MHC Class I Antigen Presentation Precursors, but Is Not Required for Most Peptidase Needed to Trim Long Antigenic Tripeptidyl Pe

Tripeptidyl Peptidase II Is the Major Peptidase Needed to Trim Long Antigenic Precursors, but Is Not Required for Most MHC Class I Antigen Presentation This information is current as of May 2, 2019. Ian A. York, Nidhi Bhutani, Sophia Zendzian, Alfred L. Goldberg and Kenneth L. Rock J Immunol 2006; 177:1434-1443; ; doi: 10.4049/jimmunol.177.3.1434 http://www.jimmunol.org/content/177/3/1434 Downloaded from

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Tripeptidyl Peptidase II Is the Major Peptidase Needed to Trim Long Antigenic Precursors, but Is Not Required for Most MHC Class I Antigen Presentation1

Ian A. York,2* Nidhi Bhutani,3† Sophia Zendzian,* Alfred L. Goldberg,† and Kenneth L. Rock*

Recent reports concluded that tripeptidyl peptidase (TPPII) is essential for MHC class I Ag presentation and that the proteasome in vivo mainly releases peptides 16 residues or longer that require processing by TPPII. However, we find that eliminating TPPII from human cells using small interfering RNA did not decrease the overall supply of peptides to MHC class I molecules and reduced only modestly the presentation of SIINFEKL from OVA, while treatment with proteasome inhibitors reduced these processes dramatically. Purified TPPII digests peptides from 6 to 30 residues long at similar rates, but eliminating TPPII in cells reduced the processing of long antigenic precursors (14–17 residues) more than short ones (9–12 residues). Therefore, TPPII appears to be the major peptidase capable of processing proteasome products longer than 14 residues. However, proteasomes in Downloaded from vivo (like purified proteasomes) release relatively few such peptides, and these peptides processed by TPPII require further trimming in the endoplasmic reticulum (ER) by ER aminopeptidase 1 for presentation. Taken together, these observations demonstrate that TPPII plays a specialized role in Ag processing and one that is not essential for the generation of most presented peptides. Moreover, these findings reveal that three sequential proteolytic steps (by proteasomes, TPPII, and then ER aminopep- sidase 1) are required for the generation of a subset of epitopes. The Journal of Immunology, 2006, 177: 1434–1443.

ntigen-specific cytotoxic CD8ϩ T lymphocytes recog- peptidase 1 (ERAP1) (9, 10) (also known as ER aminopeptidase http://www.jimmunol.org/ nize short peptides that are bound to MHC class I mol- associated with Ag processing or ERAAP (11)), an IFN-␥-induced A ecules (MHC class I) on the cell surface. The great ma- aminopeptidase in the ER. Trimming of N-extended precursors can jority of the peptides associated with MHC class I are derived from also occur in the cytosol. Although there are many cytosolic amin- cell proteins, most of which are degraded by the proteasome (re- opeptidases, whether they perform specific functions in Ag pre- viewed in Refs. 1–3). Purified 26S and 20S proteasomes degrade sentation, and their relative importance, is presently unclear. proteins to peptides of 3–22 aa long whose length follows a log Recently, several studies have also suggested an important role normal distribution (4, 5). Although 70–80% of proteasome prod- in Ag processing for tripeptidyl peptidase II (TPPII). This enzyme ϳ ucts are too small to serve as MHC class I epitopes, 10% of is an exceptionally large (2–9 megadaltons) cytosolic peptidase by guest on May 2, 2019 peptides are 8–10 residues long, the size required for strong bind- that removes groups of three residues from the N terminus of pep- ing to MHC class I complexes. Another 10–15% of peptides are tide substrates (tripeptidyl exopeptidase activity) and has also been too long to bind directly to MHC class I molecules but may serve reported to have a weak endoprotease activity (12). Recent studies as precursors for MHC class I-binding peptides (4–6). Several have suggested that TPPII may play two roles in MHC class I Ag lines of evidence have shown that proteasome cleavages generate presentation. One reported function is to make the endoproteolytic the C-terminal residue of MHC class I-binding peptides (7, 8). cleavages necessary to generate a presented peptide, as has been

However, mature epitopes can be efﬁciently generated in vivo reported in the processing for HIV Nef73–82 (13). However, it is from N-extended precursors (7, 8). This trimming process requires unlikely that TPPII frequently generates the C-terminal residues, a a free N terminus (8) and is therefore mediated by aminopepti- function normally served by proteasomes. Early studies had sug- dases. One such aminopeptidase which plays an important role in gested that TPPII may even substitute for the proteasome in the Ag presentation in vivo is endoplasmic reticulum (ER)4 amino- degradation of cell proteins and generation of most antigenic peptides (14); however, these suggestions have not been substantiated. A second role proposed for TPPII is in trimming the N-terminal *Department of Pathology, University of Massachusetts Medical School, Worcester, extensions from long precursor peptides (15). It has been sug- MA 01655; and †Department of Cell Biology, Harvard Medical School, Boston MA gested, based on studies in cultured cells with inhibitors, that 02115 among cytosolic peptides, only TPPII can trim peptides longer Received for publication February 8, 2006. Accepted for publication April 28, 2006. than ϳ16 aa (16), consistent with previous biochemical studies The costs of publication of this article were defrayed in part by the payment of page establishing that other cytosolic peptidases (e.g., thimet oligopep- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tidase (TOP) (16) and various aminopeptidases (15)) have little activity against peptides longer than ϳ13–15 residues. Remark- 1 This work was supported by grants from the National Institutes of Health (to K.L.R.). ably, in these experiments inhibiting TPPII reduced Ag presenta- 2 Address correspondence and reprint requests to Dr. Ian A. York, Department of tion to the same extent as did inhibitors of the proteasome. More- Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, over, inhibiting both TPPII and proteasomes had no greater effect, Worcester, MA 01655. E-mail address: [email protected] which led to the surprising conclusion that TPPII was essential for 3 Current address: Department of Molecular Pharmacology, Stanford University School of Medicine, Palo Alto, CA 94305. 4 ␤ ␤ Abbreviations used in this paper: ER, endoplasmic reticulum; TPPII, tripeptidyl somal entry site; TOP, thimet oligopeptidase; 2-m, 2-microglobulin; AAF-amc, peptidase II; HC, heavy chain; siRNA, small interfering RNA; IRES, internal ribo- Ala-Ala-aminomethyl coumarin; VSV, vesicular stomatitis virus.

