Pathways Accessory to Proteasomal Proteolysis Are Less Efficient in Major Histocompatibility Complex Class I Antigen Production
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m211221200
ISSN1083-351X
AutoresBenedikt M. Kessler, Xu Hong, Jelena Petrovic, Anna Borodovsky, Nico P. Dantuma, Matthew Bogyo, Herman S. Overkleeft, Hidde L. Ploegh, Rickard Glas,
Tópico(s)Toxoplasma gondii Research Studies
ResumoDegradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal β-subunits (β1, β2, and β5) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2Db molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways. Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal β-subunits (β1, β2, and β5) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2Db molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways. major histocompatibility complex interferon-γ 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinyl sulfone cytotoxic T lymphocyte green fluorescent protein ubiquitin 4-hydroxy-5-iodo-3-nitrophenylacetyl-Ala-Ala-Phe-vinyl sulfone 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Gly(cis)-vinyl sulfone adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone 7-amino-4-methylcoumarin fluorescence-activated cell sorter chloromethyl ketone antigen presenting cell Several cytosolic proteases, including the 26 S proteasome, bleomycin hydrolase, puromycin-sensitive amino peptidase and leucine-aminopeptidase, contribute to the generation of MHC1 class I ligands (1Rock K.L. Goldberg A.L. Annu. Rev. Immunol. 1999; 17: 739-779Crossref PubMed Scopus (784) Google Scholar, 2Stoltze L. Schirle M. Schwarz G. Schroter C. Thompson M.W. Hersh L.B. Kalbacher H. Stevanovic S. Rammensee H.G. Schild H. Nat. Immunol. 2000; 1: 413-418Crossref PubMed Scopus (215) Google Scholar, 3Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1590) Google Scholar). However, the 26 S proteasome, a large multicatalytic proteinase complex, carries out the bulk of both cytosolic protein degradation and MHC class I ligand production (1Rock K.L. Goldberg A.L. Annu. Rev. Immunol. 1999; 17: 739-779Crossref PubMed Scopus (784) Google Scholar, 2Stoltze L. Schirle M. Schwarz G. Schroter C. Thompson M.W. Hersh L.B. Kalbacher H. Stevanovic S. Rammensee H.G. Schild H. Nat. Immunol. 2000; 1: 413-418Crossref PubMed Scopus (215) Google Scholar). This protease has a multisubunit 20 S core structure containing two sets of three distinct catalytic sites, X (β5), Y (β1), and Z (β2), associated with one or two 19 S regulatory accessory complexes (1Rock K.L. Goldberg A.L. Annu. Rev. Immunol. 1999; 17: 739-779Crossref PubMed Scopus (784) Google Scholar, 2Stoltze L. Schirle M. Schwarz G. Schroter C. Thompson M.W. Hersh L.B. Kalbacher H. Stevanovic S. Rammensee H.G. Schild H. Nat. Immunol. 2000; 1: 413-418Crossref PubMed Scopus (215) Google Scholar). The proteasome generates a wide range of peptide cleavage products (3–24 amino acids in length) that are ultimately degraded into free amino acids (4Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. J. Biol. Chem. 1999; 274: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 5Tamura N. Lottspeich F. Baumeister W. Tamura T. Cell. 1998; 95: 637-648Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Saric T. Beninga J. Graef C.I. Akopian T.N. Rock K.L. Goldberg A.L. J. Biol. Chem. 2001; 276: 36474-36481Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In mammalian cells, a minor subset of peptides is rescued from further degradation and is translocated from the cytosol into the endoplasmic reticulum for assembly with MHC class I molecules. The MHC class I pathway is thereby assured constitutive production of ligands through cytosolic proteolysis. In the case of an immunological challenge, mammalian cells express IFN-γ-inducible proteasomal β-subunits (LMP7/β5i, LMP2/β1i, and MECL-1/β2i) that replace the constitutively expressed subunits in newly synthesized proteasomes (1Rock K.L. Goldberg A.L. Annu. Rev. Immunol. 1999; 17: 739-779Crossref PubMed Scopus (784) Google Scholar). Such replacement leads to increased proteasomal production of peptides with hydrophobic C termini, usually preferred for both TAP transport and MHC class I binding (7Fruh K. Yang Y. Curr. Opin. Immunol. 1999; 11: 76-81Crossref PubMed Scopus (224) Google Scholar). However, the majority of potential MHC class I ligands, as deduced from their primary structure, are not efficiently processed, although the correct motifs for TAP transport and MHC class I binding are contained in the protein sequence (8Yewdell J.W. Bennink J.R. Annu. Rev. Immunol. 