Declercq W. Vanden Berghe T. Vandenabeele P. Cell Death Differ. 2007; 14: 44-55Crossref PubMed Scopus (464) Google Scholar). Of the initiator apoptotic caspases, the strongest evidence for alternative non-apoptotic roles is for caspase-8. The proteolytic activity of the caspase may be dispensable for some of these processes. For instance, tumor necrosis factor (TNF)-mediated NF-κB activation in T cells, fibroblasts, and epithelial cells is dependent on caspase-8 protein but not its proteolytic activity. In contrast, NF-κB activation in response to T cell receptor ligation in T cells does require caspase-8 activity (3Su H. Bidere N. Zheng L. Cubre A. Sakai K. Dale J. Salmena L. Hakem R. Straus S. Lenardo M. Science. 2005; 307: 1465-1468Crossref PubMed Scopus (368) Google Scholar). Regarding differentiation, caspase-8 is required for maturation of monocytes into macrophages, and pan-caspase-inhibitors block this process (4Kang T.B. Ben-Moshe T. Varfolomeev E.E. Pewzner-Jung Y. Yogev N. Jurewicz A. Waisman A. Brenner O. Haffner R. Gustafsson E. Ramakrishnan P. Lapidot T. Wallach D. J. Immunol. 2004; 173: 2976-2984Crossref PubMed Scopus (323) Google Scholar, 5Sordet O. Rebe C. Plenchette S. Zermati Y. Hermine O. Vainchenker W. Garrido C. Solary E. Dubrez-Daloz L. Blood. 2002; 100: 4446-4453Crossref PubMed Scopus (275) Google Scholar). In addition, a requirement for caspase-8 activity has been demonstrated during differentiation of placental villous trophoblasts (6Black S. Kadyrov M. Kaufmann P. Ugele B. Emans N. Huppertz B. Cell Death Differ. 2004; 11: 90-98Crossref PubMed Scopus (154) Google Scholar). Targeted deletion reveals that caspase-8 protein is also required for T cell activation, formation of blood vessels, and maintenance of hemopoietic progenitor cells in mice (4Kang T.B. Ben-Moshe T. Varfolomeev E.E. Pewzner-Jung Y. Yogev N. Jurewicz A. Waisman A. Brenner O. Haffner R. Gustafsson E. Ramakrishnan P. Lapidot T. Wallach D. J. Immunol. 2004; 173: 2976-2984Crossref PubMed Scopus (323) Google Scholar, 7Salmena L. Lemmers B. Hakem A. Matysiak-Zablocki E. Murakami K. Au P.Y. Berry D.M. Tamblyn L. Shehabeldin A. Migon E. Wakeham A. Bouchard D. Yeh W.C. McGlade J.C. Ohashi P.S. Hakem R. Genes Dev. 2003; 17: 883-895Crossref PubMed Scopus (391) Google Scholar, 8Varfolomeev E.E. Schuchmann M. Luria V. Chiannilkulchai N. Beckmann J.S. Mett I.L. Rebrikov D. Brodianski V.M. Kemper O.C. Kollet O. Lapidot T. Soffer D. Sobe T. Avraham K.B. Goncharov T. Holtmann H. Lonai P. Wallach D. Immunity. 1998; 9: 267-276Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar). More significantly, caspase-8 null mice and humans manifest a complex condition, including immunodeficiency early in their life and autoimmunity as the individuals age (9Chun H.J. Zheng L. Ahmad M. Wang J. Speirs C.K. Siegel R.M. Dale J.K. Puck J. Davis J. Hall C.G. Skoda-Smith S. Atkinson T.P. Straus S.E. Lenardo M.J. Nature. 2002; 419: 395-399Crossref PubMed Scopus (586) Google Scholar, 10Salmena L. Hakem R. J. Exp. Med. 2005; 202: 727-732Crossref PubMed Scopus (70) Google Scholar). If the cell utilizes lethal pro-apoptotic proteases such as caspase-8 to perform other cellular functions, an immediate challenge it must overcome is how to survive while harboring active caspase-8. One possibility is to sequester the "apoptotic" substrates (e.g. procaspase-3, procaspase-7, and Bid) from active caspase-8, while leaving non-apoptotic substrates available for proteolysis. To test this hypothesis, we need to identify the elusive non-apoptotic caspase-8 substrates. To this end we employed a bioinformatic approach. Searching the human proteome with a matrix model based on the well defined substrate specificity of caspase-8 revealed a number of potential substrates. We formally tested three of these and confirmed that one, HDAC7, is very efficiently cleaved by caspase-8 both in vitro and in vivo. HDAC7 is a class II histone deacetylase, and like other members of the family, it represses transcription of MEF2-dependent genes in a cell type-specific manner (11Verdin E. Dequiedt F. Kasler H.G. Trends Genet. 