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1212.Full.Pdf Differences in the Expression of Human Class I MHC Alleles and Their Associated Peptides in the Presence of Proteasome Inhibitors This information is current as Chance John Luckey, Jarrod A. Marto, Megan Partridge, Ed of September 26, 2021. Hall, Forest M. White, John D. Lippolis, Jeffrey Shabanowitz, Donald F. Hunt and Victor H. Engelhard J Immunol 2001; 167:1212-1221; ; doi: 10.4049/jimmunol.167.3.1212 http://www.jimmunol.org/content/167/3/1212 Downloaded from References This article cites 60 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/167/3/1212.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differences in the Expression of Human Class I MHC Alleles and Their Associated Peptides in the Presence of Proteasome Inhibitors1 Chance John Luckey,2* Jarrod A. Marto,2† Megan Partridge,* Ed Hall,‡ Forest M. White,† John D. Lippolis,* Jeffrey Shabanowitz,† Donald F. Hunt,†§ and Victor H. Engelhard3* We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC- associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor- resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Further- more, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays Downloaded from and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides http://www.jimmunol.org/ contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed. The Journal of Immunology, 2001, 167: 1212–1221. he recognition of antigenic peptides in association with duction of known epitopes (8–10). Finally, treatment of cells with class I MHC molecules at the cell surface provides a various inhibitors of proteasome activity leads to a significant de- ϩ mechanism by which CD8 T lymphocytes gain infor- crease in presentation of several peptide epitopes as well as re- T b mation about the proteins being made within cells. In most cells, duced surface expression of H2-K and HLA-A*0201 (2). These by guest on September 26, 2021 class I-associated peptides are derived from endogenously ex- inhibitors also diminish the ability of several human class I mol- pressed intracellular proteins and are between 8 and 11 aa long (1). ecules to form stable dimers (11, 12). The enzyme complex most often implicated in the generation of Despite this breadth of evidence, other studies have suggested class I- associated peptides is the proteasome (2). Proteasome in- that proteasome involvement in class I- restricted peptide produc- volvement in class I peptide production is based on several lines of tion is more limited (13). In particular, it has been suggested that evidence. First, the expression of the catalytically active protea- ubiquitination is not involved in the production of at least some ␤ some subunits LMP2 and LMP7, as well as the proteasome class I-associated peptides (14). More directly, cell surface expres- regulator PA28, have been shown to enhance the production of sion of two murine class I alleles is only partially blocked by class I-associated peptides in vivo and in vitro (3–5). Second, ubiq- proteasome inhibitors (15, 16). Furthermore, the ability of two uitination, which targets proteins for proteasome-mediated degra- human class I molecules to form stable dimers, a property thought dation, is required for generation of class I- associated epitopes to depend upon peptide availability for binding, was not blocked from some proteins (6, 7). Third, incubation of whole proteins or when proteasome activity was inhibited (17). A few specific TAP- synthetic polypeptides with proteasomes in vitro results in the pro- dependent epitopes have also been shown to be either insensitive to proteasome inhibition (15, 18) or destroyed by proteasome ac- *Department of Microbiology and Carter Immunology Center, University of Virginia tivity (19). Finally, cells grown for extended periods in proteasome Health Sciences Center, Charlottesville, VA 22908; and Departments of †Chemistry and ‡Information Technology and Communication, Research Computing Support inhibitors continue to express stable murine class I dimers (20). Group, University of Virginia, Charlottesville, VA 22901; and §Department of Collectively these studies strongly suggest that both proteasomes Pathology, University of Virginia, Charlottesville, VA 22904 and nonproteasomal proteases can independently generate class Received for publication November 13, 2000. Accepted for publication May I-associated peptides. 18, 2001. Neither the relative contributions of different proteolytic path- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ways to class I expression, nor the sequence and protein source of with 18 U.S.C. Section 1734 solely to indicate this fact. the peptides they produce, are well understood. Here, we have 1 This work was supported by U.S. Public Health Service Grants AI 20963 and AI combined proteasome inhibitors, flow cytometry, and mass spec- 21393 (to V.H.E.), and AI33993 (to D.F.H.). C.J.L. was supported by Medical Sci- trometry to address these issues. Cells treated with acid to remove entist Training Program Grant GM 07267. surface class I peptide complexes were allowed to re-express 2 C.J.L. and J.A.M. contributed equally to this work. newly formed complexes in the presence or absence of proteasome 3 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA inhibitors. We then used flow cytometry to measure total class I 22908. E-mail address:[email protected]. surface re-expression and mass spectrometry to analyze the peptides Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1213 displayed under these conditions. Our results provide insight into both on 1 ϫ 106 cells as described above. The majority of cells were washed the relative contribution as well as the specificity of proteasome in- twice in PBS at 4°C, snap frozen in liquid nitrogen, and stored at Ϫ80°C hibitor-insensitive proteases that generate class I-associated peptides. for later use. Frozen cell pellets were then resuspended at a concentration of 1 ϫ 108 cells/ml in 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfonate, 20 mM Tris-HCl pH 8.0, 100 ␮M iodoacetamide, 5 Materials and Methods ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin A, 5 mM EDTA, Inhibitors 0.04% sodium azide, and 1 mM PMSF (lysis buffer; all from Boehringer Mannheim, Indianapolis, IN). After rocking for1hat4°C, lysates were 4 Lactacystin (LAC) (Calbiochem, La Jolla, CA) is a Streptomyces metab- clarified by centrifugation at 100,000 ϫ g for 1 h. Lysates were precleared olite that irreversibly inhibits proteasomes via covalent binding to the cat- for4hat4°Cwith recombinant protein A-Sepharose beads (Pharmacia, alytic sites of the ␤ subunits (21, 22). N-acetyl-L-leucinyl-L-leucinal-L-nor- Piscataway, NJ). Class I-specific Ab-saturated recombinant protein leucinal (LLnL; Calbiochem), also known as calpain Inhibitor I, reversibly A-Sepharose beads were then added to lysates for 12 h at 4°C. Beads were inhibits proteasomes as well as several other classes of proteases (23). spun down and lysate was removed to a separate tube. Beads saturated with Carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (z-L3VS) was a gener- Abs of other class I specificities could then be added sequentially to purify ous gift from Dr. H. Ploegh (Harvard University, Cambridge, MA) and has additional alleles. Beads were washed twice in lysis buffer, twice in 50 mM been shown to specifically inhibit proteasomes by binding to all of the Tris-HCl/1 M NaCl (pH 8.0), twice in 50 mM Tris-HCl/250 mM NaCl (pH known active sites and blocking all known in vitro measures of the pro- 8.0), and three times in 50 mM Tris-HCl (pH 8.0).
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