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MAKING PEPTIDES FOR PRESENTATION

2004 Nature Publishing Group http://www.nature.com/natureimmunology

Post-proteasomal antigen processing for major histocompatibility complex class I presentation

Kenneth L Rock1, Ian A York1 & Alfred L Goldberg2
Peptides presented by major histocompatibility complex class I molecules are derived mainly from cytosolic oligopeptides generated by proteasomes during the degradation of intracellular proteins. Proteasomal cleavages generate the final C terminus of these epitopes. Although proteasomes may produce mature epitopes that are eight to ten residues in length, they more often generate N-extended precursors that are too long to bind to major histocompatibility complex class I molecules. Such precursors are trimmed in the cytosol or in the endoplasmic reticulum by aminopeptidases that generate the N terminus of the presented epitope. 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 limiting the supply of many antigenic peptides. Thus, the extent of antigen presentation depends on the balance between several proteolytic processes that may generate or destroy epitopes.

Major histocompatibility complex (MHC) class I molecules bind a large variety of peptides that are generated from the vast majority of cellular proteins and transport these epitopes to the cell surface for display to the immune system14. This process allows CD8+ T lymphocytes to identify and eliminate cells that are synthesizing abnormal or foreign proteins, as may arise through mutations or infection by viruses. MHC class I molecules can present a large repertoire of peptides because their peptide-binding grooves interact strongly with only a few amino acid side chains of the peptide and also with aspects that are present on all peptides, such as the terminal -amino (N) and carboxy (C) groups5. A consequence of binding the N- and C-terminal ends is that MHC class I molecules stably bind only peptides of a precise and uniform length5. Thus, some MHC class I molecules bind mainly peptides of eight residues, whereas others bind mainly nineresidue or ten-residue peptides. Therefore, for complete surveillance of proteins present in the intracellular milieu, cells must generate peptides of eight to ten residues from nearly all cellular proteins, thereby creating a wide range of binding motifs that are represented among the hundreds of MHC class I alleles. This article reviews the understanding at present of how these MHC class Ipresented peptides are produced and the peptidases that act after the proteasome and influence antigen presentation.

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

Published online 28 June 2004; doi:10.1038/ni1089

The ability to synthesize and recognize MHC molecules arose early in the evolution of vertebrates. To generate the MHC class Ipresented peptides, nature took advantage of the two main cellular proteolytic systems that are phylogenetically much older than vertebrates. All eukaryotic cells continuously turn over their proteins to eliminate damaged, unfolded or incomplete proteins6, to regulate cellular processes and to adapt to new conditions. These pathways provide a convenient library of peptides derived from the great majority of cellular proteins that can be sampled by MHC class I molecules4. The degradation of most cellular proteins occurs by the ubiquitinproteasome pathway3,7,8. The initial step in this process is the conjugation of the polypeptide cofactor ubiquitin to the amino group of lysines found in the protein substrate, and subsequently other ubiquitin molecules are linked to the first ubiquitin molecule. A chain of four or more ubiquitin molecules serves as a molecular tag that marks the protein for rapid degradation by the 26S proteasome. The 26S proteasomes are composed of a core cylinder, the 20S proteasome, which in cells is capped at each end by a 19S complex of subunits that recognize and unfold ubiquinated substrates to control their access into the 20S core9. In the 20S core, six peptidase sites act together to cleave the protein into many diverse oligopeptides, although the precise location of the cleavages vary widely with each molecule degraded9. This process creates a very large number (perhaps hundreds) of different peptides, depending on the length and sequence of the protein. It is now apparent that hydrolysis of proteins by proteasomes is the key step in the generation of most antigenic peptides9. Treating cells with highly specific proteasome inhibitors not only blocks the degradation of most cellular proteins but also blocks most MHC class I