2003; 19: 286-293Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). We provide evidence that cleavage of HDAC7 is a loss of function event, abrogating its transcription repressor activity. Finally we discuss the implications of HDAC7 cleavage in both apoptotic and non-apoptotic scenarios. PoPS Search Parameters—The matrix model of caspase-8 specificity (C14.009 > Boyd > 1.2) was used to search the human proteome using the Web-based PoPS program. The model was designed based on studies using positional scanning libraries and fluorescence-quenched substrates, and each amino acid was given a score of -5.0 to +5.0 (12Thornberry 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 (1872) Google Scholar, 13Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (267) Google Scholar). Unprofiled amino acids were given a value of 0. Each subsite was given equal weighting. The threshold was set to 24 (maximum score is 25 for five independent subsites). Phylogeny—Phylogenic tree and multiple sequence alignment were generated using ClustalW at the San Diego Super-computer Center Biology Workbench. Materials and Antibodies—Benzyloxycarbonyl-Val-Ala-(O-methyl)-Asp-fluoromethyl ketone (Z-VAD-fmk) and acetyl-Asp-Glu-Val-Asp-amino-4-trifluoromethylcoumarin (Ac-DEVD-afc) were from Enzyme System Products. SuperSignal was from Pierce. DC protein assay was from Bio-Rad. Killer-TRAIL, FasL, and TNFα were from Alexis Biochemicals. Cycloheximide and MG132 were from Calbiochem. Etoposide was from Biomol. Annexin V-PE was from Caltag. Luciferase reporter assay was from Promega. Monoclonal antibodies were specific for XIAP/hILP, mouse caspase-3, and HSP90 (BD Transduction Laboratories), FLAG (M2), mouse CD8a-FITC and mouse CD4-FITC (Sigma), caspase-8 (C15, kind gift from Dr. Markus Peter, University of Chicago), and poly(ADP-ribose) polymerase and GST (Pharmingen). Polyclonal rabbit antibodies were specific for caspase-3 and hemagglutinin epitope tag (HA) (Santa Cruz Biotechnology), cleaved caspase-3 (Cell Signaling Technology), p35 (kind gift from Dr. Stan Krajewski, Burnham Institute for Medical Research, La Jolla, CA), cleaved poly(ADP-ribose) polymerase (New England Biolabs), and CrmA (kind gift from Dr. David Pickup, Duke University, Durham, NC). Polyclonal goat antibody was against HDAC7 (N-18, Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated donkey anti-rabbit IgG, donkey anti-mouse IgG (Amersham Biosciences), and donkey anti-goat IgG (Santa Cruz Biotechnology). All other chemicals were from Sigma. Plasmids—RAB9A with a C-terminal His6 tag in pET-3a was a kind gift from Dr. Suzanne Pfeiffer (Stanford University, San Francisco). Rat TRIM3/BERP with an N-terminal FLAG tag in pcDNA3 was a kind gift from Dr. Steve Vincent (University of British Columbia, Vancouver, Canada). Histone deacetylase 7 (HDAC7) splice variant 3 with a C-terminal FLAG tag in pcDNA3.1 was a kind gift from Dr. Eric Verdin (The Gladstone Institute, San Francisco, CA). Myc-XIAP plasmid was described previously (14Takahashi R. Deveraux Q. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar). Bax plasmid was already described (15Deveraux Q.L. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (684) Google Scholar). FLAG-Bid, Bcl-xL-HA (in pcDNA3), and pCMV-β-galactosidase were the kind gifts from Dr. John Reed (Burnham Institute for Medical Research, La Jolla, CA). MEF2D plasmid was a kind gift from Dr. Xiao-kun Zhang (Burnham Institute for Medical Research, La Jolla, CA). The pcDNA3.1/caspase-8 wild type and dominant-negative catalytic mutant C285A (caspase-1 numbering system) was described previously (16Boatright K.M. Renatus M. Scott F.L. Sperandio S. Shin H. Pedersen I. Ricci J.-E. Edris W.A. Sutherlin D.P. Green D.R. Salvesen G.S. Mol. Cell. 2003; 11: 529-541Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). The pcDNA3/p35-FLAG and pcDNA3/CrmA plasmid was described previously (17Ryan C.A. Stennicke H.R. Nava V.E. Lewis J. Hardwick J.M. Salvesen G.S. Biochem. J. 2002; 366: 595-601Crossref PubMed Scopus (33) Google Scholar). Luciferase reporter construct under control of the Nur77 promoter (Nur77-luc) was described previously (18Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Human Bid was PCR-amplified with primers containing flanking EcoRI sites and cloned into the EcoRI site of pGEX-4T-1. HDAC7 D375A mutant was generated by site-directed mutagenesis using QuickChange (Stratagene). FLAG-tagged HDAC7 Constructs—HDAC7-FLAG/pcDNA3.1 was cut with EcoRI to remove full-length HDAC7 and produce linearized pcDNA3.1 containing a C-terminal FLAG tag. The N-terminal (1–375) fragment (600 primer, GAGAACCCACTGCTTACTGGC, and 1700 primer, CCCGCGGAATTCTGTCTCCAGGTCTTCAGCCG) and C-terminal (376–915) fragment (1687 primer, GGCGAATTCGCCATGGGCGGGGGACCGGGCCAG, and 755 primer, TAGAAGGCACAGTCGAGG) of HDAC7 were generated by PCR, digested with EcoRI, and cloned into pcDNA3.1 with a C-terminal FLAG tag. The N-terminally FLAG-tagged HDAC7 construct, and double-tagged FLAG-HDAC7-FLAG, were PCR-amplified from HDAC7-FLAG/pcDNA3.1 and cloned into pFLAG-CMV4. GFP-tagged HDAC7 Constructs—Full-length HDAC7 was subcloned into the EcoRI site of pEGFP-N2 (Clontech). The N-terminal (1–375) fragment (600 primer and 1688 primer, CGCGCGGTACCCTGTCTCCAGGTCTTCAGCCG) was amplified by PCR and cloned into the EcoRI and KpnI sites of pEGFP-N2. The C-terminal fragment of HDAC7 (376–915) was amplified as described above and cloned into the EcoRI site of pEGFP-N2. All plasmids were verified by sequencing. Recombinant Proteins—His6-tagged wild type caspases, caspase-3 catalytic mutant (C285A, caspase-1 numbering system), p35 C2A, and RAB9A were expressed in BL21(DE3) Escherichia coli and purified by nickel-affinity chromatography as described previously (19Stennicke H.R. Salvesen G.S. Methods (San Diego). 1999; 17: 313-319Crossref PubMed Scopus (162) Google Scholar). Caspases were titrated with Z-VAD-fmk to determine the concentration of catalytic sites as described previously (20Stennicke H.R. Salvesen G.S. Methods Enzymol. 2000; 322: 91-100Crossref PubMed Google Scholar). GST-Bid was expressed and purified as described previously (14Takahashi R. Deveraux Q. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar). Recombinant HDAC7 and TRIM3 were purified from transfected HEK293. Cells were lysed with modified radioimmunoprecipitation buffer containing 200 μm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin, and 2 μm E-64 as described previously (21Scott F.L. Denault J.B. Riedl S.J. Shin H. Renatus M. Salvesen G.S. EMBO J. 2005; 24: 645-655Crossref PubMed Scopus (316) Google Scholar). Lysates were clarified by centrifugation and combined with 50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.05% (v/v) Nonidet P-40, 0.25% (w/v) gelatin at a 1:1 ratio. For every 1 ml of lysate, 20 μl of anti-FLAG M2 affinity gel beads (Sigma) were added and immunoprecipitated for up to 4 h at 4 °C, and the beads were washed three times in PBS. Beads were either used directly in caspase cleavage assays or resuspended in 50 mm Tris-Cl, 150 mm NaCl, pH 7.4, and eluted with 150 μg/ml 3xFLAG peptide (Sigma) for 30 min at 4 °C. Protein Cleavage Assay—Putative protein substrates in caspase buffer (20 mm PIPES, 100 mm NaCl, 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 10 mm dithiothreitol, 1 mm EDTA, pH 7.2) were incubated for 30 min at 37 °C with the indicated concentration of active site titrated caspase. In some instances caspase buffer was supplemented with 10% (w/v) PEG 6000. Cleavage assays were terminated by the addition of reducing Laemmli sample buffer and boiled for 5 min. Substrate cleavage was monitored by SDS-PAGE and immunoblotting. Cell Lines and Transfections—The DPK cell line is a CD4+CD8+ thymocyte precursor cell line (a kind gift from Dr. John Kaye, The Scripps Research Institute, La Jolla, CA) and was maintained in Click's medium with 10% heat-inactivated fetal bovine serum, 50 μm β-mercaptoethanol, nonessential amino acids, 20 mm Hepes, penicillin/streptomycin, and 2 mm l-glutamine. HEK293 and COS7 cells were transfected with FuGENE 6 transfection reagent (Roche Applied Science). Immature primary thymocytes were purified from the thymus of a 7-week-old FVB/N mouse. Greater than 75% of cells were CD4+CD8+. Thymocytes were cultured for up to 2 days in RPMI with 10% heat-inactivated fetal bovine serum, 50 μm β-mercaptoethanol, penicillin/streptomycin, and 2 mm l-glutamine. Induction and Monitoring of Apoptosis—HEK293 cells were treated with KillerTRAIL, TNFα with cycloheximide, etoposide, staurosporine, MG132, or transfected with pcDNA3/Bax for the indicated times and concentrations. Primary mouse thymocytes were treated with 100 ng/ml FasL with 1 μg/ml Enhancer (Alexis Biochemicals). Activation-induced cell death (AICD) was induced in primary thymocytes with 5 μg/ml biotinylated anti-mouse CD3ϵ chain (145-2C11; Pharmingen) with or without 5 μg/ml biotinylated anti-mouse CD28 (Pharmingen) and 25 μg/ml streptavidin (Southern Biotech) for 24 or 48 h. All cells were harvested, washed in ice-cold PBS, and cell pellets were stored at -20 °C. Frozen pellets were lysed in ice-cold modified radioimmunoprecipitation buffer (containing protease inhibitors, as described above), normalized for protein concentration, and analyzed by SDS-PAGE and immunoblotting. A portion of the lysate was added to caspase buffer containing 100 μm Ac-DEVD-afc, and executioner caspase activity was monitored as described previously (21Scott F.L. Denault J.B. Riedl S.J. Shin H. Renatus M. Salvesen G.S. EMBO J. 2005; 24: 645-655Crossref PubMed Scopus (316) Google Scholar). In some experiments a portion of the cells was reserved prior to storage, stained with annexin V-PE or annexin V-FITC, and analyzed by FACS (FACSort, BD Biosciences). In other experiments, the sub-G1 population was identified by harvesting cells in PBS containing 0.1% (v/v) Triton X-100, 0.1% (w/v) sodium citrate, 50 μg/ml propidium iodide, and analyzed by FACS on the FL3 channel. Electrophoresis and Immunoblotting—Samples were analyzed by 8–18% linear gradient acrylamide SDS-PAGE under reducing conditions, and immunoblotting was as described (22Denault J.B. Salvesen G.S. J. Biol. Chem. 2003; 278: 34042-34050Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Fluorescent