1999; 17: 51-88Crossref PubMed Scopus (791) Google Scholar, 9Chen W. Norbury C.C. Cho Y. Yewdell J.W. Bennink J.R. J. Exp. Med. 2001; 193: 1319-1326Crossref PubMed Scopus (217) Google Scholar). Such failure may depend on proteolysis by cytosolic proteases inefficient at generating the requisite cleavage products. Thus, it is possible that MHC class I processing may be regulated by differential participation of non-proteasomal peptidases in cytosolic protein degradation. Impaired proteasomal activity can be functionally compensated, at least in part, by another large cytosolic peptidase, tripeptidyl-peptidase II (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar, 11Geier E. Pfeifer G. Wilm M. Lucchiari-Hartz M. Baumeister W. Eichmann K. Niedermann G. Science. 1999; 283: 978-981Crossref PubMed Scopus (317) Google Scholar, 12Wang E.W. Kessler B.M. Borodovsky A. Cravatt B.F. Bogyo M. Ploegh H.L. Glas R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9990-9995Crossref PubMed Scopus (104) Google Scholar, 13Tomkinson B. Trends Biochem. Sci. 1999; 24: 355-359Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Despite covalent inhibition by NLVS (14Bogyo M. McMaster J.S. Gaczynska M. Tortorella D. Goldberg A.L. Ploegh H.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 94: 6629-6634Crossref Scopus (406) Google Scholar) or lactacystin (15Fenteany G. Schreiber S.L. J. Biol. Chem. 1998; 273: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar), EL-4 lymphoma cells adapted to growth in the presence of this inhibitor (denoted EL-4ad) maintain cytosolic proteolysis and cell viability by a mechanism that includes compensatory up-regulation of tripeptidyl-peptidase II (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar, 11Geier E. Pfeifer G. Wilm M. Lucchiari-Hartz M. Baumeister W. Eichmann K. Niedermann G. Science. 1999; 283: 978-981Crossref PubMed Scopus (317) Google Scholar, 12Wang E.W. Kessler B.M. Borodovsky A. Cravatt B.F. Bogyo M. Ploegh H.L. Glas R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9990-9995Crossref PubMed Scopus (104) Google Scholar). However, it is unknown whether the adapted state has functional consequences at the level of MHC class I ligand generation and antigen presentation to CTLs. We show that lymphoma cells with reduced reliance on proteasomal activity no longer efficiently produced MHC class I ligands, although cytosolic proteolysis continued, and proliferation was not altered compared with control cells. Assembly of H-2Db molecules was dramatically reduced, and endogenous tumor antigens were not presented efficiently under these conditions. This phenotype contributed to escape from tumor rejection in tumor graft experiments in syngeneic C57Bl/6 mice. Using a GFP reporter to measure proteolysis, we show in live cells that non-proteasomal serine peptidase activity participated in protein degradation, but inhibition of these enzymes failed to have a significant effect on the assembly of H-2Kb molecules. Continued proteolysis in proteasome-impaired cells afforded the cell the requisite housekeeping functions while preventing the full display of the usual set of MHC class I-restricted epitopes. EL-4 is a benzopyrene-induced thymoma cell line of the H-2b haplotype, derived from C57Bl/6 mice. RMA is a Rauscher's virus-induced T cell lymphoma cell line and is also derived from C57Bl/6. Adaptation to the proteasomal inhibitor NLVS was obtained by incubation of these cells in RPMI 1640 medium containing 5% fetal calf serum, 1% penicillin/streptomycin, 1% glutamine, and 10 μm NLVS. Gradually outgrowing cells were selected and cultured in 50 μm NLVS over a period of several weeks as described previously (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar). EL-4.Ub-R-GFP and EL-4.Ub-M-GFP cells were obtained by electroporation of EL-4 cells with constructs Ub-R-GFP and Ub-M-GFP (16Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (468) Google Scholar), respectively, and stable clones were selected with 0.5 mg/ml G418. Electroporation was preformed in a Bio-Rad Gene-Pulser at 250 V and 960 microfarads. NLVS (14Bogyo M. McMaster J.S. Gaczynska M. Tortorella D. Goldberg A.L. Ploegh H.