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antigen presentation of peptides derived Presented Presented epitope with Antigen expressed in cells epitope proteasome inhibitors from cytosolic proteins1013. However, those inhibitors do not reduce the presentation of Protein peptides that are already of the correct length C-extended epitope to bind to MHC molecules and that require Plasmid 11,14,15 (Fig. 1). Thus, Mature epitope no further proteolysis expressing peptide those agents inhibit antigen presentation by N-extended epitope or protein blocking the generation of either the presented peptides or longer precursor peptides. Although proteasomes are required for Figure 1 Generation of the termini of MHC class Ibinding peptides. Proteasome inhibitors block antigen presentation, they actually destroy the production of MHC class Ibinding peptides from full-length proteins and from peptides that are many more epitopes than they generate. extended at the C terminus by even one amino acid. Proteasome inhibitors do not affect presentation When purified 20S or 26S proteasomes are of mature epitopes (with no extra N- or C-terminal amino acids) or of MHC class Ibinding peptides incubated with protein substrates, most pep- with the correct C terminus but with as many as 20 amino acids at the N terminus. Therefore, tides produced range in size from about 2 to proteasomes are required for the generation of the C terminus but not the N terminus of MHC class I 25 amino acids16,17. Only a small fraction of epitopes. Green bars, amino acid sequence that binds MHC class I; blue lines, amino acid sequences that must be removed before MHC class I binding; no icons, no presented epitope. these peptides (about 15%) are the correct size for antigen presentation, and most (70%) are too short to bind to MHC class I molecules16,17. Purified viral infections when cells are exposed to IFN-. However, systematic in proteasomes, when degrading ovalbumin, destroy the immunodomi- vitro studies with other protein substrates are needed to determine if 26S nant epitope SIINFEKL (ovalbumin amino acids 257264) more than proteasomes and especially immunoproteasomes generate mainly N90% of the time18 (Fig. 2a). Therefore, the generation of antigenic extended precursors. Unfortunately, it is not possible to directly characterize and quanpeptides by proteasomes seems to be a stochastic process, and most of tify the oligopeptides produced by proteasomes in living cells, the time proteasomes cleave within epitopes. because nearly all peptides are rapidly destroyed in the cytosol5,6,22. Proteasomes produce N-extended epitope precursors Although it is dangerous to generalize from the very few quantitaOne fundamental question about the function of the proteasome in tive in vitro studies to the nature of the proteasomal products proantigen processing is whether it makes the cleavages that generate the duced from diverse proteins in vivo, the experiments described presented peptides directly or whether it generates longer precursors below make it clear that a substantial fraction of epitopes are generthat require further proteolytic processing. Many early in vitro stud- ated as longer precursors in vivo23,24. Longer precursors have been ies found that the mature epitope and extended precursors could be found in cell extracts bound to heat shock proteins, such as hsp70, generated by purified proteasomes1921, but those studies were not hsp90 and gp96 (refs. 25,26). Thus, it seems likely that in vivo, as quantitative and were done under nonphysiological conditions. in vitro, most presented peptides must be produced initially as Thus, it was unclear how often proteasomes generate long precursors N-extended precursors. versus mature epitopes. When purified 20S or 26S proteasomes are incubated with protein substrates in more native conditions and the Precursor peptides can be trimmed in vivo products are analyzed with quantitative assays, the proteasomes are As proteasomes generate many peptides that are too long to bind found to generate more long peptides than peptides of the correct stably to MHC class I molecules, an important question is whether size required for binding to MHC class I molecules16,17. Moreover, cells can actually trim and present these extended peptides. To when purified 26S proteasomes degrade denatured ovalbumin mole- investigate this, antigenic peptides with extra C- or N-terminal cules, the majority (70%) of the SIINFEKL-containing peptides residues were injected into cells or were expressed from minigenes in cells, and presentation of the mature epitope on MHC class I produced have N-terminal extensions18 (Fig. 2b). The nature of the peptides produced by proteasomes changes after molecules was measured11,14,15. Both C- and N-extended versions exposure of cells to proinflammatory cytokines that enhance overall could be efficiently trimmed to the correct epitope and presented on antigen presentation. When cells are treated with interferon- (IFN-), MHC class I molecules (Fig. 1). However, the addition of proteanew catalytic subunits are incorporated into the proteasome to gener- some inhibitors completely blocked the presentation of peptides ate immunoproteasomes9. Purified immunoproteasomes degrade from constructs with even a single extra C-terminal residue. As proteins at rates similar to those of constitutive particles, but they pro- expected, these agents failed to affect the presentation of constructs duce from ovalbumin increased amounts of N-extended SIINFEKL that were already the correct size for presentation11,14,15 (Fig. 1). peptides, whereas the amount of the mature SIINFEKL epitope pro- These results suggested proteasomes are the only proteases in cells duced by immunoproteasomes is the same as that produced by constitu- that can generate the proper C terminus of these peptides from tive proteasomes18 (Fig. 2). Similarly, in vitro experiments have shown longer precursors. These results also indicate that the cytosol lacks that immunoproteasomes generate peptides from enolase that seem, on carboxypeptidases that remove extra residues from the C terminus average, to be one residue longer than the products of constitutive pro- of peptides, and indeed C-terminal trimming activity has not been teasomes17, although such a size difference has not been noted with detected in cell extracts27. other substrates18. Similarly, for many19,20 but not all epitopes21, NIn contrast, proteasome inhibitors do not decrease the presentation extended precursors are the main products generated in vitro by 20S of peptides from constructs that have from 1 to more than 24 extra Nproteasomes from human lymphoblastoid cell lines, in which immuno- terminal residues11,15 (Fig. 1). However, presentation from Nproteasomes typically predominate. The limited data available suggest extended precursors expressed in cells from minigenes is blocked by that in all conditions, long precursors are likely to be the main form of acetylation of the terminal () amino group on their N terminus14. epitope produced by proteasomes. This tendency may be enhanced in This modification prevents cleavage by aminopeptidases but not by