MHC class I Ag presentation (16, 17). Moreover, because inhib- Small interfering RNA (siRNA) iting TPPII only affected the trimming of peptides longer than 15 siRNA was obtained from Qiagen. TPPII-specific siRNA was 5Ј-AAG residues in length, Reits et al. (16) concluded that proteasomes CAACTCACTGGCCAAATT-3Ј and 5Ј-AATTTGGCCAGTGAGTTGC- must be generating predominately precursor peptides that are 16 3Ј. As a control, we used siRNA directed against murine TOP, which does residues or longer, in clear contrast to findings on the size distri- not have a target sequence in human cells, as previously described (10). bution of peptides produced by purified proteasomes (4–6). It was siRNA directed against ERAP1 has been previously described (10). RNA oligos were annealed and prepared, and cells were transfected with siRNA therefore postulated that intracellular proteasomes behave differ- using Oligofectamine (Invitrogen Life Technologies), as previously de- ently in vivo than they do after purification. These findings led to scribed (10). Based on preliminary experiments, cells were analyzed 3 or a model in which proteasomes make the initial cleavages in Ags to 4 days after transfection. To confirm the efficacy of knockdown by PCR, generate oligopeptides of 16 or more residues, and then TPPII mRNA was collected from cells (RNeasy kit; Qiagen) and PCR was performed using TPPII-specific primers 5Ј-GGTGGGCAAGTCTCAGT plays an essential role in shortening the N-terminal extensions on GAT-3Ј and 5Ј-CATCAAAGCGGTTGATTCCT-3Ј, or control primers these peptides to a size where other aminopeptidases could trim the specific for ERAP1: 5Ј-GGGAGCTGGAGAGAGGCTAT-3Ј and products of TPPII down to mature epitopes or to individual amino 5Ј-CTTGCTTTGAAGGCAGGTTC-3Ј. acids (16, 17). Plasmids In this study, we examine the role of TPPII in trimming peptides for MHC class I Ag presentation. Initial studies on the purified Plasmid pUG1 was constructed using ubiquitin cDNA (provided by L. Whitton, The Scripps Research Institute, La Jolla, CA), modified to have enzyme showed that TPPII is in fact not selective for long pep- SfoI and BamHI sites at the 3Ј end. The internal ribosome entry sequence tides, and that it can trim both long and short ones. In intact cells, (IRES) and GFP sequence from the murine stem cell virus (23) was cloned however, TPPII plays an important role in trimming N-extended downstream of this sequence in a pcDNA6 (Invitrogen Life Technologies)

peptide precursors longer than 14 residues for MHC class I Ag backbone. Oligonucleotides encoding various peptides were cloned into the Downloaded from presentation, presumably because other peptidases cannot efﬁ- SfoI-BamHI sites to produce a ubiquitin fusion protein. The sequences of the peptides expressed in this way are listed in Table I. To produce plasmid ciently digest such long substrates. Elimination of TPPII by siRNA pIG-FLOVA, the full-length OVA gene, was subcloned into pUG1 replac- is shown to result in only a small reduction in the generation of ing ubiquitin so that the IRES-GFP cassette was downstream of OVA. SIINFEKL from OVA and does not reduce the overall supply of Cells were transfected using HeLa Monster (Mirus) as previously de- MHC class I-presented peptides from cellular proteins. Therefore, scribed (10).

TPPII is the primary enzyme that processes peptides that are Flow cytometry http://www.jimmunol.org/ longer than ϳ14 residues, but these are only a small fraction of Presentation of the immunodominant H-2Kb-binding epitope from chicken those released by the proteasome. Thus, its role contrasts sharply OVA was measured by flow cytometry. Hela-Kb cells were transfected with that of the proteasome which generates the great majority of with siRNA and, 3 days later, the cells were transfected with plasmids MHC class I-presented peptides. expressing various SIINFEKL precursors. Twenty-four hours later, the cells were analyzed by flow cytometry, gating on GFP-expressing cells. Transfection efficiency generally was between 10 and 35%, depending on Materials and Methods the construct; samples with very low transfection efficiency (Ͻ2%) were Cells, virus, and Abs not included in analysis. Hela-Kb cells have been described previously (10). The mAb W6/32 rec- Statistical analysis ␤ by guest on May 2, 2019 ognizes human MHC H chain (HC) only when associated with 2-micro- ␤ Samples were compared using two-tailed Student t analysis, and differ- globulin ( 2-m) (18). HC10 recognizes HC only when not associated with ␤ b ␤ ences were deemed statistically significant for p values Ͻ0.05. 2-m (19). B8.24.3 recognizes H-2K associated with 2-m (20). A poly- b ␤ clonal rabbit antiserum that recognizes H-2K not associated with 2-m Immunoprecipitations was a gift from S. Nathenson (Albert Einstein College of Medicine, New York, NY). 25.D1.16 recognizes H-2Kb only in association with SIIN- Cells were starved for1hincysteine/methionine-free medium, metaboli- FEKL (21). cally labeled with 35S, and chased with a 10-fold excess of normal medium. Recombinant adenovirus expressing the HSV TAP-blocking protein In some cases, the proteasome inhibitor MG132 (Calbiochem) was added ICP47, and control virus AdBHG10, have been previously described (22). to medium at 10 ␮M throughout the starve, label, and chase periods. Cells

Table I. Plasmids used in these experiments

Plasmid Name Peptide Sequence Notesa

pUG-S8L SIINFEKL 8-mer; mature epitope pUG-M-S8L M SIINFEKL 9-mer pUG-N2-S8L LE SIINFEKL 10-mer; natural OVA sequence pUG-N3-S8L QLE SIINFEKL 11-mer; natural OVA sequence pUG-N4-S8L EQLE SIINFEKL 12-mer; natural OVA sequence pUG-N5-S8L LEQLE SIINFEKL 13-mer; natural OVA sequence pUG-N6-S8L G LEQLE SIINFEKL 14-mer; natural OVA sequence pUG-N7-S8L SG LEQLE SIINFEKL 15-mer; natural OVA sequence pUG-N8-S8L VSG LEQLE SIINFEKL 16-mer; natural OVA sequence pUG-N9-S8L EVSG LEQLE SIINFEKL 17-mer; natural OVA sequence pUG-N10-S8L DEVSG LEQLE SIINFEKL 18-mer; natural OVA sequence pUG-N1NP-S8L L SIINFEKL 9-mer; upstream sequence from VSV NP pUG-N2NP-S8L DL SIINFEKL 10-mer; upstream sequence from VSV NP pUG-N3NP-S8L SDL SIINFEKL 11-mer; upstream sequence from VSV NP pUG-N4NP-S8L LSDL SIINFEKL 12-mer; upstream sequence from VSV NP pUG-N5NP-S8L SLSDL SIINFEKL 13-mer; upstream sequence from VSV NP pIG-FLOVA Full-length OVA