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 94: 6629-6634Crossref Scopus (406) Google Scholar) covalently modifies all catalytically active subunits of the proteasome, but with preference for the β-subunits with chymotryptic specificity. Several derivatives of NLVS were obtained by variations in the peptide scaffold: 4-hydroxy-5-iodo-3-nitrophenylacetyl-Ala-Ala-Phe-vinyl sulfone (AAF-VS) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Gly(cis)-vinyl sulfone (LLG(cis)-VS). LLG-VS was obtained in thecis- and trans-isomers due to the absence of a side chain on the P1 glycine. Whereas the trans-form of LLG-VS modifies proteasomal β-subunits, the cis-form modifies yet uncharacterized targets in the cytosol distinct from the proteasome. Adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone (Ada-Ahx3-Leu3-VS) is an N-terminally extended vinyl sulfone inhibitor that blocks all proteasomal β-subunits in a covalent manner (17Kessler B.M. Tortorella D. Altun M. Kisselev A.F. Fiebiger E. Hekking B.G. Ploegh H.L. Overkleeft H.S. Chem. Biol. 2001; 8: 913-929Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). To assay the activity of the proteasome, we used the fluorogenic substrates succinyl-LLVY-AMC, benzyloxycarbonyl-GGL-AMC,t-butyloxycarbonyl-LRR-AMC, and benzyloxycarbonyl-YVAD-AMC (Sigma). To assay tripeptidyl-peptidase II activity, we used AAF-AMC (Sigma). Cell extracts or proteasome-enriched fractions and substrate (100 μm) were mixed in 50 mm Tris (pH 7.5), 5 mm MgCl2, 1 mm dithiothreitol, and 2 mm ATP in a final volume of 100 μl. Peptide hydrolysis was monitored by fluorescence spectroscopy (PerSeptive Biosystems, Framingham, CT) with excitation at 380 nm and fluorescence reading at 460 nm. Preparation of proteasome-enriched fractions was performed using 0.5–1 × 109 control or adapted EL-4 cells and C57Bl/6 livers. Cells were washed with phosphate-buffered saline and lysed by vortexing with glass beads in 50 mm Tris base (pH 7.5), 250 mm sucrose, 5 mm MgCl2, 1 mm dithiothreitol, and 2 mm ATP. Glass beads and cell debris were removed by sequential centrifugations at 3000 and 14,000 rpm, respectively. Microsomes were removed by centrifugation for 1 h at 100,000 × g, and large cytosolic proteins or protein complexes containing proteasomes and tripeptidyl-peptidase II were then sedimented at 100,000 × g for 5 h. The resulting pellet was dissolved in 50 mm Tris base (pH 7.5), 5 mm MgCl2, 1 mm dithiothreitol, 2 mm ATP, and 30% glycerol. Cells were starved in methionine/cysteine-deficient medium for 45–60 min, pulsed with [35S]methionine for 15 min, and chased for the indicated times. Cells were collected by centrifugation and lysed in 0.5% Nonidet P-40 lysis buffer, and MHC class I molecules were immunoprecipitated with rabbit anti-p8 serum (H-2Kbcytoplasmic tail), monoclonal antibody Y3 (H-2Kbα1α2), or antibody B22.249.1 (H-2Db α1α2) as described previously (18Machold R.P. Andree S. Van Kaer L. Ljunggren H.-G. Ploegh H.L. J. Exp. Med. 1995; 181: 1111-1122Crossref PubMed Scopus (69) Google Scholar). Viral peptide epitopes are known to stabilize the MHC class I complex at 4 °C. We added Db-binding peptide ASNENMDAM (influenza NT60, amino acids 366–374) at 10 μm to detect the presence of unfolded heavy chains. Immune complexes were removed by adsorption to staphylococcus A and analyzed by SDS-PAGE as previously described (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar). For the generation of CTLs specific for tumor antigens expressed by EL-4 cells, we primed B6 mice two to three times with control or adapted EL-4 cells transfected with B7.1. Priming of responses with EL-4 cells not expressing B7.1 led to very low CTL responses or no response at all. To generate RMA cell-specific CTLs, we primed C57BL/6 mice with irradiated RMA cells two to three times; and for the subsequent generation of in vitro effector cells, 25 × 106 splenocytes were restimulated in vitro with 1–2 × 106 irradiated tumor cells for 5 days. The H-2Db restricted CTL clone ln17 was acquired from Elisabeth Wolpert and Vanoohi Fredriksson (MTC, Karolinska Institutet). The conditions for generation and analysis of this CTL have been described previously (23van Hall T. van Bergen J. van Veelen P.A. Kraakman M. Heukamp L.C. Koning F. Melief C.J. Ossendorp F. Offringa R. J. Immunol. 2000; 165: 869-877Crossref PubMed Scopus (41) Google Scholar). Control EL-4 and EL-4ad cells (cultured in RPMI medium 1640 supplemented with 5% fetal calf serum) were washed with phosphate-buffered saline and resuspended in 200 μl/inoculate. The cells were inoculated into the right flanks of syngeneic C57Bl/6 mice at 104 or 106cells/animal. Some of the mice were irradiated with 400 rads prior to tumor inoculation to inhibit antitumor immune responses. Outgrowth of the tumors was monitored by palpations weekly. EL-4 cells can adapt to proliferate in the presence of high concentrations of NLVS, a covalent proteasomal inhibitor (denoted EL-4ad cells) (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar). MHC class I molecules show allelic variation in their ability to undergo assembly and transport during proteasomal inhibition, and H-2Db is one allele that fails to assemble in cells that are treated with proteasomal inhibitors for short-term periods (19Benham A.M. Gromme M. Neefjes J. J. Immunol. 