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proteasomes or other endopeptidases. The presentation of peptides derived from N-extended constructs is as rapid as that for mature peptides that do not require trimming. Thus, there must be intracellular aminopeptidases that rapidly trim these N-extended precursors to the proper length for MHC binding. Peptide trimming in the ER A small fraction of the peptides produced by proteasomes escape destruction in the cytosol and is transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), where these peptides can bind to MHC class I molecules. TAP transports peptides that range in length from about 7 to more than 20 amino acids and can therefore import into the ER both mature epitopes and longer precursors28. In some cases, TAP transports the longer precursors more rapidly than the mature epitope2931. Thus, in principle, precursor peptides can be trimmed in the cytosol and/or the ER. Experimental evidence suggests that N-terminal trimming occurs in both of these subcellular compartments27,3238. Secreted or membrane proteins are normally targeted to the ER by N-terminal signal sequences, which direct their transport through a transmembrane channel, the SEC61 complex, after which the signal sequence is usually removed by the ER enzyme signal peptidase39. Similarly, minigenes that express peptides with N-terminal signal sequences are delivered through SEC61 directly into the lumen of the ER, in a TAP-independent process. Such ER-targeted peptides can be presented even if they are transported as N-extended precursors11,35,37,38,40. In addition to these model constructs, TAP-negative cell lines naturally present some peptides that are generated from signal peptides. Although the signal peptidase liberates the signal peptide, these epitopes have N-terminal flanking residues that must be removed, almost certainly by trimming in the ER41,42. Isolated microsomes contain luminal aminopeptidase activities that can trim N-terminal residues from peptides38,4345. One such microsomal peptidase was purified23,46 and was found to be identical to an aminopeptidase that had been isolated before but whose immunological relevance had not been recognized. This peptidase is a zinc-containing 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 or puromycin-insensitive leucine aminopeptidase49 and was renamed ER aminopeptidase 1 (ERAP1)23,50 or ER-associated aminopeptidase (ERAAP)46 to more accurately reflect its properties. As would be expected of a peptidase involved in MHC class I antigen presentation, ERAP1 is expressed in most cells32,49. Moreover, its expression is increased in cells treated with IFN-, a potent stimulator of MHC class I presentation46,50. Like many other aminopeptidases, ERAP1 is able to remove from the N terminus of peptides nearly all amino acids except those followed by a proline46. One of its unexpected properties is that ERAP1 strongly prefers peptide substrates that are 916 residues in length (S.-C. Chang, N. Bhutani and A.L.G., unpublished data), which corresponds exactly to the lengths of peptides transported selectively by TAP28,51. ERAP1 also has the unique property of rapidly trimming N-extended antigenic peptides to eight or nine residues, and then further cleavages occur much more slowly or cease completely23. The peptides produced by purified ERAP1 are therefore precisely the length of epitopes that are bound by most MHC class I molecules1. In contrast, known aminopeptidases continually cleave Nterminal residues from small peptides (preferentially those less than five residues) until only free amino acids remain. ERAP1, unlike other aminopeptidases, monitors the nature of the C-terminal residue and somehow measures the distance from this C terminus to the cleaved N terminus. ERAP1 strongly prefers peptides with hydrophobic C-terminal residues (S.-C. Chang, N. Bhutani and A.L.G., unpublished data). Exactly how this enzymes active sites sequentially remove N-terminal residues that are 916 residues away from the C terminus remains a mystery. Further insight into the structure if ERAP1 should elucidate this unusual length dependence and substrate specificity, which are of immunological importance because these preferences influence which peptides are trimmed and available for presentation. Decreasing ERAP1 abundance with small interfering RNAs (siRNAs) considerably reduces antigen presentation of N-extended SIINFEKL constructs targeted to the ER23,46. Therefore, at least for SIINFEKL precursors, ERAP1 seems to provide the only important peptide-trimming enzymatic activity in the ER found in human HeLa