a VSV NP, vesicular stomatitis virus nucleoprotein. 1436 SPECIALIZED ROLE OF TPPII IN Ag PRESENTATION were lysed with 1% Nonidet P-40 and 0.5% deoxycholic acid in TBS, with Table II. Peptides used to measure trimming by TPPII a protease inhibitor mixture (Roche). Lysates were incubated overnight at 4°C, then clarified and immunoprecipitated with appropriate Abs. Because Peptide Sequence Length folding is inefficient in Hela-Kb cells (as with many nonlymphoid cells), three times as much lysate was use for the “Folded” immunoprecipitate TNKTRRFSAGQASRFSAIDASIDSARFGQY 30 (mAb W6/32) as for the “Unfolded”) (mAb HC10). Immunoprecipitates TNKTRRFAGQARFAIDAIDARFGQY 25 were separated using SDS-PAGE and autoradiographs were scanned and TNKTRRFGQRFIDIDRFGQY 20 quantified using Quantity One software (Bio-Rad). “Percent folded” MHC TNKTRGQRFIDIDRFGQY 18 class I was calculated by adding the intensity of the “Folded” band, to the TNKTRGQRFIDRFGQY 16 intensity of “Unfolded” band multiplied by three. TNKTRGQIDRFGQY 14 TNKTRIDRFGQY 12 Acid wash recovery assay TNKTRIDGQY 10 TNKTRGQY 8 Cells were transfected with siRNA for 3 days, and with plasmid expressing TNKTRY 6 either full-length OVA or SIINFEKL for 2 days. Cells were trypsinized and EVSGLEQLESIINFEKL (“N9-SIINFEKL”) 17 washed, and then treated with acid (1:1 mixture of 0.163 M citric acid and GLEQLESIINFEKL (“N6-SIINFEKL”) 14

0.32 M NaH2PO4 (pH 2.6)) for 60 s, then transferred to a 100-fold excess QLESIINFEKL (“N3-SIINFEKL”) 11 of RPMI 1640 medium with 10% FCS, buffered with HEPES. Cells were SIINFEKL 8 pelleted and resuspended in warm medium, and incubated for various times before analysis by ﬂow cytometry. In some cases, MG132 (10 ␮M) was added to the medium for 90 min before the trypsinization and subsequently through the recovery period. Results

Purified TPPII trims both long and short precursor peptides Downloaded from Purification of TPPII It has been previously suggested that TPPII preferentially degrades TPPII was purified from young rabbit muscle, using high concentrations of peptides greater than 16 residues in length (27). However, this DTT and ammonium sulfate for stabilizing its structure as reported for the Drosophila homolog (24). After the muscles were minced to small pieces, conclusion was based on indirect data obtained with inhibitors and they were homogenized in a buffer containing 50 mM sodium phosphate fluorescent peptides injected into cells (16). The size range of pep-