1998; 161: 83-89PubMed Google Scholar, 20Luckey C.J. Marto J.A. Partridge M. Hall E. White F.M. Lippolis J.D. Shabanowitz J. Hunt D.F. Engelhard V.H. J. Immunol. 2001; 167: 1212-1221Crossref PubMed Scopus (73) Google Scholar, 21Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2193) Google Scholar). We therefore investigated H-2Db ligand production in EL-4ad cells, which proliferate with low proteasomal activity. In control EL-4 cells, almost all folded H-2Db molecules were transported from the endoplasmic reticulum within 120 min after onset of the chase, as judged from their acquisition of Golgi-specific glycan modifications (Fig. 1 a, left panel). In EL-4 cells treated with NLVS (50 μm, 3 h), only a minimal fraction of H-2Db heavy chains were transported even after long chase times (Fig. 1 a,middle panel), as reported previously (21Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2193) Google Scholar). Stabilization of H-2Db molecules in cell lysates of NLVS-treated EL-4 cells by addition of the influenza nucleoprotein-(366–374) peptide confirmed that most of these H-2Db heavy chains were devoid of peptide ligand (+ lanes) (22Ljunggren H.-G. Stam N.J. Ohlen C. Neefjes J.J. Hoglund P. Heemels M.T. Bastin J. Schumacher T.N. Townsend A. Karre K. Ploegh H.L. Nature. 1990; 346: 476-480Crossref PubMed Scopus (789) Google Scholar). In EL-4ad cells, a fraction of H-2Db resumed folding, although at much lower levels compared with control EL-4 cells. The majority of H-2Db heavy chains in EL-4ad cells remained unassembled in the endoplasmic reticulum devoid of peptide, as indicated by the stabilizing effect of nucleoprotein-(366–374) added to lysates of these cells (Fig. 1 a, right panel). In RMAad cells, which were similarly adapted to NLVS, maturation of H-2Db molecules was comparable to that observed in EL-4ad cell (Fig. 1 b). Despite normal proliferation, tumor cells can therefore avoid production of most H-2Db ligands by reduced reliance on proteasomal activity. Tumor cells often acquire deficiencies in MHC class I antigen presentation to escape from host immune detection. To test whether reduced reliance on proteasomal activity has functional consequences, we tested presentation of endogenous tumor antigens to CTLs by EL-4ad and RMAad cells. Both EL-4 and RMA cells express H-2Kb-restricted (gagL75–83) as well as H-2Db-restricted (env189–196) murine leukemia virus-derived peptides and, in addition, an endogenous H-2Db-restricted tumor epitope (23van Hall T. van Bergen J. van Veelen P.A. Kraakman M. Heukamp L.C. Koning F. Melief C.J. Ossendorp F. Offringa R. J. Immunol. 2000; 165: 869-877Crossref PubMed Scopus (41) Google Scholar). We generated antitumor CTLs by priming C57Bl/6 mice and subsequent in vitrorestimulation of splenocytes with B7.1-transfected EL-4 cells. We found that tumor antigen-specific CTLs performed efficient killing of control EL-4 cells, whereas EL-4ad cells were not efficiently recognized, although the latter were killed significantly better than C4.4-25−, a β2-microglobulin-deficient variant of EL-4 (Fig. 2 a and data not shown). We also found that RMAad cells likewise had a reduced ability to present endogenous tumor antigens compared with control RMA cells. Although RMAad target cells were killed at higher levels than TAP-deficient RMA-S cells, 5–10 times more CTLs were required to obtain the same degree of killing as seen on RMA target cells (Fig.2 b). Even more pronounced differences were obtained using CTL clone ln17, specific for tumor antigen-specific peptide NKGENAQAI restricted by H-2Db (20Luckey C.J. Marto J.A. Partridge M. Hall E. White F.M. Lippolis J.D. Shabanowitz J. Hunt D.F. Engelhard V.H. J. Immunol. 2001; 167: 1212-1221Crossref PubMed Scopus (73) Google Scholar). In line with previous data, we found that ln17 detected the presence of the tumor-specific epitope on RMA cells, but failed to recognize RMA-S cells (Fig. 2 c). Furthermore, no recognition of RMAad cells was observed. MHC class I-restricted presentation of a tumor antigen-specific peptide can thereby be inhibited when proteasomal proteolysis is inhibited in a suitable manner. Because we used IFN-γ secretion as readout for antigen detection by ln17, these data also exclude that the differences in CTL killing were due merely to differences in target cell apoptosis when comparing control and NLVS-adapted target cells. These data support the conclusion that EL-4ad cells fail to display the full repertoire of MHC class I-associated antigens at the cell surface. The chymotrypsin-like activity of the proteasome is required for the production of most MHC class I ligands and is normally rate-limiting for intracellular proteolysis (1Rock K.L. Goldberg A.L. Annu. Rev. Immunol. 