Microscopy—COS7 cells grown on glass coverslips in 24-well plates were transfected with FuGENE 6. Cells transfected with C-terminal GFP-tagged HDAC7 constructs were washed in PBS and fixed with 4% paraformaldehyde. Cells co-transfected with 200 ng of C-terminal FLAG-tagged HDAC7, 200 ng of caspase-8, and 1.6 μg of XIAP-myc were washed twice with PBS, fixed with ice-cold methanol for 2 min, blocked with 5% nonfat powdered milk (w/v) in PBS for 1 h at 37 °C, and incubated with mouse anti-FLAG monoclonal antibody (M2, 10 μg/ml) and a FITC-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR). Samples were stained with 250 nm 4′,6-diamidino-2-phenylindole and mounted with VectaShield (Burlingame, CA). Images were captured with a Color CCD SPOT RT Camera (Diagnostic Instruments Inc.) attached to an Inverted TE300 Nikon microscope. Nur77 Promoter Activity Assays—HEK293 cells in 6-well trays were transfected with the indicated plasmids for 48 h. The amount of DNA was kept constant by balancing with empty pcDNA3.1 plasmid. Cells were lysed in 100 μl of Passive Lysis Buffer (Promega), and luciferase activity was assessed using the luciferase assay system (Promega) according to the manufacturer's instruction. β-Galactosidase activity was assayed to monitor transfection efficiency and viability of cells (hydrolysis of 800 μg/ml o-nitrophenyl β-d-galactopyranoside in PBS with 1 mm dithiothreitol, 1 μm MgCl2, emission at 405 nm). Luciferase activity was normalized to β-galactosidase activity. For experiments involving transfection of caspase-8, if the β-galactosidase activity for a given experiment had a standard deviation of more than 10%, it was assumed that expression of caspase-8 had killed too many cells and that data set was excluded. Quantitative RT-PCR—Total RNA was purified from primary thymocytes with TRIzol (Invitrogen) according to the manufacturer's instructions. Residual genomic DNA was removed with DNA-free (Ambion), according to manufacturer's instructions. 1–5 μg of RNA was reverse-transcribed with Superscript First Strand Synthesis System for RT-PCR (Invitrogen). Nur77 mRNA was quantified with the SYBR green fluorogenic detection system on a Stratagene Mx3000p. PCRs were performed in duplicate with the following primers: Nur77 sense (5′-CTTGAGTTCGGCAAGCCTAC-3′) and Nur77 antisense (5′-CGAGGATGAGGAAGAAGACG-3′). Standard curves were plotted, and Nur77 expression was normalized to cyclophilin A. Identification of HDAC7 as a Potential Caspase-8 Substrate in Silico—The distinct specificity of caspases has been established using positional scanning substrate approaches (12Thornberry 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 (1872) Google Scholar, 13Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (267) Google Scholar). Empirical data from these studies were used to generate a matrix model of caspase-8 specificity for the P4-P3-P2-P1-P1′ sub-sites. This model was used to search the human proteome for potential caspase-8 substrates using PoPS, a web-based program for prediction of protease substrates. Using this five subsite model (number C14.009 > Boyd > 1.2 in the model data base at the PoPS website), the maximum score for a potential caspase-8 substrate is 25. Extracellular proteins were eliminated from further consideration, as were candidate substrates whose predicted cleavage sites were within extracellular domains (supplemental Table 1). Three high scoring substrates, whose cleavage may have interesting biological consequences, were selected for in vitro analysis with recombinant caspase-8: HDAC7, RAB9A, and tripartite motif-containing 