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Major histocompatibility complex (MHC) class I molecules bind alarge variety of peptides that are generated from the vast majority ofcellular proteins and transport these epitopes to the cell surface for dis-play to the immune system1–4. This process allows CD8+ T lympho-cytes to identify and eliminate cells that are synthesizing abnormal or‘foreign’ proteins, as may arise through mutations or infection byviruses. MHC class I molecules can present a large repertoire of pep-tides because their peptide-binding grooves interact strongly with onlya few amino acid side chains of the peptide and also with aspects thatare present on all peptides, such as the terminal α-amino (N) and car-boxy (C) groups5. A consequence of binding the N- and C-terminalends is that MHC class I molecules stably bind only peptides of a pre-cise and uniform length5. Thus, some MHC class I molecules bindmainly peptides of eight residues, whereas others bind mainly nine-residue or ten-residue peptides. Therefore, for complete surveillanceof proteins present in the intracellular milieu, cells must generate pep-tides of eight to ten residues from nearly all cellular proteins, therebycreating a wide range of binding motifs that are represented among thehundreds of MHC class I alleles. This article reviews the understand-ing at present of how these MHC class I–presented peptides are pro-duced and the peptidases that act after the proteasome and influenceantigen presentation.

The ability to synthesize and recognize MHC molecules arose earlyin the evolution of vertebrates. To generate the MHC class I–presentedpeptides, nature took advantage of the two main cellular proteolyticsystems that are phylogenetically much older than vertebrates. Alleukaryotic cells continuously turn over their proteins to eliminatedamaged, unfolded or incomplete proteins6, to regulate cellularprocesses and to adapt to new conditions. These pathways provide aconvenient library of peptides derived from the great majority of cellu-lar proteins that can be sampled by MHC class I molecules4.

The degradation of most cellular proteins occurs by the ubiquitin-proteasome pathway3,7,8. The initial step in this process is the conju-gation of the polypeptide cofactor ubiquitin to the ε amino group oflysines found in the protein substrate, and subsequently other ubiqui-tin molecules are linked to the first ubiquitin molecule. A chain offour or more ubiquitin molecules serves as a molecular ‘tag’ thatmarks the protein for rapid degradation by the 26S proteasome. The26S proteasomes are composed of a core cylinder, the 20S protea-some, which in cells is capped at each end by a 19S complex of sub-units that recognize and unfold ubiquinated substrates to controltheir access into the 20S core9. In the 20S core, six peptidase sites acttogether to cleave the protein into many diverse oligopeptides,although the precise location of the cleavages vary widely with eachmolecule degraded9. This process creates a very large number (per-haps hundreds) of different peptides, depending on the length andsequence of the protein.

It is now apparent that hydrolysis of proteins by proteasomes is thekey step in the generation of most antigenic peptides9. Treating cellswith highly specific proteasome inhibitors not only blocks the degra-dation of most cellular proteins but also blocks most MHC class I

1Department of Pathology, University of Massachusetts Medical Center,Worcester MA 01655, USA. 2Department of Cell Biology, Harvard MedicalSchool, Boston 02115, Massachusetts, USA. Correspondence should beaddressed to I.A.Y. (ian.york@umassmed.edu).

Published online 28 June 2004; doi:10.1038/ni1089

Post-proteasomal antigen processing for major histocompatibility complex class I presentationKenneth L Rock1, Ian A York1 & Alfred L Goldberg2

Peptides presented by major histocompatibility complex class I molecules are derived mainly from cytosolic oligopeptidesgenerated by proteasomes during the degradation of intracellular proteins. Proteasomal cleavages generate the final C terminusof these epitopes. Although proteasomes may produce mature epitopes that are eight to ten residues in length, they more oftengenerate N-extended precursors that are too long to bind to major histocompatibility complex class I molecules. Such precursorsare trimmed in the cytosol or in the endoplasmic reticulum by aminopeptidases that generate the N terminus of the presentedepitope. Peptidases can also destroy epitopes by trimming peptides to below the size needed for presentation. In the cytosol,endopeptidases, especially thimet oligopeptidase, and aminopeptidases degrade many proteasomal products, thereby limitingthe supply of many antigenic peptides. Thus, the extent of antigen presentation depends on the balance between severalproteolytic processes that may generate or destroy epitopes.

MAKING PEPTIDES FOR PRESENTATION©

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antigen presentation of peptides derivedfrom cytosolic proteins10–13. However, thoseinhibitors do not reduce the presentation ofpeptides that are already of the correct lengthto bind to MHC molecules and that requireno further proteolysis11,14,15 (Fig. 1). Thus,those agents inhibit antigen presentation byblocking the generation of either the pre-sented peptides or longer precursor peptides.