(pH 7.5), 0.25 M sucrose, 5 mM DTT, 5 mM MgCl2, 2 mM ATP, and 10% tides that are hydrolyzed by TPPII has not been carefully analyzed. ϫ glycerol. The homogenate was centrifuged for 15 min at 10,000 g to To investigate this issue, we analyzed the ability of purified mam- http://www.jimmunol.org/ ϫ remove cell debris and then was centrifuged for1hat100,000 g to malian TPPII to hydrolyze peptides of varying lengths. We first remove membranous fractions. The supernatants were loaded onto a DE-52 column and after washing, were eluted with a linear gradient of 0–400 mM compared the ability of purified TPPII to degrade a homologous ammonium sulfate. The TPPII peak was identified by butabindide-sensitive series of peptides that vary in length from 6 to 30 residues, but activity against Ala-Ala-aminomethyl coumarin (AAF-amc). Active frac- contain the same 5 (or more) N-terminal residues. TPPII is a trip- tions were collected, loaded on a resource Q column (Bio-Rad) after di- eptidyl peptidase that releases with each cleavage tripeptides from the luting the ammonium sulfate concentration to 10 mM and eluted with a linear gradient of 300–0 mM ammonium sulfate. Fractions from the N terminus of the substrate. Therefore, all the peptides tested contain ResourceQ column containing TPPII (i.e., AAF-amc activity that was in- the same initial cleavage site. In addition, all except the shortest sub- hibited by 4 nM butabindide) were concentrated to 1 ml and loaded on a strates share the same three C-terminal residues. The rate of hydro- 38-ml glycerol gradient (23–37% glycerol in 25 mM HEPES (pH 7.5), 5 lysis of these substrates was assayed by measuring the initial rate of by guest on May 2, 2019 mM DTT, 0.5 mM ATP, 5 mM MgCl ). After centrifugation for 22 h at 2 the appearance of new N termini using fluorescamine (25). 100,000 ϫ g, the gradient was fractionated, and the fractions active against AAF-amc were pooled and concentrated. The resulting preparations were TPPII was able to digest peptides in this series that were 16–30 free of proteasomes as shown by the lack of activity against the proteaso- residues in length, in agreement with the in vivo findings of Reits mal substrate Succinyl-Leu-Leu-Val-Tyr-aminomethyl coumarin (Suc- et al. (16). However, the enzyme showed no consistent preference LLVY-amc), and of aminopeptidase activity because the activity against for these long substrates (Fig. 1A). Although there were some dif- AAF-amc was not affected by the general aminopeptidase inhibitor, bestatin. ferences in cleavage rates, they were presumably due to the substrates’ internal sequences. The important point is that all peptides TPPII enzyme assay To measure TPPII activity, cell extracts were prepared by lysing HeLa cells in ice-cold 50 mM sodium phosphate (pH 7.5), 5 mM MgCl2, 2 mM ATP, 1 mM DTT, and 10% glycerol using Dounce homogenization. This homogenate was spun at 10,000 ϫ g for 15 min to remove nuclear and membrane fractions, and contained active proteasomes and TPPII, as tested by activity against specific fluorogenic substrates, Suc-LLVY-amc and AAF-amc. The resulting supernatant fractions were aliquoted, snap-frozen in liquid nitrogen and stored at Ϫ80°C until use. Ten micrograms of Hela extract, in the absence or presence of butabindide (4 nM), were incubated with 100 ␮M of each peptide at 37°C for 2 h, and the new N termini of the peptide products generated were assayed by using fluorescamine as described previously (25, 26). The extracts were preincubated with or without butabindide for 20 min at room temperature. Percent inhibition in each case is calculated from the amount of N termini generated (nanomoles) in the absence of butabindide. To measure activity of TPPII against specific peptides, 100 ␮M of each peptide was incubated with 4 nM of purified TPPII, at 37°C for 1 h, and the FIGURE 1. Pure TPPII cleaves peptides from 6 to 30 aa long. A, TPPII new N termini of the peptides generated were assayed by using fluores- was purified as described in the text, and the degradation of a peptide camine. One set of peptides used for these experiment have five identical N-terminal residues and three C-terminal residues (except for the six-res- library ranging in size from 6 to 30 aa was measured by the generation of idue peptide that has the same five N-terminal residues and the final com- new N termini. B, The degradation of peptides SIINFEKL, QLESIINFEKL mon C-terminal residue) (Table II). Another set of peptides consisted of the (“N3-SIINFEKL”), GLEQLESIINFEKL (“N6-SIINFEKL”), and OVA epitope SIINFEKL with 0, 3, 6, or 9 residues of natural N-terminal EVSGLEQLESIINFEKL (“N9-SIINFEKL”) by purified TPPII was mea- flanking residues (Table II). sured as in A. Data shown are one representative experiment of three. The Journal of Immunology 1437 ranging from 6 to 30 residues were cleaved reasonably well by uitin-N(x)-SIINFEKL fusion protein was followed by an IRES and TPPII. GFP, whose intensity was used to identify cells expressing similar To confirm and extend these results, we also analyzed a series of levels of peptide. SIINFEKL- H-2Kb complex formation was N-extended precursors of the MHC class I-presented OVA epitope quantitatively measured by flow cytometry, using the mAb SIINFEKL that contained 0–9 residues of the natural flanking se- 25.D1.16 (21). We found that, as predicted by Reits et al. (16), quence. Again, TPPII was able to trim precursors that were 17 TPPII knockdown reduced by 60–80% the presentation of residues in length but also ones that were shorter, and showed no SIINFEKL from N-extended SIINFEKL precursors longer than 16 consistent preference for longer peptides (Fig. 1B). In fact, with aa (Fig. 3A). By contrast, TPPII knockdown had little or no effect both these series of substrates, a moderate preference for peptides on the presentation of SIINFEKL when the mature epitope itself 8–14 residues long was observed. was expressed as a ubiquitin fusion protein (Fig. 3A). This finding siRNA reduces levels of TPPII indicates that TPPII knockdown did not affect TAP transport, MHC class I assembly, or other steps in the Ag presentation path- To study the role of TPPII in MHC class I Ag presentation, we way downstream of peptide production, and also confirms that prevented its expression with siRNA. Hela-Kb cells were treated the siRNA treatment effectively reduced TPPII levels. The obser- with control siRNA or with siRNA specific for TPPII. RNA levels vation that TPPII knockdown does not increase the presentation of A were markedly reduced by 3 or 4 days after transfection (Fig. 2 ). SIINFEKL even though in vitro TPPII can trim SIINFEKL (Fig. Although Abs suitable for measuring protein levels of TPPII were 1B) suggests that TPPII is not an important (i.e., rate-limiting) not available to us, we showed that TPPII’s enzymatic activity, enzyme in the degradation of such peptides, which is also consis- assayed with the fluorometric substrate AAF-amc, was reduced by tent with the findings of Reits et al. (16). By contrast, we previ- ϳ90% (Fig. 2B). (This siRNA-mediated reduction in TPPII was Downloaded from ously showed that knockdown of the endopeptidase TOP, which is substantially greater than that achieved by Reits et al. (16) in their a major enzyme for degradation of SIINFEKL in the cytosol, led study of Ag presentation.) The cells remained viable and continued to grow over the 4-day period, and the activities of two other critical to enhanced presentation of SIINFEKL when SIINFEKL or N- cellular proteolytic activities, the proteasome, (measured by Suc- extended precursors were expressed in cells (29). LLVY-amc), and TOP (assayed using McC-PLGPK-Dnp), were not To learn whether TPPII also generates presented peptides from precursors that are shorter than 16 residues, we then tested the affected by the treatment with TPPII-specific siRNA (Fig. 2B). http://www.jimmunol.org/ effect of TPPII knockdown of the processing and presentation of TPPII plays an important role in trimming long antigenic SIINFEKL from precursors extended by 1 to 6 amino acids at the precursors in vivo N terminus (9–15 mer). Although TPPII knockdown had little or Previous studies in cultured cells suggested that TPPII plays a no effect on the presentation of SIINFEKL itself (Fig. 3) or of critical role in processing peptides longer than ϳ16 residues (16) 1ϩSIINFEKL (Fig. 3B), surprisingly, TPPII knockdown consis- to MHC class I-presented peptides. To determine whether TPPII tently reduced presentation from precursors that were shorter than trimming of these long precursors was in fact important in MHC 16 residues. Presentation of SIINFEKL from 14 and 15 mer were class I Ag presentation, we transfected siRNA-treated cells with significantly reduced to about the same extent as 16 mer, and in the plasmids expressing the immunodominant H-2Kb-restricted absence of TPPII presentation of SIINFEKL from the 12 mer by guest on May 2, 2019 epitope from OVA, SIINFEKL, extended by 8 or 9 residues on its (EQLE-SIINFEKL) was significantly reduced by 30–40% below N terminus to yield 16 and 17 mer, fused to the C terminus of levels in controls (Fig. 3B). Interestingly, as with SIINFEKL itself, ubiquitin. Ubiquitin C-terminal hydrolases in cells efficiently re- TPPII knockdown did not significantly increase or decrease the move ubiquitin moieties from such fusion proteins, liberating pep- presentation of SIINFEKL from E-SIINFEKL or LE-SIINFEKL. tides with defined N termini (28). The sequence encoding the ubiq- It was possible that the amino acids upstream of SIINFEKL in these constructs are, for some reason, poorly processed by other cellular aminopeptidases so that TPPII is unusually important in their processing. To address this possibility, we studied presentation of SIINFEKL with N-terminal extensions consisting of the sequence upstream of the immunodominant H-2Kb-restricted peptide RGYVYQGL from the vesicular stomatitis virus (VSV) nucleoprotein. Previous studies have implicated puromycin-specific aminopeptidase and bleomycin hydrolase in generating RGYVYQGL from N-extended precursors (30). However, TPPII knockdown affected the presentation of SIINFEKL from precursors extended by 1–5 aa (L-SIINFEKL through SLSDL-SIINFEKL) to a very similar extent as it did with native SIINFEKL sequence. Presentation of SIINFEKL from 12- and 13-residue precursors LSDL- and SLSDL-SIINFEKL was reduced by ϳ30–40% below control levels (Fig. 3B). Again, TPPII knockdown did not enhance the pre- FIGURE 2. TPPII is specifically knocked down by siRNA treatment. A, sentation of SIINFEKL from precursor peptides as short as 10 Hela-Kb cells were treated with siRNA specific for TPPII (lanes marked residues, although it may have slightly reduced the presentation “TPPII”), or with irrelevant control siRNA (“Ctrl”). After 3 days, mRNA from DL-SIINFEKL for reasons that are unclear. Therefore, while was collected and RT-PCR was performed using primers for TPPII or, as TPPII is particularly important in the processing of peptides longer a control, for ERAP1. B, Hela-Kb cells were treated with siRNA as in A. ϳ After 3 days, cell lysates were prepared and the activities of TPPII and, as than 14 residues, it also plays a role in processing of shorter controls, of proteasome and TOP, were assayed as described in Materials N-extended peptides. These findings in intact cells are consistent and Methods. The activities are expressed as a percent of control siRNA- with our observations that purified TPPII trims both short and long treated cells. Data shown are one representative experiment of three. peptides. 1438 SPECIALIZED ROLE OF TPPII IN Ag PRESENTATION