2004 Nature Publishing Group http://www.nature.com/natureimmunology

a
Ovalbumin 6% 94% Proteasome SIINFEKL IFN- Destroyed SIINFEKL SIINFEKL & N-extended precursors

b
Peptide (pmol produced/ 100 pmol ovalbumin digested)

12 10 8 6 4 2 0 7+S 6+S 4+S 3+S 2+S 1+S 7+S 6+S 4+S 3+S 2+S

1+S

SI IN FE KL N -e xt SI e IN nd FE ed KL

Proteasomes

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

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tumor cells and mouse fibroblast cells, as has also been suggested by biochemical studies of rat liver50. Reducing ERAP1 abundance in vivo also decreases the presentation of epitopes generated from several antigens that are known or presumed to be initially cleaved by proteasomes23,46. For example, loss of ERAP1 reduces the presentation of SIINFEKL from full-length ovalbumin by about 70% (ref. 23). This finding correlates well with the in vitro observation that about 70% of SIINFEKL-containing precursors are N extended27. The overall influence of ERAP1 on peptide supply for antigen presentation can be estimated by measuring the transport of MHC class I alleles to the cell surface, as this transport is dependent on peptide binding. For some alleles, for example, H-2Kk and H-2Ld, elimination of ERAP1 reduces the peptide supply by 2045% (ref. 46). However, the expression of several other MHC class I alleles is not reduced. For example, in constitutive conditions, H-2Kb is unaffected by ERAP1 silencing23. However, in IFN--treated cells, ERAP1 silencing does reduce the surface expression of H-2Kb molecules23. This finding indicates that after IFN- stimulation, the lack of ERAP1 causes a reduction in the overall supply of antigenic peptides, presumably because there are more N-extended peptides in the ER that must be trimmed by ERAP1 to the proper size to bind to MHC class I molecules. An increase of N-extended precursors in the ER may occur because IFN- increases the content of immunoproteasomes, which produce more N-extended precursors, and of TAP, which preferentially transports peptides more than eight or nine residues in length28,51. However, other possible mechanisms may also account for the greater importance of ERAP1 after IFN- treatment. In addition to ERAP1, there may be other aminopeptidases in the ER that are involved in peptide trimming. Additional aminopeptidase peaks have been found in microsomal preparations50. The source of one such ER enzymatic activity has recently been identified, leukocyte-derived arginine aminopeptidase (L-RAP, also called ER-aminopeptidase 2 (ERAP2)), an aminopeptidase highly homologous to ERAP1 that is also induced by IFN- stimulation52. ERAP2 preferentially cleaves basic dipeptides52 but can remove nearly all N-terminal residues from oligopeptides (S.-C. Chang and A.L.G., unpublished data). Thus, ERAP2 may also be important in antigen processing, although its function remains to be determined. ERAP2 has a much more limited pattern of expression in tissues than ERAP1 (ref. 52). In the absence of IFN- stimulation, ERAP2 is absent from HeLa cells, whereas ERAP1 is abundant50. The identity of the other microsomal peptidases, whether they are genuinely localized in the ER or represent cytosolic contaminants and whether they are involved in antigen processing or degradation remain to be determined. After the discovery that MHC class I molecules bound peptides of a uniform size, it was proposed that MHC class I molecules might first bind precursor peptides and then serve as a template that limits the trimming of the bound peptide to the proper size53,54. Evidence has been presented in support of this model44; however, it is difficult to distinguish whether trimming occurs on the MHC class I molecule or whether the MHC class I molecules simply bind and protect peptides of the proper size from destruction. It is not apparent that the active site of a peptidase is able to access and trim a long peptide bound to an MHC class I molecule55,56. Moreover, the discovery that ERAP1 rapidly trims peptides to a length of eight or nine residues and