Although proteasomes are required forantigen presentation, they actually destroymany more epitopes than they generate.When purified 20S or 26S proteasomes areincubated with protein substrates, most pep-tides produced range in size from about 2 to25 amino acids16,17. Only a small fraction ofthese peptides (about 15%) are the correctsize for antigen presentation, and most(∼ 70%) are too short to bind to MHC class I molecules16,17. Purifiedproteasomes, when degrading ovalbumin, destroy the immunodomi-nant epitope SIINFEKL (ovalbumin amino acids 257–264) more than90% of the time18 (Fig. 2a). Therefore, the generation of antigenicpeptides by proteasomes seems to be a stochastic process, and most ofthe time proteasomes cleave within epitopes.

Proteasomes produce N-extended epitope precursorsOne fundamental question about the function of the proteasome inantigen processing is whether it makes the cleavages that generate thepresented peptides directly or whether it generates longer precursorsthat require further proteolytic processing. Many early in vitro stud-ies found that the mature epitope and extended precursors could begenerated by purified proteasomes19–21, but those studies were notquantitative and were done under nonphysiological conditions.Thus, it was unclear how often proteasomes generate long precursorsversus mature epitopes. When purified 20S or 26S proteasomes areincubated with protein substrates in more native conditions and theproducts are analyzed with quantitative assays, the proteasomes arefound to generate more long peptides than peptides of the correctsize required for binding to MHC class I molecules16,17. Moreover,when purified 26S proteasomes degrade denatured ovalbumin mole-cules, the majority (∼ 70%) of the SIINFEKL-containing peptidesproduced have N-terminal extensions18 (Fig. 2b).

The nature of the peptides produced by proteasomes changes afterexposure of cells to proinflammatory cytokines that enhance overallantigen presentation. When cells are treated with interferon-γ(IFN-γ),new catalytic subunits are incorporated into the proteasome to gener-ate immunoproteasomes9. Purified immunoproteasomes degradeproteins at rates similar to those of constitutive particles, but they pro-duce from ovalbumin increased amounts of N-extended SIINFEKLpeptides, whereas the amount of the mature SIINFEKL epitope pro-duced by immunoproteasomes is the same as that produced by constitu-tive proteasomes18 (Fig. 2). Similarly, in vitro experiments have shownthat immunoproteasomes generate peptides from enolase that seem, onaverage, to be one residue longer than the products of constitutive pro-teasomes17, although such a size difference has not been noted withother substrates18. Similarly, for many19,20 but not all epitopes21, N-extended precursors are the main products generated in vitro by 20Sproteasomes from human lymphoblastoid cell lines, in which immuno-proteasomes typically predominate. The limited data available suggestthat in all conditions, long precursors are likely to be the main form ofepitope produced by proteasomes. This tendency may be enhanced in

viral infections when cells are exposed to IFN-γ. However, systematic invitro studies with other protein substrates are needed to determine if 26Sproteasomes and especially immunoproteasomes generate mainly N-extended precursors.

Unfortunately, it is not possible to directly characterize and quan-tify the oligopeptides produced by proteasomes in living cells,because nearly all peptides are rapidly destroyed in the cytosol5,6,22.Although it is dangerous to generalize from the very few quantita-tive in vitro studies to the nature of the proteasomal products pro-duced from diverse proteins in vivo, the experiments describedbelow make it clear that a substantial fraction of epitopes are gener-ated as longer precursors in vivo23,24. Longer precursors have beenfound in cell extracts bound to heat shock proteins, such as hsp70,hsp90 and gp96 (refs. 25,26). Thus, it seems likely that in vivo, as in vitro, most presented peptides must be produced initially as N-extended precursors.

Precursor peptides can be trimmed in vivoAs proteasomes generate many peptides that are too long to bindstably to MHC class I molecules, an important question is whethercells can actually trim and present these extended peptides. Toinvestigate this, antigenic peptides with extra C- or N-terminalresidues were injected into cells or were expressed from ‘minigenes’in cells, and presentation of the mature epitope on MHC class Imolecules was measured11,14,15. Both C- and N-extended versionscould be efficiently trimmed to the correct epitope and presented onMHC class I molecules (Fig. 1). However, the addition of protea-some inhibitors completely blocked the presentation of peptidesfrom constructs with even a single extra C-terminal residue. Asexpected, these agents failed to affect the presentation of constructsthat were already the correct size for presentation11,14,15 (Fig. 1).These results suggested proteasomes are the only proteases in cellsthat can generate the proper C terminus of these peptides fromlonger precursors. These results also indicate that the cytosol lackscarboxypeptidases that remove extra residues from the C terminusof peptides, and indeed C-terminal trimming activity has not beendetected in cell extracts27.