1999; 17: 739-779Crossref PubMed Scopus (784) Google Scholar, 4Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. J. Biol. Chem. 1999; 274: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 7Fruh K. Yang Y. Curr. Opin. Immunol. 1999; 11: 76-81Crossref PubMed Scopus (224) Google Scholar). To visualize protein turnover in EL-4ad cells, we performed pulse-chase experiments with [35S]methionine and displayed labeled protein by SDS-PAGE. As expected from the proliferation rates of these cell lines (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar), we observed a similar rate of decay of labeled proteins when comparing control EL-4 and EL-4ad cells (Fig. 2 d). We conclude that NLVS-adapted cells have a severely inhibited chymotryptic proteasomal activity, as deduced from experiments employing active site-directed covalent probes. When complemented by the induction of other cytosolic proteases (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar, 11Geier E. Pfeifer G. Wilm M. Lucchiari-Hartz M. Baumeister W. Eichmann K. Niedermann G. Science. 1999; 283: 978-981Crossref PubMed Scopus (317) Google Scholar, 12Wang E.W. Kessler B.M. Borodovsky A. Cravatt B.F. Bogyo M. Ploegh H.L. Glas R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9990-9995Crossref PubMed Scopus (104) Google Scholar), the remaining proteasomal activity is adequate for normal protein turnover, but not for production of all class I ligands. The activity of the ubiquitin-specific protease USP14 is associated with the 19 S cap proteasome and is up-regulated when the proteasome is inhibited (24Borodovsky A. Kessler B.M. Casagrande R. Overkleeft H.S. Wilkinson K.D. Ploegh H.L. EMBO J. 2001; 20: 5187-5196Crossref PubMed Scopus (403) Google Scholar). More generally, inhibition of proteasomal proteolysis should lead to accumulation of ubiquitin-conjugated substrates. Adaptation to proteasomal inhibitors might well include increased activity of deubiquitinating enzymes to deal with such accumulation. We therefore examined whether this was the case also in EL-4ad cells using 125I-labeled ubiquitin-vinyl sulfone (24Borodovsky A. Kessler B.M. Casagrande R. Overkleeft H.S. Wilkinson K.D. Ploegh H.L. EMBO J. 2001; 20: 5187-5196Crossref PubMed Scopus (403) Google Scholar). Cellular fractions of control EL-4 and EL-4ad cells (cytosolic as well as proteasome-enriched fractions) were incubated with ubiquitin-vinyl sulfone, and covalently modified polypeptides were separated by SDS-PAGE. We found increased labeling of IsoT1, USP14, and UCH-L1 in EL-4ad cells compared with control EL-4 cells (Fig. 3 a), in line with what was observed in acutely treated EL-4 cells (24Borodovsky A. Kessler B.M. Casagrande R. Overkleeft H.S. Wilkinson K.D. Ploegh H.L. EMBO J. 2001; 20: 5187-5196Crossref PubMed Scopus (403) Google Scholar). More active ubiquitin removal could prepare these substrates for degradation by other proteases. Two additional active-site probes with different peptide scaffolds were used (25Bogyo M. Shin S. McMaster J.S. Ploegh H.L. Chem. Biol. 1998; 5: 307-320Abstract Full Text PDF PubMed Scopus (157) Google Scholar), [125I]AAF-VS and [125I]LLG-VS, to examine whether residual proteasomal activity is mediated by the β5/β5i-subunits (X/LMP7). None of these probes labeled β5/β5i-subunits (X/LMP7) in lysates of EL-4ad cells, whereas strong labeling was detected in lysates of control EL-4 cells (Fig. 3 b). This confirms that virtually no catalytic activity remains for the β5/β5i-subunits (X/LMP7) in EL-4ad cells, which is important in view of the fact that small amounts of peptide ligand suffice to load MHC class I molecules with peptide (26Yewdell J.W. Trends Cell Biol. 2001; 11: 294-297Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Interestingly, using [125I]LLG(cis)-VS, we detected a series of modified polypeptides distinct from proteasomal β-subunits. Because vinyl sulfones are mechanism-based probes (14Bogyo M. McMaster J.S. Gaczynska M. Tortorella D. Goldberg A.L. Ploegh H.