FIGURE 3. TPPII knockdown reduces Ag presentation from long peptide precursors. Hela-Kb cells were transfected with siRNA specific for TPPII, or Downloaded from with control siRNA. Two days later, the cells were transfected with plasmids expressing, as ubiquitin C-terminal fusions, the peptide SIINFEKL or N-terminally extended precursors of SIINFEKL as well as GFP. One day later, the cells were stained with 25.D1.16 (specific for SIINFEKL bound to H-2Kb) and analyzed by flow cytometry, gating on GFP. A, Traces of GFP-positive cells treated with TPPII siRNA (thick lines) or control siRNA (thin lines) transfected with plasmids expressing SIINFEKL (top panel), EVSGLEQLE-SIINFEKL (N8-SIINFEKL, a 16 mer; middle panel) EVSGLEQLE-SIINFEKL (N9-SIINFEKL, a 17 mer; bottom panel). Shaded traces: cells transfected with empty vector. Data shown are representative of at least three experiments. B, Hela-Kb cells were transfected with siRNA and plasmids as in A. The plasmids expressed SIINFEKL with the indicated N-terminal extensions. Shown Ϯ are mean fluorescent intensities as a percent of control-transfected cells (average SD for at least three experiments per sample). Statistically significant http://www.jimmunol.org/ .(p Ͻ 0.01, Student t test) ءء 0.01Ͻp Ͻ 0.05; Student t test) or) ء differences between TPPII knockdown and control siRNA are indicated with

ERAP1 and TPPII both process SIINFEKL precursors 16 residues (31), these findings are consistent with a model in Although the data above show TPPII contributes to the generation which TPPII is the major enzyme that processes longer peptides to of presented peptides, it is unclear whether it produces the mature a length that other aminopeptidases like ERAP1 can then epitopes or simply generates shorter precursors that are then hydrolyze. trimmed further by other aminopeptidases. To test this possibility, Role of TPPII in generating SIINFEKL from OVA we compared the effects on SIINFEKL presentation of TPPII by guest on May 2, 2019 knockdown to that of knockdown of ERAP1, the IFN-induced Reits et al. (16) interpreted their data to indicate that most MHC aminopeptidase in the endoplasmic reticulum that trims longer pre- class I-presented peptides were initially generated in vivo by pro- cursors to the 8–9 residue presented epitopes. As previously teasomes as long precursors that are predominately longer than 16 shown (10), ERAP1 knockdown has little effect when the mature residues, and therefore required trimming by TPPII. This conclu- SIINFEKL epitope itself is expressed in cells, but it reduces pre- sion is in sharp disagreement with studies of the peptide products sentation of SIINFEKL when even a single amino acid is added to of purified 26S and 20S proteasomes, which mainly yield peptides its N terminus (Fig. 4). ERAP1 was considerably more important shorter than 8 residues (4, 5, 32, 33). It was therefore of interest to than TPPII in the presentation of peptides 13 residues and shorter, evaluate the effect of the TPPII knockdown on the presentation of but the effect of ERAP1 knockout on longer peptides (a 14 mer and SIINFEKL from full-length OVA. We found that TPPII knock- 16mer) was about equal to knockout of TPPII. down only inhibited the presentation of SIINFEKL from the full- We then knocked down ERAP1 and TPPII simultaneously. The length protein by ϳ20–30%, as compared with control-transfected double knockdown reduced levels of ERAP1 and TPPII mRNA cells (Fig. 5A). These results are consistent with our earlier find- about as much as single knockdown (data not shown). Presentation ings that N-extended SIINFEKL precursors are generated during of SIINFEKL from all precursors was slightly less in the double Ag processing (10) and demonstrate that TPPII does play a role in knockdown than when ERAP1 alone was knocked down (Fig. 4). trimming some of these peptides for Ag presentation. However, However, the double knockdown reduced presentation from ma- the silencing of TPPII has a much smaller effect on the presenta- ture SIINFEKL by ϳ10%, slightly more than knockdown of either tion of SIINFEKL from OVA, than it does on presentation from TPPII or ERAP1 alone, although this was not significant (Fig. 4). precursors longer than 15 residues (Fig. 3). The important impli- Because neither knockdown alone affected presentation of cation of this observation is that the majority of SIINFEKL pre- SIINFEKL, this suggests that the double knockdown may have cursors that are generated from OVA in vivo are Ͻ16 residues in more nonspecific effects than either single knockdown, although length. These results are at variance with the models proposed by the nonspecific effects are still quite modest. Even if this is not Reits et al. (16) and Kloetzel (17), but completely consistent with taken into account, there was no significant difference in the effect earlier biochemical studies on isolated proteasomes (4–6). of silencing TPPII with ERAP1 over silencing ERAP1 by itself. Therefore, these peptidases almost certainly act sequentially on the Peptide supply to newly synthesized MHC class I is not reduced same substrate, rather than acting in parallel, redundant pathways. by TPPII knockdown In other words, in the cytosol TPPII must be generating a shorter The finding that TPPII knockdown had quite modest effects on the precursor that is then trimmed in the ER to the mature form by presentation of SIINFEKL from full-length OVA prompted us to ERAP1. Because ERAP1 poorly trims peptides that are longer than ask whether TPPII is generally important in generating peptides The Journal of Immunology 1439