In contrast, proteasome inhibitors do not decrease the presentationof peptides from constructs that have from 1 to more than 24 extra N-terminal residues11,15 (Fig. 1). However, presentation from N-extended precursors expressed in cells from minigenes is blocked byacetylation of the terminal (α) amino group on their N terminus14.This modification prevents cleavage by aminopeptidases but not by

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Figure 1 Generation of the termini of MHC class I–binding peptides. Proteasome inhibitors block the production of MHC class I–binding peptides from full-length proteins and from peptides that areextended at the C terminus by even one amino acid. Proteasome inhibitors do not affect presentationof mature epitopes (with no extra N- or C-terminal amino acids) or of MHC class I–binding peptideswith the correct C terminus but with as many as 20 amino acids at the N terminus. Therefore,proteasomes are required for the generation of the C terminus but not the N terminus of MHC class Iepitopes. Green bars, amino acid sequence that binds MHC class I; blue lines, amino acid sequencesthat must be removed before MHC class I binding; ‘no’ icons, no presented epitope.

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proteasomes or other endopeptidases. The presentation of peptidesderived from N-extended constructs is as rapid as that for maturepeptides that do not require trimming. Thus, there must be intracel-lular aminopeptidases that rapidly trim these N-extended precursorsto the proper length for MHC binding.

Peptide trimming in the ERA small fraction of the peptides produced by proteasomes escapedestruction in the cytosol and is transported by the transporter asso-ciated with antigen processing (TAP) into the endoplasmic reticulum(ER), where these peptides can bind to MHC class I molecules. TAPtransports peptides that range in length from about 7 to more than 20amino acids and can therefore import into the ER both mature epi-topes and longer precursors28. In some cases, TAP transports thelonger precursors more rapidly than the mature epitope29–31. Thus, inprinciple, precursor peptides can be trimmed in the cytosol and/orthe ER. Experimental evidence suggests that N-terminal trimmingoccurs in both of these subcellular compartments27,32–38.

Secreted or membrane proteins are normally targeted to the ER byN-terminal signal sequences, which direct their transport through atransmembrane channel, the SEC61 complex, after which the signalsequence is usually removed by the ER enzyme signal peptidase39.Similarly, minigenes that express peptides with N-terminal signalsequences are delivered through SEC61 directly into the lumen of theER, in a TAP-independent process. Such ER-targeted peptides can bepresented even if they are transported as N-extended precur-sors11,35,37,38,40. In addition to these model constructs, TAP-negativecell lines naturally present some peptides that are generated from sig-nal peptides. Although the signal peptidase liberates the signal pep-tide, these epitopes have N-terminal flanking residues that must beremoved, almost certainly by trimming in the ER41,42.

Isolated microsomes contain luminal aminopeptidase activities thatcan trim N-terminal residues from peptides38,43–45. One such microso-mal peptidase was purified23,46 and was found to be identical to anaminopeptidase that had been isolated before but whose immunologi-cal relevance had not been recognized. This peptidase is a zinc-contain-ing metalloprotease that is most closely related to the ‘M1/gluzincin’family of peptidases. It had been called adipocyte-derived leucine

aminopeptidase47, aminopeptidase regulator of TNFR1 shedding48 orpuromycin-insensitive leucine aminopeptidase49 and was renamed ERaminopeptidase 1 (ERAP1)23,50 or ER-associated aminopeptidase(ERAAP)46 to more accurately reflect its properties. As would beexpected of a peptidase involved in MHC class I antigen presentation,ERAP1 is expressed in most cells32,49. Moreover, its expression isincreased in cells treated with IFN-γ, a potent stimulator of MHC class Ipresentation46,50.