3 (TRIM3). Recombinant proteins were purified and incubated with recombinant caspase-8. Neither RAB9A nor TRIM3 was cleaved by caspase-8, despite the high scoring prediction (data not shown). For RAB9A it is clear why it was not cleaved; crystal structures show the predicted cleavage site is in a rigid tight turn, with P4–P2 within a β-strand, making it inaccessible to the protease active site (23Wittmann J.G. Rudolph M.G. FEBS Lett. 2004; 568: 23-29Crossref PubMed Scopus (22) Google Scholar). There is no structural information about TRIM3 to explain why it was not cleaved by caspase-8. HDAC7 was cleaved by caspase-8, generating a C-terminal FLAG-tagged fragment of ∼63 kDa (Fig. 1A). This fragment is in accordance with that predicted by PoPS. Although we initially set our threshold at 24 to focus on proteins most likely to be caspase-8 substrates, using this matrix model for caspase-8 specificity, which is based on data using small synthetic substrates, PoPS attributes much lower scores for well characterized cleavage sites within in vivo caspase-8 substrates (supplemental Table 1). According to the PoPS prediction, HDAC7 may also harbor additional cleavage sites with lower PoPS score (Asp228, PoPS 15.31; Asp589, PoPS 14.57; Asp385, PoPS 14.33). To confirm the cleavage site allocated ant the highest PoPS score was indeed the one utilized by caspase-8 in vitro, we generated recombinant HDAC7 with Asp375 mutated to alanine. This mutant was not cleaved by caspase-8 (Fig. 1A). Nucleocytoplasmic shuttling regulates the transcription repressor function of HDAC7. Phosphorylation by protein kinase D or Ca2+/CaM-dependent protein kinase I promote 14-3-3 binding and cytoplasmic accumulation of HDAC7, such that it no longer functions as a transcription repressor (24Dequiedt F. Van Lint J. Lecomte E. Van Duppen V. Seufferlein T. Vandenheede J.R. Wattiez R. Kettmann R. J. Exp. Med. 2005; 201: 793-804Crossref PubMed Scopus (149) Google Scholar, 25Kao H.Y. Verdel A. Tsai C.C. Simon C. Juguilon H. Khochbin S. J. Biol. Chem. 2001; 276: 47496-47507Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Dephosphorylation by myosin phosphatase promotes nuclear import and transcription repression (26Parra M. Mahmoudi T. Verdin E. Genes Dev. 2007; 21: 638-643Crossref PubMed Scopus (69) Google Scholar). Cleavage of HDAC7 at Asp375 would yield fragments containing distinct functional domains; the N-terminal transcription factor binding elements and nuclear localization signal (NLS) would be separated from the histone deacetylase (HDAC) domain and nuclear export signal (NES; Fig. 1B). Cleavage may also disrupt the 14-3-3-binding site. Phylogenic and primary sequence analysis demonstrate that the caspase-8 cleavage site is conserved in other mammalian HDAC7 proteins, although it is not found in the more distantly related HDAC7 from chickens (Fig. 1, C and D). Indeed the chicken HDAC7 protein appears to be more closely related to the other class IIa histone deacetylase family members and the ancestral yeast class II histone deacetylase gene product (yHDA1), which do not contain the predicted cleavage site. Caspase-8 Cleaves HDAC7 with Physiological Efficiency—Procaspase-3, procaspase-7, and Bid are physiological caspase-8 substrates, whose cleaved counterparts have integral roles as pro-apoptotic cell death mediators. We therefore determined the catalytic efficiency for caspase-8 against HDAC7 compared with these physiological substrates. HDAC7 was cleaved more efficiently than either Bid or procaspase-3. Under these experimental conditions, the EC50 value for HDAC7 cleavage