FIGURE 4. ERAP1 is more important than TPPII in trimming N-extended SIINFEKL precursors. Hela-Kb cells were transfected with siRNA specific for TPPII, ERAP1, or both, or with control siRNA. After 2 days, the cells were transfected with plasmids expressing as ubiquitin fusions SIINFEKL, or N-extended SIINFEKL precursors as indicated. One day Downloaded from later, the cells were stained with 25.D1.16 (anti-H-2Kb-SIINFEKL) and analyzed by flow cytometry. Shown are mean fluorescent intensities as a FIGURE 5. TPPII is not essential for Ag presentation. A, Hela-Kb cells percent of control-transfected cells (average Ϯ SD for at least three ex- were transfected with siRNA specific for TPPII, ERAP1, or both, or with periments per sample). Statistically significant differences between TPPII control siRNA. After 2 days, the cells were transfected with a plasmid Ͻ p Ͻ 0.05, Student t test) expressing full-length OVA. One day later, the cells were stained with 0.01) ء and control siRNA are indicated with p Ͻ 0.01); statistically significant differences between TPPII and b) ءء or 25.D1.16 (anti-H-2K -SIINFEKL) and analyzed by flow cytometry. http://www.jimmunol.org/ ERAP1 siRNA are indicated with “†” (p Ͻ 0.01). There was no statistically Shown are mean fluorescent intensities as a percent of control-transfected significant difference in presentation between ERAP1 siRNA, and ERAP1 cells (average Ϯ SD for at least five experiments). Presentation was sta- ϩ TPPII siRNA (p Ͼ 0.05). tistically different between control siRNA and TPPII, between TPPII and ERAP1, and between TPPII and ERAP1 ϩ TPPII siRNA (p Ͻ 0.01, Stu- dent t test), but was not significantly different between ERAP1 and ERAP1 ϩ TPPII siRNA (p Ͼ 0.05). B and C, Hela-Kb cells were transfected with siRNA specific for TPPII, or with control siRNA (“Ctrl”). One day later, for MHC class I molecules. MHC class I HC and ␤ -m normally 2 cells were infected with a recombinant adenovirus expressing the TAP associate in the ER before the antigenic peptide is bound; however, blocker ICP47 (“ICP47”) or mock-infected (“Ctrl”). Three days after this dimeric complex without a bound peptide is relatively unsta- siRNA transfection, the cells were metabolically labeled with [35S]methi- by guest on May 2, 2019 ␤ ble, so that HC and 2-m dissociate at elevated temperatures or onine and cysteine for 15 min, and chased with nonradioactive medium for after prolonged times (e.g., overnight incubations) at 4°C. After 30, 60, or 90 min. Cells were lysed and the lysates were incubated at 4°C the peptide associates with MHC class I to form a mature trimeric overnight, then one-fourth of the lysate was immunoprecipitated with ␤ complex, the association between HC and ␤ -m becomes much HC10 (specific for MHC class I H chains not associated with 2-mn) (“Un- 2 folded”), and three-fourths was immunoprecipitated with W6/32 (specific more stable. Therefore, the amount of stable MHC class I can be ␤ for H chains associated with 2-m; “Folded”). B, Immunoprecipitates were used as a measure of overall peptide supply. separated using SDS-PAGE and autoradiographed. C, Autoradiographs To quantify peptide supply to MHC class I molecules, we met- were scanned and quantified using Quantity One software. Data shown are abolically labeled cells with 35S and lysed them after various chase representative of at least three experiments. periods. To allow dissociation of unstable complexes, we incubated the lysates overnight at 4°C (22), and then immunoprecipitated MHC class I with W6/32 (specific for HC associated with Proteasomes play a much greater role than TPPII in generating ␤ ␤ 2-m) or HC10 (specific for HC not associated with 2-m). As a MHC class I-presented peptides control to confirm that a reduction in peptide supply could be de- It was recently suggested that TPPII was as important as the protected under these conditions, we performed the same procedure teasome in generating MHC class I-presented peptides (16). This on Hela-Kb cells infected for 24 h with a recombinant adenovirus suggestion seemed inconsistent with many prior observations in- expressing ICP47 (22), a herpes simplex protein that binds to TAP dicating a predominant role of the proteasome, as well as with our and blocks peptide transport into the ER (22, 34). As expected, in observation that TPPII has only a modest effect on the presentation cells expressing ICP47, little stable W6/32-reactive MHC class I of SIINFEKL from OVA (Fig. 5A), or of peptides from cellular was present, even though free HC levels were similar to control- proteins generally (Fig. 5, B and C). To directly evaluate the rel- infected cells in which W6/32 reactive (“folded”) MHC class I was ative contributions of proteasomes and TPPII in the presentation of readily detected (Fig. 5, B and C). TPPII siRNA and control SIINFEKL from OVA, we compared the effect of inhibiting pro- siRNA-transfected Hela-Kb expressed very similar levels of W6/ teasomes and of TPPII silencing in HeLa-Kb cells. Because cells 32-reactive and HC10-reactive HC (Fig. 5, B and C). In fact, in cannot be treated with proteasome inhibitors for the lengths of time TPPII knockdown cells, a moderate, but consistent, increase in necessary to measure Ag presentation after transfection (24–48 h) peptide-loaded MHC class I molecules was observed (Fig. 5, B and we used an alternate protocol where surface MHC class I mole- C), presumably because TPPII normally destroys some peptides (9 cules were stripped from Ag-transfected cells by acid treatment, residues or longer) that could otherwise serve as antigenic precur- and allowed to recover in cells lacking either TPPII or proteasome sors or bind to MHC class I molecules. activity. Hela-Kb cells were treated with TPPII-specific, or control, 1440 SPECIALIZED ROLE OF TPPII IN Ag PRESENTATION siRNA for 1 day, then transfected with plasmids expressing vari- detect reduced peptide supply resulting for formation of MHC ous SIINFEKL precursors and incubated for a further 2 days. Cells class I complexes, this finding demonstrates that proteasomes play were then treated with the proteasome inhibitor MG132 followed a much more important role in generating peptides for MHC class by citric acid (pH 2.5) to denature pre-existing MHC class I mol- I than does TPPII. ecules, and allowed to generate new H-2Kb-SIINFEKL complexes. Discussion As expected, the recovery of surface H-2Kb-SIINFEKL on cells It is well-established that proteasomes catalyze the degradation of expressing a SIINFEKL minigene was not affected by MG132 most cytosolic proteins, and that most peptides presented on MHC treatment (Fig. 6A) (because this minigene does not require pro- class I molecules are a byproduct of the continual degradation of teolysis for presentation), or by TPPII knockdown (which did not cell proteins by the ubiquitin-proteasome pathway (1–3). The great affect SIINFEKL presentation: Fig. 3). Therefore, components of majority of the peptides produced by proteasomes are rapidly hy- the Ag presentation pathway other than the proteolysis machinery drolyzed to amino acids by cytosolic peptidases. However, it is were not affected by these treatments. In contrast, MG132 treat- still not well-understood how a small fraction escape complete ment abolished the recovery of H-2Kb-SIINFEKL at the surface of degradation and are processed to the 8–10 residue peptides that cells expressing full-length OVA, while knockdown of TPPII had bind to MHC class I molecules. Proteasomes can directly generate little or no effect (Fig. 6B). the mature epitopes, but isolated proteasomes and especially “im- We conclude that generation of SIINFEKL from full-length munoproteasomes” appear to generate preferentially N-extended OVA is absolutely dependent on proteasomes, but that TPPII is not precursors (35). A number of studies with model peptides have required for this processing. To confirm that this is generally true shown that cellular aminopeptidases are able to process rapidly

for most MHC class I-presented peptides, we analyzed the effect such N-extended peptides to the mature epitopes, and several Downloaded from on overall peptide supply of proteasome inhibition vs TPPII aminopeptidases that can carry out this processing in vivo have knockdown. Assembled vs free MHC class I molecules were im- been identiﬁed (9, 11, 36–38). Nevertheless, it remains unclear munoprecipitated from lysates of radiolabeled cells treated with how important various cellular aminopeptidases are in generating MG132 or TPPII siRNA and peptide occupancy was tested as MHC class I epitopes, or whether they may mainly destroy described above. Again, TPPII knockdown did not reduce (and in epitopes that could otherwise be presented.