Like many other aminopeptidases, ERAP1 is able to remove fromthe N terminus of peptides nearly all amino acids except those fol-lowed by a proline46. One of its unexpected properties is that ERAP1strongly ‘prefers’ peptide substrates that are 9–16 residues in length(S.-C. Chang, N. Bhutani and A.L.G., unpublished data), which cor-responds exactly to the lengths of peptides transported selectively byTAP28,51. ERAP1 also has the unique property of rapidly trimmingN-extended antigenic peptides to eight or nine residues, and thenfurther cleavages occur much more slowly or cease completely23.The peptides produced by purified ERAP1 are therefore preciselythe length of epitopes that are bound by most MHC class I mole-cules1. In contrast, known aminopeptidases continually cleave N-terminal residues from small peptides (preferentially those less thanfive residues) until only free amino acids remain. ERAP1, unlikeother aminopeptidases, monitors the nature of the C-terminalresidue and somehow measures the distance from this C terminus tothe cleaved N terminus. ERAP1 strongly prefers peptides withhydrophobic C-terminal residues (S.-C. Chang, N. Bhutani andA.L.G., unpublished data). Exactly how this enzyme’s active sitessequentially remove N-terminal residues that are 9–16 residuesaway from the C terminus remains a mystery. Further insight intothe structure if ERAP1 should elucidate this unusual length depen-dence and substrate specificity, which are of immunological impor-tance because these preferences influence which peptides aretrimmed and available for presentation.

Decreasing ERAP1 abundance with small interfering RNAs(siRNAs) considerably reduces antigen presentation of N-extendedSIINFEKL constructs targeted to the ER23,46. Therefore, at least forSIINFEKL precursors, ERAP1 seems to provide the only importantpeptide-trimming enzymatic activity in the ER found in human HeLa

Figure 2 Proteasomes generate MHC class I–binding peptides inefficiently. (a) Most ovalbumin molecules degraded by purified proteasomes do not yieldthe H-2Kb-binding peptide SIINFEKL or its precursor peptides. Degradation by IFN-γ-induced immunoproteasomes produces SIINFEKL or its precursorsmore often, but still destroys nearly 90% of potential SIINFEKL epitopes. Green bars, amino acid sequence that binds MHC class I; blue lines, amino acidsequences that must be removed before MHC class I binding. (b) Purified proteasomes produce more peptides containing SIINFEKL (S) with N-terminalextensions of one to seven amino acids (1 + S to 7 + S) than SIINFEKL itself. Immunoproteasomes produce about the same number of SIINFEKLmolecules, but make many more N-extended SIINFEKL-containing peptides18.

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tumor cells and mouse fibroblast cells, as has also been suggested bybiochemical studies of rat liver50. Reducing ERAP1 abundance in vivoalso decreases the presentation of epitopes generated from severalantigens that are known or presumed to be initially cleaved by protea-somes23,46. For example, loss of ERAP1 reduces the presentation ofSIINFEKL from full-length ovalbumin by about 70% (ref. 23). Thisfinding correlates well with the in vitro observation that about 70% ofSIINFEKL-containing precursors are N extended27.

The overall influence of ERAP1 on peptide supply for antigenpresentation can be estimated by measuring the transport of MHCclass I alleles to the cell surface, as this transport is dependent onpeptide binding. For some alleles, for example, H-2Kk and H-2Ld,elimination of ERAP1 reduces the peptide supply by 20–45% (ref.46). However, the expression of several other MHC class I alleles isnot reduced. For example, in constitutive conditions, H-2Kb isunaffected by ERAP1 silencing23. However, in IFN-γ-treated cells,ERAP1 silencing does reduce the surface expression of H-2Kb mol-ecules23. This finding indicates that after IFN-γ stimulation, thelack of ERAP1 causes a reduction in the overall supply of antigenicpeptides, presumably because there are more N-extended peptidesin the ER that must be trimmed by ERAP1 to the proper size tobind to MHC class I molecules. An increase of N-extended precur-sors in the ER may occur because IFN-γ increases the content ofimmunoproteasomes, which produce more N-extended precur-sors, and of TAP, which preferentially transports peptides morethan eight or nine residues in length28,51. However, other possiblemechanisms may also account for the greater importance of ERAP1after IFN-γ treatment.

In addition to ERAP1, there may be other aminopeptidases in theER that are involved in peptide trimming. Additional aminopepti-dase peaks have been found in microsomal preparations50. Thesource of one such ER enzymatic activity has recently been identi-fied, leukocyte-derived arginine aminopeptidase (L-RAP, alsocalled ER-aminopeptidase 2 (ERAP2)), an aminopeptidase highlyhomologous to ERAP1 that is also induced by IFN-γ stimulation52.ERAP2 preferentially cleaves basic dipeptides52 but can removenearly all N-terminal residues from oligopeptides (S.-C. Chang andA.L.G., unpublished data). Thus, ERAP2 may also be important inantigen processing, although its function remains to be deter-mined. ERAP2 has a much more limited pattern of expression intissues than ERAP1 (ref. 52). In the absence of IFN-γ stimulation,ERAP2 is absent from HeLa cells, whereas ERAP1 is abundant50.The identity of the other microsomal peptidases, whether they aregenuinely localized in the ER or represent cytosolic contaminantsand whether they are involved in antigen processing or degradationremain to be determined.