then stops23 indicates that MHC class I molecules are not needed for the generation of peptides of the proper size. Nevertheless, it remains to be determined whether there is any cooperation between ERAP1 and MHC class I molecules in peptide trimming. In addition to ERAP1 and ERAP2, the protease furin has been linked to the generation of some MHC-presented peptides in the secretory compartments57,58. This endoprotease is a member of the subtilysin family that cleaves after polybasic motifs in the processing of neuropeptides and hormones in the Golgi appartus59. Such a motif is present in the secretory form of the hepatitis B virus C gene product57,58 . Epitopes fused with the C-terminal region of this antigen are presented independently of TAP and proteasome function57,58. Presentation of such epitopes is enhanced by overexpression of furin58 and is inhibited by the furin-specific inhibitor decRVKRCMK57. As the presentation of most epitopes requires proteasomes and TAP, furin presumably has only a minor function in the generation of antigenic peptides. Whether furin is involved in the trimming of TAP-transported peptides is unknown, although the limited sequence specificity and subcellular location of furin must limit the number of possible substrates59. Peptide trimming in the cytosol Certain N-extended antigenic precursors are trimmed in the cytosol before being transported by TAP into the ER. This conclusion was first suggested by the observation that the mature SIINFEKL epitope is efficiently presented on MHC class I molecules when SIINFEKL is linked to a 25-residue N-terminal extension, even when proteasome function is eliminated11. Because TAP transports peptides of this length poorly if at all60, it is most likely that the 32residue peptide was trimmed before uptake into the ER. Subsequently, it was found that the presentation of some antigens is reduced by protease inhibitors that block the activity of peptidases in the cytosol but not in the ER32,6163. In cytosolic extracts there are several peptidases that can trim antigenic peptides. These enzymes seem to exist free in solution, rather than as a large multienzyme complex. An IFN--inducible peptidase that could trim N-extended SIINFEKL was purified from HeLa cytosol and was identified as leucine aminopeptidase27. This zinc metallopeptidase is normally present in many tissues but is synthesized in increased amounts after IFN- stimulation. Puromycin-sensitive aminopeptidase, a zinc metallopeptidase, and bleomycin hydrolase, a cysteine protease, are two other cytosolic aminopeptidases that were shown to trim a vesicular stomatitis virus nucleoprotein precursor peptide in cytosolic extracts derived from human Epstein-Barr virustransformed B lymphoblastoid cell lines32. Puromycin-sensitive aminopeptidase and bleomycin hydrolase are constitutively expressed in most cells and are not induced by IFN-. Tripeptidyl peptidase II (TPPII) has been linked to the generation of some antigenic peptides9,24,64. This proteolytic enzyme is a very large (greater than 1 MDa) multimeric complex that that removes three residues at a time from the N terminus of substrates, provided the N terminus is not blocked65, and may also have some endoproteolytic activity66. In cell extracts, TPPII acts sequentially with puromycin-sensitive aminopeptidase to trim a long precursor to the final mature epitope67. Because TPPII has a strong preference for peptides more than 15 residues in length (ref. 24 and P. Venkatraman and A.L.G., unpublished data), it may have a specific function in the processing of very long proteasomal products. An unresolved question is whether these different cytosolic aminopeptidases have general functions in trimming peptides for antigen presentation in vivo. At present there are insufficient data to draw firm conclusions. Injection of leucine aminopeptidase into cells results in increased trimming of peptides22. Puromycin-sensitive aminopeptidase and bleomycin hydrolase are inhibited by Ala-AlaPhe-chloromethylketone, and in intact cells this agent reduces the