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 94: 6629-6634Crossref Scopus (406) Google Scholar,27Palmer J.T. Rasnick D. Klaus J.L. Bromme D. J. Med. Chem. 1995; 38: 3193-3196Crossref PubMed Scopus (482) Google Scholar), we conclude that these polypeptides correspond to additional, yet to be identified, proteases. This activity is not inhibited by NLVS, further supporting the alteration in proteolytic specificity in EL-4ad cells (Fig. 3 b, middle panel). Labeling of the β5/β5i- subunits (X/LMP7) with [125I]NLVS was likewise inhibited in RMAad cells when tested with these peptide vinyl sulfones (Fig. 3 c). We next made stable EL-4 transfectants expressing Ub-R-GFP to monitor proteasomal degradation of a protein substrate in live cells (16Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (468) Google Scholar). GFP was converted into an N-end rule substrate and was degraded in EL-4 cells due to its N-terminal arginine, whereas Ub-M-GFP was comparatively stable (Fig.4, a–c) because of the presence of a methionine residue. NLVS treatment of EL-4.Ub-R-GFP cells led to accumulation of fluorescence, as detected by FACS. In line with data from yeast mutants (28Heinemeyer W. Kleinschmidt J.A. Saidowsky J. Escher C. Wolf D.H. EMBO J. 1991; 10: 555-562Crossref PubMed Scopus (357) Google Scholar, 29Seufert W. Jentsch S. EMBO J. 1992; 11: 3077-3080Crossref PubMed Scopus (121) Google Scholar), efficient inhibition of primarily the chymotryptic proteasomal activity was sufficient for accumulation of fluorescence (Fig. 4, c and d). In addition, the accumulation of R-GFP fluorescence observed in cells exposed to 10 μm NLVS also correlated with the induction of cellular toxicity and subsequent cell death (data not shown). These data further confirm that NLVS is indeed an efficient inhibitor of proteasomal protein degradation in live cells. We next tested whether inhibition of accessory pathways has any effect on cytosolic proteolysis. To do this, we accumulated high levels of the R-GFP substrate in live EL-4.Ub-R-GFP cells and then blocked protein synthesis to study changes in the steady state of the substrate (Fig.5, a–d). This revealed residual substrate degradation in the continued presence of 10 μm NLVS because a substantial fraction of the substrate was removed after 8 h. However, this was inhibited by treatment with AAF-CMK, an efficient inhibitor of tripeptidyl-peptidase II and other serine oligopeptidases (Fig. 5, c and d). Although inhibition of oligopeptidases by AAF-CMK had minor effects on untreated EL-4.Ub-R-GFP cells, we observed a significant effect on R-GFP accumulation upon treating EL-4.Ub-R-GFP cells with both NLVS and AAF-CMK in combination (Fig. 5 e). These data show that non-proteasomal oligopeptidase activity indeed contributes to cytosolic proteolysis, especially during situations of limiting or insufficient proteasomal activity. To further study whether oligopeptidases inhibitable by AAF-CMK are important in generating MHC class I ligands, we performed a pulse-chase experiment with [35S]methionine metabolic labeling and precipitation of H-2Kb molecules. A substantial fraction of H-2Kb molecules continue to assemble in EL-4ad cells (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar). We examined whether this may be due to ligands produced by oligopeptidases inhibitable by AAF-CMK. We found that this treatment had minor effects on the assembly and transport of H-2Kbmolecules in EL-4ad cells and also in control EL-4 cells with active proteasomes (Fig. 5 f). We conclude that pathways accessory to proteasomal proteolysis that are inhibited by AAF-CMK support protein degradation, but reveal poor yields of MHC class I ligands. EL-4ad cells continue to depend on proteasomal β-subunit activity, at least to some extent (30Princiotta M.F. Schubert U. Chen W. Bennink J.R. Myung J. Crews C.M. Yewdell J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 513-518Crossref PubMed Scopus (77) Google Scholar). NLVS fails to block β2- and β2i-subunits (Z/MECL-1) in vivo, a pattern of inhibition that is shared between NLVS and other covalent proteasomal inhibitors such as lactacystin (15Fenteany G. Schreiber S.L. J. Biol. Chem. 1998; 273: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar) and epoxomicin (31Meng L. Mohan R. Kwok B.H. Elofsson M. Sin N. Crews C.