After the discovery that MHC class I molecules bound peptides of auniform size, it was proposed that MHC class I molecules might firstbind precursor peptides and then serve as a template that limits thetrimming of the bound peptide to the proper size53,54. Evidence hasbeen presented in support of this model44; however, it is difficult todistinguish whether trimming occurs on the MHC class I molecule orwhether the MHC class I molecules simply bind and protect peptidesof the proper size from destruction. It is not apparent that the activesite of a peptidase is able to access and trim a long peptide bound toan MHC class I molecule55,56. Moreover, the discovery that ERAP1rapidly trims peptides to a length of eight or nine residues and thenstops23 indicates that MHC class I molecules are not needed for thegeneration of peptides of the proper size. Nevertheless, it remains tobe determined whether there is any cooperation between ERAP1 andMHC class I molecules in peptide trimming.

In addition to ERAP1 and ERAP2, the protease furin has beenlinked to the generation of some MHC-presented peptides in thesecretory compartments57,58. This endoprotease is a member of thesubtilysin family that cleaves after polybasic motifs in the processingof neuropeptides and hormones in the Golgi appartus59. Such a motifis present in the secretory form of the hepatitis B virus C gene prod-uct57,58 . Epitopes fused with the C-terminal region of this antigen arepresented independently of TAP and proteasome function57,58.Presentation of such epitopes is enhanced by overexpression offurin58 and is inhibited by the furin-specific inhibitor decRVKR-CMK57. As the presentation of most epitopes requires proteasomesand TAP, furin presumably has only a minor function in the genera-tion of antigenic peptides. Whether furin is involved in the trimmingof TAP-transported peptides is unknown, although the limitedsequence specificity and subcellular location of furin must limit thenumber of possible substrates59.

Peptide trimming in the cytosolCertain N-extended antigenic precursors are trimmed in the cytosolbefore being transported by TAP into the ER. This conclusion wasfirst suggested by the observation that the mature SIINFEKL epi-tope is efficiently presented on MHC class I molecules when SIIN-FEKL is linked to a 25-residue N-terminal extension, even whenproteasome function is eliminated11. Because TAP transports pep-tides of this length poorly if at all60, it is most likely that the 32-residue peptide was trimmed before uptake into the ER.Subsequently, it was found that the presentation of some antigens isreduced by protease inhibitors that block the activity of peptidasesin the cytosol but not in the ER32,61–63.

In cytosolic extracts there are several peptidases that can trim anti-genic peptides. These enzymes seem to exist free in solution, ratherthan as a large multienzyme complex. An IFN-γ-inducible peptidasethat could trim N-extended SIINFEKL was purified from HeLa cytosoland was identified as leucine aminopeptidase27. This zinc metallopep-tidase is normally present in many tissues but is synthesized inincreased amounts after IFN-γ stimulation. Puromycin-sensitiveaminopeptidase, a zinc metallopeptidase, and bleomycin hydrolase, acysteine protease, are two other cytosolic aminopeptidases that wereshown to trim a vesicular stomatitis virus nucleoprotein precursorpeptide in cytosolic extracts derived from human Epstein-Barrvirus–transformed B lymphoblastoid cell lines32. Puromycin-sensitiveaminopeptidase and bleomycin hydrolase are constitutively expressedin most cells and are not induced by IFN-γ. Tripeptidyl peptidase II(TPPII) has been linked to the generation of some antigenic pep-tides9,24,64. This proteolytic enzyme is a very large (greater than 1 MDa)multimeric complex that that removes three residues at a time fromthe N terminus of substrates, provided the N terminus is notblocked65, and may also have some endoproteolytic activity66. In cellextracts, TPPII acts sequentially with puromycin-sensitive aminopep-tidase to trim a long precursor to the final mature epitope67. BecauseTPPII has a strong preference for peptides more than 15 residues inlength (ref. 24 and P. Venkatraman and A.L.G., unpublished data), itmay have a specific function in the processing of very long proteaso-mal products.