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and I.A.Y. and K.L.R., unpublished data), perhaps by proteasomes alone or with other cytosolic or ER aminopeptidases. The finding that inhibition of TPPII reduces the presentation of certain antigens also indicates that at least some of the initial trimming of peptides 16 residues or more in length occurs in the cytosol. Another unresolved issue is whether these various cytosolic aminopeptidases and those in the ER serve unique or redundant functions in peptide trimming. Destruction of antigenic peptides and precursors Aside from their occasional function in antigen presentation, the small fragments generated by proteasomes during protein hydrolysis are not useful except as a source of amino acids. Moreover, their accumulation in cells could be harmful, for example, by functioning as dominant inhibitors of protein-protein interactions. To prevent peptide accumulation, cells have evolved a variety of exopeptidases and endopeptidases that together rapidly hydrolyze the peptides to free amino acids, which can then be reused in synthesis of new proteins. Consequently, peptides have half-lives of only a few seconds when microinjected into living cells22. It has been estimated that more than 99% of proteasome products are destroyed before binding to TAP6,69. Thus, although peptides are continuously produced in vivo, they are essentially undetectable in cells unless they are protected by binding to an intracellular protein22,25,54,70. In cell extracts, the enzyme that seems responsible for the initial cleavage of most antigenic peptides is a ubiquitous cytosolic metalloendopeptidase, thimet oligopeptidase (TOP)71. TOP has broad substrate specificity. Specific TOP inhibitors decrease the hydrolysis of many (but not all) antigenic peptides in cell extracts71. In addition, the products of casein degradation by the proteasome that ranged from 8 to 17 residues in length were digested rapidly by endoproteolytic cleavages, mainly by TOP, yielding fragments of intermediate size (four to seven residues), which were then rapidly converted to single amino acids by aminopeptidases (T. Saric and A.L.G., unpublished data). Increasing TOP content in cells by transfection and thereby accelerating the degradation of peptides in the cytosol notably reduces the overall number of presented peptides generated from cellular proteins72,73. In contrast, silencing the expression of TOP with siRNA has the opposite effect, increasing the number of presented peptides, apparently because the peptides (or their precursors) survive longer in the cytosol and are more likely to reach the TAP transporter intact72,73. Cells also contain many other endopeptidases and exopeptidases that probably also contribute to the degradation of proteasome products, including some with very narrow specificities that act on specific peptides (for example, post-prolyl oligopeptidase or proline aminopeptidase). Unfortunately, few of the other endopeptidases in the cytosol have been characterized in depth, and their precise functions have not yet been systematically studied. The degradation or processing of longer proteasome products (1525 residues) seems to be a particularly important function of TPPII, as TOP and aminopeptidases fail to digest such long peptides (ref. 24 and T. Saric and A.L.G., unpublished data). In addition to trimming precursors and digesting short peptides to amino acids, aminopeptidases can also contribute substantially to the destruction of some antigenic peptides in cell extracts. For example, overexpression of leucine aminopeptidase in the cytosol reduces the overall supply of peptides for MHC class I molecules22. Also, the Sendai virus nucleoprotein peptide FAPGNYPAL (amino acids 324332) is hydrolyzed more rapidly in HeLa extracts by puromycin-sensitive aminopeptidase than by TOP71. Moreover, many microinjected peptides are stabilized in vivo if their terminal

Substrates 2- to 7-mer 8-mer 50% 9-mer 50%

Trimming by ERAP1 Slow Slow Slow

Immune consequences None (not epitopes) Minimal epitope destruction Minimal destruction of 9-mer epitope 9-mer epitope destroyed 8-mer epitope presented

Rapid to 8-mer

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10- to 18-mer

Rapid to 8- to 9-mers

8- & 9-mer epitopes presented Some destruction of 9-mer epitopes?

Figure 3 The size preference of ERAP1 leads to different effects on presentation by different MHC class I alleles. ERAP1 trims peptides with eight or fewer amino acids (2- to 7-mers) slowly and does not reduce presentation of peptide for MHC class I alleles that preferentially bind eight-residue peptides (8-mers). Approximately 50% of peptides with nine residues (9-mers) are rapidly destroyed, leading to moderate reduction of presentation by MHC class I alleles that preferentially bind nine-residue peptides, 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.