fact somewhat increased) the amount of peptide-loaded MHC class Because peptides in intact cells are extremely short-lived (39), http://www.jimmunol.org/ I (Fig. 6, C and D). In contrast, cells treated with MG132 showed most studies of peptide generation and hydrolysis have been con- a marked reduction in W6/32-reactive MHC class I molecules. In ducted in cell lysates or with puriﬁed enzymes. Such studies have addition to further conﬁrming that this experimental approach can demonstrated that 26S and 20S proteasomes generate peptides by guest on May 2, 2019

FIGURE 6. Proteasomes are more important than TPPII in Ag presentation. A and B, Hela-Kb cells were transfected with siRNA specific for TPPII (round symbols) or with control siRNA (square symbols). One day later, the cells were transfected with plasmids expressing (A) SIINFEKL as a ubiquitin fusion or (B) full-length OVA. Two days later, the cells were pretreated for 90 min with proteasome inhibitor MG132 (solid symbols), or mock-treated with DMSO (open symbols), acid-stripped as described in the text, and allowed to recover for 90 min. At intervals, the cells were stained with 25.D1.16 (anti-H-2Kb-SIINFEKL) and analyzed by flow cytometry. Shown are the mean fluorescent intensities (MFI), expressed as a percentage of the maximum (pre-acid-strip) MFI. Maximum H-2Kb-SIINFEKL levels were almost identical in TPPII- or control-siRNA-treated cells expressing SIINFEKL, while on OVA-expressing cells maximum H-2Kb-SIINFEKL levels on TPPII siRNA-transfected cells were ϳ70% those of control siRNA-transfected cells. C and D, Hela-Kb cells were transfected with TPPII-specific or control siRNA. Three days later, the cells were pretreated with proteasome inhibitor MG132 or mock-treated with DMSO, labeled with [35S]methionine and cysteine for 15 min, and chased with nonradioactive medium for 90 min; MG132 or DMSO was present throughout the label and chase periods. Cells were lysed and the lysates were incubated at 4°C overnight, then immunoprecipitated with HC10 ␤ ␤ (specific for MHC class I H chains not associated with 2-m) (“Unfolded”) or with W6/32 (specific for H chains associated with 2-m) (“Folded”). C, Immunoprecipitates were separated using SDS-PAGE and autoradiographed. D, Autoradiographs were scanned and quantified using Quantity One software. Data shown are representative of at least three experiments. The Journal of Immunology 1441 ranging from 3 to ϳ24 residues long, with the great majority being Hela-Kb cells did not cause any reduction in the presentation of Ͻ8 aa long and ϳ90% smaller than 15 residues. This size distri- SIINFEKL from a 16-mer precursor (N8-SIINFEKL) (data not bution appears to result from a kinetic competition between further shown), although presentation from this precursor was markedly cleavages by the active sites and the ability of small peptides to reduced by TPPII siRNA (e.g., Fig. 3A), and in the experiments of diffuse out the gated exit channel in its outer ␣-ring (40, 41) (4–6). Reits et al. (16), 16-mer degradation was dependent on TPPII (16). Recently, Reits et al. (16) reported that inhibitors of TPPII activity Yet another difference is that we used different cell types. Al- reduced surface levels of MHC class I molecules to the same de- though it is perhaps possible that proteasomes in different cell gree as did proteasome inhibitors. The important implication of types generate peptides with different lengths, this seems quite this finding was that the proteasome and TPPII were both required unlikely and is inconsistent with these particles being highly con- to generate most presented peptides. Because the same study also served between cell types and our present understanding of their found that TPPII was essential in vivo to cleave injected fluores- function. cent peptides that were longer than 16 residues, but not shorter The substrate size preference of TPPII had not been previously ones, it was also proposed that in intact cells (as opposed to in defined. We find that purified mammalian TPPII efficiently digests vitro) proteasomes predominately generate peptides that are longer both short and long peptides. In fact, TPPII hydrolyzes short pep- than 16 residues, which are then trimmed by TPPII for Ag pre- tides somewhat better than longer ones (Fig. 1). This contrasts with sentation (16, 17). Here, we show that while TPPII may contribute our findings and those of others (16) that reducing TPPII activity moderately to Ag presentation from some proteins (e.g., OVA), in cells affects the trimming of longer peptides more than shorter TPPII is not essential for the bulk of MHC class I Ag presentation, ones. This difference probably has nothing to do with the proper- and that proteasomes in intact cells most likely generate shorter ties of TPPII, but arises because other peptidases in cells can trim

peptides, as seen with the purified particles. rapidly short peptides but not long ones. For example, the primary Downloaded from Consistent with the observations of Reits et al. (16), we find that endopeptidase, TOP, has little activity against peptides longer than knocking down TPPII with siRNA does markedly reduce Ag pre- 15 mer (42), and known aminopeptidases primarily hydrolyze pep- sentation from long precursors: in our experiments, peptides longer tides that are ϳϽ6 residues long (26, 31, 43). In other words, in than ϳ14 residues show significant dependence on TPPII. We do vivo TPPII is the rate-limiting enzyme for hydrolyzing long pep- not know whether the remaining presentation of these peptides tides but not shorter ones. These data are consistent with previous ϳ