An unresolved question is whether these different cytosolicaminopeptidases have general functions in trimming peptides forantigen presentation in vivo. At present there are insufficient data todraw firm conclusions. Injection of leucine aminopeptidase into cellsresults in increased trimming of peptides22. Puromycin-sensitiveaminopeptidase and bleomycin hydrolase are inhibited by Ala-Ala-Phe-chloromethylketone, and in intact cells this agent reduces the

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presentation of several peptides32. However, this inhibitor is notvery specific and inactivates other enzymes, including TPPII (ref.68). In living cells, TPPII hydrolyzes N-terminal residues from pep-tides 16 residues or more in length. Moreover, treatment of cellswith a TPPII inhibitor or a specific siRNA reduces MHC class Iexpression, presumably because there is a reduction in the supply ofpeptides for MHC class I molecules24. As TPPII hydrolyzes mainlypeptides more than 15 residues in length and contributes to antigenpresentation, it has been suggested that proteasomes might generatemainly precursor peptides that are more than 15 residues inlength24. TPPII may also be essential in the production of some ofthe rarer presented peptides not generated by proteasomes64. Invitro, TPPII can generate the HLA-A3 (or HLA-A11)–restrictedimmunodominant peptide from human immunodeficiency virusNef protein, and silencing of TPPII with siRNA blocks the presenta-tion of this antigen in cells64. Unfortunately, many of the ‘enzymo-logical’ properties of TPPII remain unclear or controversial and, aswith the other aminopeptidases, further systematic studies areneeded to define its precise function in vivo.

A critical related question concerns the relative importance ofpeptide trimming in the cytosol versus the ER. Insight into this hascome from experiments with HeLa cells in which ERAP1 has beensilenced by treatment with siRNA. Although these cells are unableto present N-extended SIINFEKL targeted into the ER, they are ableto trim and present N-extended SIINFEKL constructs expressed inthe cytosol, although at about 30% of the amount in control cells23.These results suggest that for SIINFEKL, when the N-extended pre-cursors are generated in the cytosol from minigenes, most of thepresented peptides are generated by trimming in the ER and, atmost, 30% of the trimming to the mature epitope occurs in thecytosol. In contrast, a peptide derived from amino acids 34–41 ofthe RU1 human tumor antigen seems to be trimmed mainly in thecytosol of human renal cell carcinoma cells67. The effect of ERAP1silencing on the overall supply of antigenic peptides seems todepend on the MHC class I allele, with some alleles showing astrong dependence on ERAP1 trimming and others seeming to relymainly on peptides generated independently of ERAP1 (refs. 23,46

and I.A.Y. and K.L.R., unpublished data), perhaps by proteasomesalone or with other cytosolic or ER aminopeptidases. The findingthat inhibition of TPPII reduces the presentation of certain anti-gens also indicates that at least some of the initial trimming of pep-tides 16 residues or more in length occurs in the cytosol. Anotherunresolved issue is whether these various cytosolic aminopepti-dases and those in the ER serve unique or redundant functions inpeptide trimming.

Destruction of antigenic peptides and precursorsAside from their occasional function in antigen presentation, thesmall fragments generated by proteasomes during protein hydrolysisare not useful except as a source of amino acids. Moreover, their accu-mulation in cells could be harmful, for example, by functioning asdominant inhibitors of protein-protein interactions. To prevent pep-tide accumulation, cells have evolved a variety of exopeptidases andendopeptidases that together rapidly hydrolyze the peptides to freeamino acids, which can then be reused in synthesis of new proteins.Consequently, peptides have half-lives of only a few seconds whenmicroinjected into living cells22. It has been estimated that more than99% of proteasome products are destroyed before binding to TAP6,69.Thus, although peptides are continuously produced in vivo, they areessentially undetectable in cells unless they are protected by bindingto an intracellular protein22,25,54,70.

In cell extracts, the enzyme that seems responsible for the initialcleavage of most antigenic peptides is a ubiquitous cytosolic metal-loendopeptidase, thimet oligopeptidase (TOP)71. TOP has broad sub-strate specificity. Specific TOP inhibitors decrease the hydrolysis ofmany (but not all) antigenic peptides in cell extracts71. In addition, theproducts of casein degradation by the proteasome that ranged from 8to 17 residues in length were digested rapidly by endoproteolytic cleav-ages, mainly by TOP, yielding fragments of intermediate size (four toseven residues), which were then rapidly converted to single aminoacids by aminopeptidases (T. Saric and A.L.G., unpublished data).Increasing TOP content in cells by transfection and thereby accelerat-ing the degradation of peptides in the cytosol notably reduces theoverall number of presented peptides generated from cellular pro-teins72,73. In contrast, silencing the expression of TOP with siRNA hasthe opposite effect, increasing the number of presented peptides,apparently because the peptides (or their precursors) survive longer inthe cytosol and are more likely to reach the TAP transporter intact72,73.