was achieved with 10- and 20-fold less caspase-8 than for Bid and procaspase-3, respectively (Fig. 2A). Accordingly, HDAC7 is the best caspase-8 substrate described to date. Considering all caspases can cleave substrates after aspartic acid residues, it was important to determine whether caspases other than caspase-8 can also cleave HDAC7. In standard caspase buffer, HDAC7 was also cleaved by the executioner caspases-3, -6, and -7 (Fig. 2B). An inactive mutant of p35, a baculovirus protein with broad reactivity with caspases, served as a positive control for caspase activity. Recombinant initiator caspases-2 and -8–10 are in a dynamic equilibrium between an inactive monomer and an active dimer. In a buffer that promotes full initiator caspase activity (caspase buffer plus 10% (w/v) PEG 6000), caspase-9 could also cleave HDAC7 to some degree, with caspases-8 and -10 being most efficient (Fig. 2C). When comparing the efficiency with which other caspases cleave HDAC7, the ranking is caspase-8 = caspase-7 > caspase-6 > caspase-3 (Fig. 2D). Exogenous HDAC7 Is Cleaved in Cells Undergoing Caspase-8-mediated Apoptosis—To determine whether endogenously activated caspase-8 can cleave HDAC7, we transfected HEK293 cells with HDAC7-FLAG and induced apoptosis with TRAIL. Greater than 50% of HDAC7-FLAG was cleaved within 2 h of TRAIL treatment (Fig. 3A). Indeed HDAC7-FLAG cleavage was evident before significant PARP cleavage, indicating it is an early event in TRAIL-mediated apoptosis. Only one C-terminal FLAG-tagged fragment was detected at ∼63 kDa, consistent with a single cleavage at Asp375. Transfection of HDAC7-FLAG D375A confirmed that cleavage was because of proteolysis at Asp375 (Fig. 3B). Disappearance of procaspase-3 and -8, increase in executioner caspase activity (Ac-DEVD-afc hydrolysis), and annexin V staining are controls for apoptosis. In contrast with TRAIL-induced apoptosis, HDAC7-FLAG was not significantly processed in cells undergoing apoptosis induced by etoposide (Fig. 3, A and C) or other intrinsic death pathway activators, including MG132, staurosporine, and ectopic Bax expression (Fig. 3C). The reduced HDAC7 expression in conjunction with Bax expression is because cells undergo apoptosis before HDAC7 expression is maximal. Finally, to confirm HDAC7-FLAG was directly processed by caspase-8 as opposed to caspase-7, which is activated downstream from caspase-8, and can efficiently cleave HDAC7 in a purified system (Fig. 2D), we employed various apoptosis inhibitors. We used natural proteinaceous inhibitors because they are more specific than small synthetic peptidyl inhibitors (27McStay G.P. Salvesen G.S. Green D.R. Cell Death Differ. 2007; 15: 322-331Crossref PubMed Scopus (261) Google Scholar). The catalytic mutant, caspase-8 C285A (caspase-1 numbering system), acts as a dominant-negative, preventing activation of caspase-8 at the death-inducing signaling complex, whereas Cowpox response modifier A (CrmA) directly and irreversibly inhibits the catalytic activity of caspase-8 (28Zhou Q. Snipas S. Orth K. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1997; 273: 7797-7800Abstract Full Text Full Text PDF Scopus (490) Google Scholar). XIAP specifically inhibits the activity of caspases-3, -7, and -9, leaving caspase-8 activity intact, whereas p35 inhibits all human caspases (15Deveraux Q.L. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (684) Google Scholar, 29Zhou Q. Krebs J.F. Snipas S.J. Price A. Alnemri E.S. Tomaselli K.J. Salvesen G.S. Biochemistry. 1998; 37: 10757-10765Cros

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