after TPPII knockdown is due to residual TPPII activity ( 10% of findings (16), although it is important to note that these observa- http://www.jimmunol.org/ control-treated cells) or whether other peptidases can inefficiently tions do not imply that TPPII preferentially digests longer peptides process these peptides. However, peptide supply to newly synthe- (17) but only that TPPII is the major enzyme that can trim these sized MHC class I is not generally reduced when TPPII is knocked peptides, while most cell peptidases strongly prefer shorter ones, down (Fig. 5). In fact, there is a small, but consistent, increase in consistent with the length of most peptides that are generated by peptide supply to MHC class I after TPPII knockdown, presum- proteasomes. ably because TPPII (like other peptidases (10, 29)) destroys some We also observed some effect of TPPII knockdown on relatively peptides (e.g., 8, 9, or 10 mer) that could otherwise bind to MHC short (11–13 residue; 3–5 residue N-terminal extensions) precur- class I by trimming them until they are too short to bind to MHC sors of SIINFEKL. Loss of TPPII reduced their presentation mod- class I molecules. Therefore, the generation of most peptides does estly, and this reduction did not depend on the sequence of the by guest on May 2, 2019 not require TPPII either in place of the proteasome (16, 17) or N-terminal extension. This result is consistent with the ability of downstream of this particle. purified TPPII to hydrolyze peptides in this size range (Fig. 1). We In addition to analyzing overall peptide supply to multiple HLA also note a very small effect on precursors with a 2-residue N- class I molecules, we examined the effect of TPPII knockdown on terminal extension (a reduction of ϳ15%, p ϭ 0.045 for the DL- the presentation of a specific peptide, SIINFEKL, the immuno- SIINFEKL precursor; not statistically significant for the LE- dominant H-2Kb-binding peptide from OVA. Reducing TPPII lev- SIINFEKL precursor). This is not consistent with studies of els reduced the presentation of SIINFEKL from full-length OVA, purified TPPII: because TPPII removes N-terminal amino acids in but the effect was quite modest, in contrast to its large effect on 14 groups of three, it converts these 10 mer in vitro to IINFEKL (data mer and longer precursor peptides. This is consistent with the pre- not shown), which does not bind to H-2Kb. One possibility is that vious finding that ϳ80% of potential SIINFEKL precursors pro- in intact cells TPPII has different activities that have not been duced by purified proteasomes from OVA are shorter than 15 res- detected as yet, perhaps because of accessory factors that might be idues, and virtually all are shorter than 16 residues (35). In lost during purification. It is also possible that TPPII directly or contrast, the proteasome inhibitor MG132 dramatically reduced indirectly alters the effects of other peptidases in intact cells. In any SIINFEKL presentation from full-length OVA. Taken together, case, the magnitude of this effect is quite small. these observations strongly suggest that proteasomes in intact Somewhat surprisingly also, TPPII knockdown in intact cells ex- cells, as in vitro, generate relatively short peptides, that are not pressing SIINFEKL itself did not alter presentation of SIINFEKL dependent on TPPII for further processing. itself. Because purified TPPII is able to digest SIINFEKL efficiently, Why might our findings differ from those of Reits et al. (16)? we had expected that knocking down TPPII activity would en- One difference is that we tested peptide supply to MHC class I by hance SIINFEKL presentation by reducing its destruction in the measuring the stability of newly synthesized MHC class I and by cytosol. The lack of effect argues that TPPII is not a rate-limiting Ag presentation assays, both more direct tests of peptide supply activity for the destruction of SIINFEKL, consistent with the no- than the measurement of cell surface expression of MHC class I tion that TPPII is not rate-limiting for peptides shorter than ϳ14 used by Reits et al. (16). Another difference is in the methods of mer. Previous experiments have shown that TOP normally de- TPPII inhibition. It is possible that the TPPII inhibitor butabindide, stroys a significant amount of SIINFEKL (29), and that amino- used by Reits et al. (16) in most experiments or the treatments to peptidases (44) may also contribute to SIINFEKL destruction. facilitate its entry (e.g., serum deprivation), may affect cells in The present studies have further documented the important role ways unrelated to TPPII inhibition, especially at the very high in processing SIINFEKL precursors played by ERAP1. Although concentrations required to treat intact cells. In our hands, multiple knockdown of ERAP1 did not alter presentation of SIINFEKL experiments using butabindide as described by Reits et al. (16) on from the mature epitope, it drastically reduces presentation from 1442 SPECIALIZED ROLE OF TPPII IN Ag PRESENTATION peptides with even a single N-terminal extension (10) (Fig. 4). In 7. Craiu, A., T. Aklopian, A. L. Goldberg, and K. L. Rock. 1997. Two distinct fact, ERAP1 knockdown affected presentation of SIINFEKL from proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. Proc. Natl. Acad. Sci. USA 94: 10850–10855. a 9 mer and an 11 mer much more than did TPPII knockdown, 8. Mo, X. Y., P. Cascio, K. Lemerise, A. L. Goldberg, and K. Rock. 1999. Distinct while the effect on a 13 mer or a 16 mer (Fig. 4) or 17 mer (N9- proteolytic processes generate the C and N termini of MHC class I-binding pep- S8L) (data not shown) was similar for both, consistent with the tides. J. Immunol. 163: 5851–5859. 9. Saric, T., S. C. Chang, A. Hattori, I. A. York, S. Markant, K. L. Rock, hypothesis that TPPII is particularly important for trimming rela- M. Tsujimoto, and A. L. Goldberg. 2002. An IFN-␥-induced aminopeptidase in tively long peptides (16), and that ERAP1 only trims peptides from the ER, ERAP1, trims precursors to MHC class I-presented peptides. Nat. Im- 9 to 16 mer if they are transported into the ER. These experiments munol. 3: 1169–1176. 10. York, I. A., S. C. Chang, T. Saric, J. A. Keys, J. M. Favreau, A. L. Goldberg, and also resolved whether TPPII and ERAP1 might act sequentially on K. L. Rock. 2002. The ER aminopeptidase ERAP1 enhances or limits antigen longer peptides, with TPPII in the cytosol first converting longer presentation by trimming epitopes to 8–9 residues. Nat. Immunol. 3: 1177–1184. peptides into shorter N-extended peptides that ERAP1 could then 11. Serwold, T., F. Gonzalez, J. Kim, R. Jacob, and N. Shastri. 2002. ERAAP cus- tomizes peptides for MHC class I molecules in the endoplasmic reticulum. Na- trim, and ERAP1 making the final cuts to generate SIINFEKL in ture 419: 480–483. the ER or, alternatively, whether TPPII and ERAP1 acted in 12. Geier, E., G. Pfeifer, M. Wilm, M. Lucchiari-Hartz, W. Baumeister, parallel trimming a distinct set of peptides. In the latter case, K. Eichmann, and G. Niedermann. 1999. A giant protease with potential to substitute for some functions of the proteasome. 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L. Peitrequin, N. Levy, A. Bachi, U. Hellman, the mature epitopes. B. J. Van den Eynde, and C. Servis. 2002. The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in The present and previous work together suggest the following the cytosol is mediated by two peptidases. J. Immunol. 169: 4161–4171. model. Peptides generated by proteasomes are predominately Ͻ16 16. Reits, E., J. Neijssen, C. Herberts, W. Benckhuijsen, L. Janssen, J. W. Drijfhout, aa long. A smaller fraction of peptides produced that are longer and J. Neefjes. 2004. A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 20: 495–506.

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