Cells also contain many other endopeptidases and exopeptidasesthat probably also contribute to the degradation of proteasomeproducts, including some with very narrow specificities that act on specific peptides (for example, post-prolyl oligopeptidase or prolineaminopeptidase). Unfortunately, few of the other endopeptidases inthe cytosol have been characterized in depth, and their precise func-tions have not yet been systematically studied. The degradation orprocessing of longer proteasome products (15–25 residues) seems tobe a particularly important function of TPPII, as TOP and aminopep-tidases fail to digest such long peptides (ref. 24 and T. Saric andA.L.G., unpublished data).

In addition to trimming precursors and digesting short peptidesto amino acids, aminopeptidases can also contribute substantiallyto the destruction of some antigenic peptides in cell extracts. Forexample, overexpression of leucine aminopeptidase in the cytosolreduces the overall supply of peptides for MHC class I molecules22.Also, the Sendai virus nucleoprotein peptide FAPGNYPAL (aminoacids 324–332) is hydrolyzed more rapidly in HeLa extracts bypuromycin-sensitive aminopeptidase than by TOP71. Moreover,many microinjected peptides are stabilized in vivo if their terminal

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Figure 3 The size preference of ERAP1 leads to different effects onpresentation by different MHC class I alleles. ERAP1 trims peptides with eight or fewer amino acids (2- to 7-mers) slowly and does not reducepresentation of peptide for MHC class I alleles that preferentially bind eight-residue peptides (8-mers). Approximately 50% of peptides with nineresidues (9-mers) are rapidly destroyed, leading to moderate reduction ofpresentation by MHC class I alleles that preferentially bind nine-residuepeptides, as well as to increased production of eight-residue epitopes. Most peptides of ten or more residues (10- to 18-mers) are rapidly trimmed,leading to increased production of nine-residue and eight-residue epitopes.

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α-amino group is modified to prevent hydrolysis by aminopepti-dases22. That study22, however, used peptides with internal residuesmodified by fluorescent and quenching groups. Those modifica-tions may inhibit cleavages by endopeptidases such as TOP and leadto an underestimation of their in vivo importance. Together, theseexperiments indicate that there is cooperation between aminopepti-dases and endopeptidases and that their relative importance mustdepend on the sequence of the peptide.

Mechanisms may also have evolved to protect antigenic peptides ortheir precursors from destruction in the cytosol before they are takenup into the ER. It has often been hypothesized that cytosolic or ERchaperones may have such a function in protecting antigenic peptidesfrom proteases and helping to deliver them to MHC class I molecules.Antigenic peptides and their precursors have been detected in cellextracts bound to molecular chaperones originating in the cytosol orER25,55 as well as in chromatin in the nucleus22. Although such bind-ing may in principle protect the peptides from destruction, in mostcases there are no data to indicate that chaperones act in vivo to pro-mote the delivery of peptides for presentation.

Silencing of the chaperonin TRiC (the eukaryotic analog of thebacterial GroEL-GroES complex) by siRNA reduces antigen presenta-tion and the amount of MHC class I molecules on the cell surface70. Itremains to be determined whether this effect of TRiC depletion isbecause of its binding of antigenic peptides or some other effect of theloss of this chaperone. For example, TRiC is essential for the produc-tion of major cell constituents such as actin and tubulin74, and therelated chaperonin in bacteria, GroEL-GroES, is essential in thedegradation of abnormal proteins75.

Whether or not cytosolic chaperones act to protect antigenic pep-tides from destruction, it is apparent from the experiments in whichpeptidases in cells are overexpressed or inhibited that destruction ofproteasome products by cytosolic peptidases is an important factorlimiting the supply of peptides for antigen presentation. Therefore,peptides generated in the cell confront a kinetic competition betweenproteolytic destruction, proteolytic trimming, protection by chaper-ones and successful binding to TAP, and only a small fraction of theprecursors or mature epitopes released by proteasomes are likely toescape complete destruction.