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10403-10408Crossref PubMed Scopus (819) Google Scholar). To examine if residual proteasomal activity influences the viability of EL-4ad cells, we used Ada-Ahx3-Leu3-VS, a cell-permeable tripeptide vinyl sulfone that covalently modifies all proteasomal β-subunits with comparable efficiency (17Kessler B.M. Tortorella D. Altun M. Kisselev A.F. Fiebiger E. Hekking B.G. Ploegh H.L. Overkleeft H.S. Chem. Biol. 2001; 8: 913-929Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). We found that proteasome-enriched fractions from control EL-4 or EL-4ad cells treated with either NLVS or Ada-Ahx3-Leu3-VS had almost completely blocked chymotryptic and trypsin-like proteasomal activities, whereas the caspase-like activity was 70% inhibited (Fig.6 a). Initially, at early time points, we observed an induction of the trypsin- and caspase-like specificities during inhibitor treatment, possibly due to allosteric effects on the proteasome upon binding of the inhibitor to the X/LMP7 site (32Kisselev A.F. Akopian T.N. Castillo V. Goldberg A.L. Mol. Cell. 1999; 4: 395-402Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Consistent with the enzyme assays using fluorogenic peptide substrates, labeling of β-subunits with Ada-[125I-Tyr]Ahx3-Leu3-VS in cell lysates followed by separation of the β-subunits by SDS-PAGE confirmed that all proteasomal active sites were covalently modified during treatment of live cells with the Ada-Ahx3-Leu3-VS inhibitor (Fig. 6 a,lower panel). Furthermore, EL-4ad cells proliferated regardless of the presence of Ada-Ahx3-Leu3-VS, whereas control EL-4 cells died within 48 h (Fig. 6 b). To confirm that proteasomes of proliferating EL-4ad cells were indeed modified, we prepared proteasome-enriched fractions from cells treated for several days with Ada-Ahx3-Leu3-VS. This analysis revealed results similar to those observed in acutely treated cells. Essentially no residual tryptic and chymotryptic activities and inhibited caspase-like activity were detected (data not shown). Thus, tumor cells may adapt and proliferate normally even when proteasomal β-subunit activity is almost absent. In vivo tumors frequently acquire mutations in genes encoding proteins of the MHC class I-processing pathway, and these are believed to be the result of immune selection (33Pawelec G. Heinzel S. Kiessling R. Muller L. Ouyang Q. Zeuthen J. Crit. Rev. Oncog. 2000; 11: 97-133PubMed Google Scholar). Therefore, we explored whether an adapted phenotype similar to that of EL-4ad cells was selected during growthin vivo. We inoculated control EL-4 and EL-4ad cells into syngeneic C57Bl/6 mice and observed tumors in most mice inoculated with EL-4ad cells at both 106 and 104 cells/animal, whereas control EL-4 cells failed to grow and produce tumors (Fig.7 a). The tumor-forming ability of EL-4ad cells was dependent, at least in part, on escape from immune recognition because both EL-4ad and control EL-4 cells formed tumors in mice with a deficiency of perforin and RAG-1 (PKOB/RAG−/−) (Fig. 7 a). Furthermore, after low dose irradiation (400 rads) of the C57Bl/6 mice, commonly used to reduce in vivo transplantation barriers (34Peng L. Krauss J.C. Plautz G.E. Mukai S. Shu S. Cohen P.A. J. Immunol. 2000; 165: 7116-7124Crossref PubMed Scopus (45) Google Scholar), both control EL-4 and EL-4ad cells were able to grow after inoculation of 106 cells/animal, whereas only EL-4ad cells grew at 104 cells/animal (Fig. 7 b). In conclusion, we have shown that the ability of NLVS-adapted cells to proliferate independently of proteasomal β-subunit activities leads to reduced immune recognition in vivo. This study shows that tumor cells may avoid efficient production of MHC class I ligands and hence immune recognition by modulation of proteasomal activity. Pathways accessory to proteasomal proteolysis can reduce the extent to which cells depend on proteasomal activity. In our case, cells adapted to growth in the presence of proteasomal inhibitors were unable to maintain normal levels of MHC class I ligand production. In addition, using the vinyl sulfone inhibitor Ada-Ahx3-Leu3-VS, we showed that inhibition of all catalytic β-subunit activities of the proteasome (more efficiently than achieved with NLVS) was compatible with continued cell growth of EL-4ad cells. These results indicate that it is possible for mammalian cells to partly escape from production of MHC class I ligands by aversion to pathways of protein degradation involving proteases other than the proteasome. Although MHC class I processing is a rather inefficient process overall, in which most (>99%) of the cleaved peptides are never displayed at the cell surface, it is an adequate method for screening of the bulk of cellular protein content for the presence of foreign antigens (26Yewdell J.W. Trends Cell Biol. 2001; 11: 294-297Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The steady-state level of MHC class I at the surface of cells depends on both its transport and removal from the cell surface (35Su R.C. Miller R.G. J. Immunol. 