presentation of several peptides32. However, this inhibitor is not very specific and inactivates other enzymes, including TPPII (ref. 68). In living cells, TPPII hydrolyzes N-terminal residues from peptides 16 residues or more in length. Moreover, treatment of cells with a TPPII inhibitor or a specific siRNA reduces MHC class I expression, presumably because there is a reduction in the supply of peptides for MHC class I molecules24. As TPPII hydrolyzes mainly peptides more than 15 residues in length and contributes to antigen presentation, it has been suggested that proteasomes might generate mainly precursor peptides that are more than 15 residues in length24. TPPII may also be essential in the production of some of the rarer presented peptides not generated by proteasomes64. In vitro, TPPII can generate the HLA-A3 (or HLA-A11)restricted immunodominant peptide from human immunodeficiency virus Nef protein, and silencing of TPPII with siRNA blocks the presentation of this antigen in cells64. Unfortunately, many of the enzymological properties of TPPII remain unclear or controversial and, as with the other aminopeptidases, further systematic studies are needed to define its precise function in vivo. A critical related question concerns the relative importance of peptide trimming in the cytosol versus the ER. Insight into this has come from experiments with HeLa cells in which ERAP1 has been silenced by treatment with siRNA. Although these cells are unable to present N-extended SIINFEKL targeted into the ER, they are able to trim and present N-extended SIINFEKL constructs expressed in the cytosol, although at about 30% of the amount in control cells23. These results suggest that for SIINFEKL, when the N-extended precursors are generated in the cytosol from minigenes, most of the presented peptides are generated by trimming in the ER and, at most, 30% of the trimming to the mature epitope occurs in the cytosol. In contrast, a peptide derived from amino acids 3441 of the RU1 human tumor antigen seems to be trimmed mainly in the cytosol of human renal cell carcinoma cells67. The effect of ERAP1 silencing on the overall supply of antigenic peptides seems to depend on the MHC class I allele, with some alleles showing a strong dependence on ERAP1 trimming and others seeming to rely mainly on peptides generated independently of ERAP1 (refs. 23,46

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-amino group is modified to prevent hydrolysis by aminopeptidases22. That study22, however, used peptides with internal residues modified by fluorescent and quenching groups. Those modifications may inhibit cleavages by endopeptidases such as TOP and lead to an underestimation of their in vivo importance. Together, these experiments indicate that there is cooperation between aminopeptidases and endopeptidases and that their relative importance must depend on the sequence of the peptide. Mechanisms may also have evolved to protect antigenic peptides or their precursors from destruction in the cytosol before they are taken up into the ER. It has often been hypothesized that cytosolic or ER chaperones may have such a function in protecting antigenic peptides from proteases and helping to deliver them to MHC class I molecules. Antigenic peptides and their precursors have been detected in cell extracts bound to molecular chaperones originating in the cytosol or ER25,55 as well as in chromatin in the nucleus22. Although such binding may in principle protect the peptides from destruction, in most cases there are no data to indicate that chaperones act in vivo to promote the delivery of peptides for presentation. Silencing of the chaperonin TRiC (the eukaryotic analog of the bacterial GroEL-GroES complex) by siRNA reduces antigen presentation and the amount of MHC class I molecules on the cell surface70. It remains to be determined whether this effect of TRiC depletion is because of its binding of antigenic peptides or some other effect of the loss of this chaperone. For example, TRiC is essential for the production of major cell constituents such as actin and tubulin74, and the related chaperonin in bacteria, GroEL-GroES, is essential in the degradation of abnormal proteins75. Whether or not cytosolic chaperones act to protect antigenic peptides from destruction, it is apparent from the experiments in which peptidases in cells are overexpressed or inhibited that destruction of proteasome products by cytosolic peptidases is an important factor limiting the supply of peptides for antigen presentation. Therefore, peptides generated in the cell confront a kinetic competition between proteolytic destruction, proteolytic trimming, protection by chaperones and successful binding to TAP, and only a small fraction of the precursors or mature epitopes released by proteasomes are likely to escape complete destruction. Antigenic peptides can also be destroyed in the ER. ERAP1 removes the N-terminal residues from about 50% of nine-residue peptides tested23 and thus makes them too small to bind stably to the MHC class I molecules that present nine- to ten-residue peptides (Fig. 3). Accordingly, silencing ERAP1 enhances the presentation of some nine-residue peptides46. Similarly, in the absence of IFN-, silencing of ERAP1 in human cells enhances the overall supply of nine-residue peptides to MHC class I molecules, whereas ERAP1 overexpression reduces peptide supply and thus MHC class I assembly23. Thus, in these situations, ERAP1 destroys more peptides than it produces. As opposite effects are seen in cells treated with IFN-23, which, as described above, generate more longer N-extended precursors with enhanced uptake into the ER, it is likely that whether ERAP1 helps produce antigenic peptides or destroys them also depends on whether the epitope is generated mainly as a mature epitope of nine or ten residues, which can only be destroyed, or from longer precursors that can be trimmed productively. Because ERAP1 hydrolyzes poorly peptides eight residues in length, it does not inhibit the supply of peptides to MHC class I molecules, such as H-2Kb, that preferentially bind eight-residue peptides23 (Fig. 3). Unlike ERAP1, ERAP2 shows no clear preference for peptides more than eight or nine residues in length, and thus in vitro it destroys antigenic peptides and does not generate them quantitatively from longer precursors (S.-C. Chang, N. Bhutani and A.L.G., unpublished data). In living cells, ERAP2 seems to reduce mainly MHC class I antigen presentation, presumably by trimming antigenic peptides in the ER to products that are too small to bind to MHC class I (I.A.Y. and K.L.R., unpublished data). Implications and unresolved issues These various observations establish that many presented peptides are first generated as N-extended precursors. As a principal effect of the induction of immunoproteasomes is increased production of longer Nextended peptides, there must be some advantage in generating these larger precursors. One such advantage may be the generation of a larger number of different peptides available for presentation. In fact, there are examples of protein sequences encoding overlapping epitopes, including cases in which N-terminal trimming of the nine-residue peptide reveals a new eight-residue epitope76,77. In addition, peptides more