Antigenic peptides can also be destroyed in the ER. ERAP1 removesthe N-terminal residues from about 50% of nine-residue peptidestested23 and thus makes them too small to bind stably to the MHCclass I molecules that present nine- to ten-residue peptides (Fig. 3).Accordingly, silencing ERAP1 enhances the presentation of somenine-residue peptides46. Similarly, in the absence of IFN-γ, silencingof ERAP1 in human cells enhances the overall supply of nine-residuepeptides to MHC class I molecules, whereas ERAP1 overexpressionreduces peptide supply and thus MHC class I assembly23. Thus, inthese situations, ERAP1 destroys more peptides than it produces. Asopposite effects are seen in cells treated with IFN-γ23, which, asdescribed above, generate more longer N-extended precursors withenhanced uptake into the ER, it is likely that whether ERAP1 helpsproduce antigenic peptides or destroys them also depends on whetherthe epitope is generated mainly as a mature epitope of nine or tenresidues, which can only be destroyed, or from longer precursors thatcan be trimmed productively. Because ERAP1 hydrolyzes poorly pep-tides eight residues in length, it does not inhibit the supply of peptidesto MHC class I molecules, such as H-2Kb, that preferentially bindeight-residue peptides23 (Fig. 3). Unlike ERAP1, ERAP2 shows noclear preference for peptides more than eight or nine residues inlength, and thus in vitro it destroys antigenic peptides and does notgenerate them quantitatively from longer precursors (S.-C. Chang, N.Bhutani and A.L.G., unpublished data). In living cells, ERAP2 seemsto reduce mainly MHC class I antigen presentation, presumably bytrimming antigenic peptides in the ER to products that are too smallto bind to MHC class I (I.A.Y. and K.L.R., unpublished data).

Implications and unresolved issuesThese various observations establish that many presented peptides arefirst generated as N-extended precursors. As a principal effect of theinduction of immunoproteasomes is increased production of longer N-extended peptides, there must be some advantage in generating theselarger precursors. One such advantage may be the generation of a largernumber of different peptides available for presentation. In fact, thereare examples of protein sequences encoding overlapping epitopes,including cases in which N-terminal trimming of the nine-residue pep-tide reveals a new eight-residue epitope76,77. In addition, peptides more

ERAP1 8-mer9-mer

TAP

Extendedprecursors

Peptides(2- to 7-mers)

MHC class I

Endoplasmic reticulum

26S proteasome

Epitope

Antigen

Aminoacids

Mature epitopes

9-mer8-mer

Cytosol

~25%~10%

~65%

Peptides(2- to 7-mers)

Aminoacids

AP

TOPAP

TOP

AP

Figure 4 MHC class I antigen presentationreflects a balance between destruction andproduction of peptides. Cytosolic proteins aredegraded to peptides mainly by the proteasome.Most of these peptides are too small to bind toMHC class I molecules, but a minority of peptidesare long enough to bind to MHC class I moleculesor are longer7. All peptides can be degraded byaminopeptidases and endopeptidases to generateamino acids. Some mature epitopes escapedestruction in the cytosol and are transported to the ER lumen by TAP, where they may bedegraded by ER aminopeptidases, such asERAP1, or escape further destruction and bind toMHC class I molecules. N-extended precursors ofMHC class I–binding peptides may be trimmed byaminopeptidases in the cytosol (or, after transportby TAP, in the ER lumen) to generate MHC classI–binding peptides. N-extended peptides mayalso be destroyed by the same mechanisms. AP, aminopeptidase. Green bars, amino acidsequence that binds MHC class I; blue lines,amino acid sequences that must be removedbefore MHC class I binding.

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than 13 residues in length are relatively resistant to degradation byTOP78, which should favor their transport and binding to MHC class Imolecules. Finally, TAP tends to bind longer peptides with higher affin-ity29–31, which should help longer precursors escape destruction in thecytosol and lead to their preferential uptake into the ER.

In the past few years, there have been great strides in understandingthe importance of different proteolytic enzymes in antigen presenta-tion, in defining the precise functions of proteasomes and in identify-ing the peptidases that act on proteasome products and determinewhich epitopes are generated or destroyed (Fig. 4). These enzymes areessential in determining whether and to what extent immuneresponses will occur. However, understanding of these proteolyticprocesses is still incomplete. More knowledge is needed about thenature of the peptides that are produced by different forms of the pro-teasome complex, the contributions of other proteases and the iden-tity of the endopeptidases or exopeptidases that can generate ordestroy MHC class I–binding peptides from these products. The largenumber of potential trimming enzymes raises the question ofwhether these different peptidases are redundant or act sequentiallyor on distinct subsets of precursors. The specificity of these hydrolasesand their respective contributions to peptide cleavage need to be fur-ther defined. In addition, whether peptide-binding proteins do in factreduce peptide destruction and enhance their delivery to the ER (andif so, how) needs to be clarified. Ultimately, elucidation of these fac-tors should permit accurate prediction of what epitopes are presentedand the specificity of immune responses, both in basal conditions andduring infection, when the content and importance of these pepti-dases can vary.

ACKNOWLEDGMENTSWe thank E. Bishop for assistance in preparation of this manuscript. Supported by grants from the National Institute of General Medical Sciences (A.L.G.) andNational Institutes of Health (K.L.R.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureimmunology/

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