2001; 167: 4869-4877Crossref PubMed Scopus (28) Google Scholar), and transport of H-2Db is substantially inhibited in EL-4ad cells. Despite this, the cell-surface H-2Db (and also H-2Kb) levels detected by FACS are almost normal, suggesting that the rate of decay at the cell surface may be reduced when transport is slow (data not shown). Earlier data on MHC class II transport in cathepsin S−/− mice have revealed a similar feature; and also in this case, an absence of T cell detection is observed for certain antigens (36Driessen C. Bryant R.A. Lennon-Dumenil A.M. Villadangos J.A. Bryant P.W. Shi G.P. Chapman H.A. Ploegh H.L. J. Cell Biol. 1999; 147: 775-790Crossref PubMed Scopus (199) Google Scholar, 37Riese R.J. Mitchell R.N. Villadangos J.A. Shi G.P. Palmer J.T. Karp E.R. De Sanctis G.T. Ploegh H.L. Chapman H.A. J. Clin. Invest. 1998; 101: 2351-2363Crossref PubMed Scopus (261) Google Scholar). In the course of an immune response, the proteolytic specificity in antigen processing has profound influence on the generation of MHC class I ligands, as illustrated by the IFN-γ-dependent substitution of proteasomal β-subunits (7Fruh K. Yang Y. Curr. Opin. Immunol. 1999; 11: 76-81Crossref PubMed Scopus (224) Google Scholar). 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When the proteasome is blocked, the removal of ubiquitin from ubiquitin-conjugated substrates may be a crucial step to engage alternative proteolytic pathways. Reduced expression of several components of the MHC class I antigen-processing pathway is often observed in human tumors. This includes down-regulation of the IFN-γ-inducible proteasomal β-subunits (β1i, β2i, and β5i) (40Frisan T. Levitsky V. Polack A. Masucci M.G. J. Immunol. 1998; 160: 3281-3289PubMed Google Scholar), important for production of MHC class I ligands, as well as down-regulation of other gene products involved in antigen processing (41Delp K. Momburg F. Hilmes C. Huber C. Seliger B. Bone Marrow Transplant. 2000; 25 Suppl. 2: 88-95Crossref PubMed Scopus (65) Google Scholar, 42Algarra I. Cabrera T. Garrido F. Hum. Immunol. 2000; 61: 65-73Crossref PubMed Scopus (141) Google Scholar, 43Wang Z. Seliger B. Mike N. Momburg F. Knuth A. Ferrone S. Cancer Res. 1998; 58: 2149-2157PubMed Google Scholar). Tumors may fail to produce certain immunodominant ligands due to altered proteasomal specificity (40Frisan T. Levitsky V. Polack A. Masucci M.G. J. Immunol. 1998; 160: 3281-3289PubMed Google Scholar, 44Morel S. et al.Immunity. 2000; 12: 107-117Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Our data show that reduced reliance on proteasomal proteolysis biases cytosolic proteolysis to produce peptides that are less fit for MHC class I assembly, thereby down-regulating the pool of potential MHC class I-restricted epitopes. Such down-regulation is stably retained in rapidly proliferating cells and can be induced at fairly high frequency (10Glas R. Bogyo M. McMaster J.S. Gaczynska M. Ploegh H.L. Nature. 1998; 392: 618-622Crossref PubMed Scopus (238) Google Scholar). Furthermore, EL-4ad cells appear to use this phenotype to avoid immunological rejection during the formation of tumors in vivo. An EL-4ad-like phenotype may be preferentially selected in tumors that are poorly antigenic, a trait observed in many types of tumors. Interestingly, in Burkitt's lymphomas, the oncogene c-myc is known to induce down-regulation of a number of components of the MHC class I-processing pathway, including down-regulation of proteasomal chymotryptic activity and up-regulation of tripeptidyl-peptidase II, thus linking the deficiency in antigen processing directly to oncogene expression (45Gavioli R. Frisan T. Vertuani S. Bornkamm G.W. Masucci M.G. Nat. Cell. Biol. 2001; 3: 283-288Crossref PubMed Scopus (104) Google Scholar, 46Staege M.S. Lee S.P. Frisan T. Mautner J. Scholz S. Pajic A. Rickinson A.B. Masucci M.G. Polack A. Bornkamm G.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4550-4555Crossref PubMed Scopus (59) Google Scholar). This study reveals a new strategy for regulation of MHC class I processing: reduced reliance on proteasomal activity to down-regulate generation of MHC class I ligands We thank members of the Ploegh laboratory for support and discussions and Elisabeth Wolpert for CTL clone ln17. We also thank Maria Masucci and Hans-Gustaf Ljunggren for discussions and suggestions on the manuscript.
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