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Figure 4 MHC class I antigen presentation reflects a balance between destruction and production of peptides. Cytosolic proteins are degraded to peptides mainly by the proteasome. Most of these peptides are too small to bind to MHC class I molecules, but a minority of peptides are long enough to bind to MHC class I molecules or are longer7. All peptides can be degraded by aminopeptidases and endopeptidases to generate amino acids. Some mature epitopes escape destruction in the cytosol and are transported to the ER lumen by TAP, where they may be degraded by ER aminopeptidases, such as ERAP1, or escape further destruction and bind to MHC class I molecules. N-extended precursors of MHC class Ibinding peptides may be trimmed by aminopeptidases in the cytosol (or, after transport by TAP, in the ER lumen) to generate MHC class Ibinding peptides. N-extended peptides may also be destroyed by the same mechanisms. AP, aminopeptidase. Green bars, amino acid sequence that binds MHC class I; blue lines, amino acid sequences that must be removed before MHC class I binding.

Cytosol

Antigen Epitope 26S proteasome ~25% ~65% ~10% Mature epitopes 8-mer 9-mer AP TOP TAP Peptides (2- to 7-mers)

Amino acids

Extended precursors AP TOP AP

Peptides (2- to 7-mers)

Amino acids

ERAP1

8-mer 9-mer

MHC class I

Endoplasmic reticulum

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than 13 residues in length are relatively resistant to degradation by TOP78, which should favor their transport and binding to MHC class I molecules. Finally, TAP tends to bind longer peptides with higher affinity2931, which should help longer precursors escape destruction in the cytosol and lead to their preferential uptake into the ER. In the past few years, there have been great strides in understanding the importance of different proteolytic enzymes in antigen presentation, in defining the precise functions of proteasomes and in identifying the peptidases that act on proteasome products and determine which epitopes are generated or destroyed (Fig. 4). These enzymes are essential in determining whether and to what extent immune responses will occur. However, understanding of these proteolytic processes is still incomplete. More knowledge is needed about the nature of the peptides that are produced by different forms of the proteasome complex, the contributions of other proteases and the identity of the endopeptidases or exopeptidases that can generate or destroy MHC class Ibinding peptides from these products. The large number of potential trimming enzymes raises the question of whether these different peptidases are redundant or act sequentially or on distinct subsets of precursors. The specificity of these hydrolases and their respective contributions to peptide cleavage need to be further defined. In addition, whether peptide-binding proteins do in fact reduce peptide destruction and enhance their delivery to the ER (and if so, how) needs to be clarified. Ultimately, elucidation of these factors should permit accurate prediction of what epitopes are presented and the specificity of immune responses, both in basal conditions and during infection, when the content and importance of these peptidases can vary.
ACKNOWLEDGMENTS We thank E. Bishop for assistance in preparation of this manuscript. Supported by grants from the National Institute of General Medical Sciences (A.L.G.) and National Institutes of Health (K.L.R.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
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