generation of major histocompatibility complex class i antigens

Review

Generation of major histocompatibility complex class I antigens

YoungYang *

Johnson & Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121, USA

Abstract

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cellsis an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machineryis required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defectiveribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss thegeneration of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Immune; MHC; PA28; Proteasome; TAP

1. Introduction

Presentation of major histocompatibility complex (MHC)class I antigens on antigen-presenting cells to cytotoxic Tlymphocytes plays a crucial role in the body’s defensesagainst intracellular pathogens [1,2]. The primary function ofMHC class I molecules is to continuously present intracellu-lar antigen-derived peptides to cytotoxic T lymphocytes, aprocess that initiates the adaptive immune response whennon-self-peptides are presented [1,2]. It has been proposedthat to ensure a rapid MHC class I-restricted cytotoxic Tlymphocyte response after infection, peptides presented byMHC class I molecules are mainly derived from newly syn-thesized defective ribosomal products of non-self antigens[3]. To efficiently present MHC class I antigens, expressionof several sets of immunological genes appears to be coordi-nately upregulated during infection [1,2,4,5]. Among theseupregulated genes,MHC class I heavy chains, transporterassociated with antigen processing (TAP) subunitsTAP1 andTAP2, tapasin and proteasome catalytic subunitsLMP2 andLMP7 are encoded in theMHC region [2,5]. The extremepolymorphism ofMHC [1], which is most likely driven byresistance to infectious pathogens, appears to have evolved toallow MHC class I molecules to efficiently display a broadspectrum of pathogen-derived peptides [1,2]. TheMHC re-gion does not encode theMHC class I light chainb2-

microglobulin (b2M), proteasome regulatorPA28, molecularchaperoneGp96, proteasome maturating factorUmp1p andproteasome catalytic subunitMECL1 genes, but they arefound to be coordinately upregulated withMHC-encodedgenes, and during infection, are required for efficient presen-tation of MHC class I antigens [2,5]. As a consequence ofincreased surface expression of functional MHC class I mol-ecules during infection, the immune system is elicited toeliminate pathogen-infected cells and to regain control overpathogen infections [1,2].

Based on the currently understood molecular, biochemicaland cellular mechanisms, a model for an ordered pathway forthe assembly of newly synthesized MHC class I molecules isproposed (Fig. 1). With the assistance of several endoplasmicreticulum (ER)-resident molecular chaperones [2,5], nascentMHC class I heavy chain binds to the light chainb2M, firstforming a heterodimer in the ER [1,2]. MHC class I het-erodimers then acquire eight- to 11-amino acid peptides[2,5], which are generated by the intracellular proteolyticmachinery that includes, but is not limited to, proteasomecomplexes, tripeptidyl peptidase II and several peptidases[2,5–7]. The MHC class I-presented peptides are transportedby TAP complexes [2,5,8] into the ER, where their bindingand loading to MHC class I heterodimers are mediated by thephysical interaction of TAP complexes and MHC class Iheterodimers via the ER-resident tapasin [8,9]. Once trimo-lecular complexes of MHC class I molecules are formed,MHC class I molecules exit the ER via an exocytic pathwayto the cell surface for inspection by cytotoxic T lymphocytes

* Corresponding author. Tel.: +1-858-450-2023; fax: +1-858-450-2094.E-mail address: [email protected] (Y. Yang)

Microbes and Infection 5 (2003) 39–47

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(Fig. 1). It is commonly believed that surfaces of antigen-presenting cells are adorned with peptide-occupied MHCclass I molecules, with ~0.3–1 × 106 MHC class I moleculesper cell [1,5]. Most peptide species bound to MHC class Imolecules have hundreds of copies on the surface of eachcell. Consequently, each cell is likely to display a heteroge-

neous collection of peptides derived from either self- ornon-self-antigens [1,5]. Pathogen-derived antigenic pep-tides, which form complexes with MHC class I moleculesthat are found on nearly all nucleated cells except neurons,are recognized by cytotoxic T lymphocytes [1,10]. T-cellreceptors on cytotoxic T lymphocytes are capable of distin-guishing the fine differences in these peptides, because thestructure of the bound peptide in a particular MHC class Imolecule appears to be the primary focus of T-cell receptorrecognition [10,11]. Studies of the crystal structures of T-cellreceptor/MHC class I complexes (Fig. 2) show that the hy-pervariable loop of the T-cell receptor interacts with apeptide-bound MHC class I molecule through the sidechains, and some backbone, of the peptide [10,11]. Upondetecting and recognizing pathogen-derived peptides, cyto-

Fig. 1. MHC class I antigen processing and presentation. During assemblyof MHC class I molecules, MHC class I heavy chains (H) initially bind alight chain b2-microglobulin (b2M) in the ER. The complexes of MHC classI heavy chain–b2M heterodimers then interact with transporter-associatedwith antigen processing (TAP) via tapasin, which binds independently toTAP and MHC class I heavy chain-b2M complexes in the ER. The prerequi-site for releasing MHC class I molecules from MHC class I peptide-loadingcomplexes is the binding of eight- to 11-amino acid peptides (P), which aretranslocated across the ER membrane by TAP complexes. Only after for-ming trimolecular complexes can MHC class I heavy chain-b2M-peptidecomplexes be expressed on the surface of antigen-presenting cells via anexocytic pathway for inspection by cytotoxic T lymphocytes. If MHC classI binding peptides are not optimal, MHC class I molecules are retrieved fromthe Golgi to the ER for another round of peptide loading. Intracellularproteasome-dependent proteolytic machinery is required to generate antige-nic peptides of eight- to 11-amino acids and N-terminal extended precursorpeptides, both of which can be transported into the ER by TAP complexes.The ATPase complex (AC) and proteasome regulator PA28 bind indepen-dently to either one or both ends of proteasomes. The ATPase complexrecognizes, recruits and unfolds polyubiquitinated antigens to proteasomes.Binding of PA28 to proteasomes alters the catalytic activities of proteaso-mes, controls the release of peptides from proteasomes and recruits pro-teasomes to TAP complexes. Cytoplasm- or ER-resident aminopeptidases(AP) are responsible for further trimming of the N-terminal extensions ofprecursor peptides.

Fig. 2. Crystal structure of a T-cell receptor/MHC class I/peptide complex.A backbone tube representation of a T-cell receptor (top, a-chain in blue andb-chain in yellow), showing its orientation bound to an MHC class Imolecule (bottom, a1-a3 domains of heavy chain in green and b2M in pink)and presenting an octamer peptide (middle, in orange) with the P1 residuepositioned toward the left. The T-cell receptor covers the MHC classI-binding groove such that the Va and Vb complementarity-determiningregions 1 and 2 are positioned over the N- and C-terminal regions of thebound peptide, respectively. The Va and Vb complementarity-determiningregion 3 straddles the peptide between the helices around the central positionof the peptide (see [50] for details).

40 Y. Yang / Microbes and Infection 5 (2003) 39–47

toxic T lymphocytes are activated and undergo a cytotoxicprocess to eliminate pathogen-infected cells [1].

2. MHC class I molecules

The gene family of human MHC class I heavy chainsconsists of a minimum of 36 open reading frames, includingthe well-characterized HLA-A, -B and -C heavy chains,which collectively, have over 250 alleles [1,12]. The molecu-lar structure of MHC class I heavy chains consists of threeextracellular domains, designated a1, a2 and a3 [1,13]. Theextracellular a1–3 domains are attached to a transmembranesegment that is connected to a highly basic cytoplasmic tail[1,2]. One of the most unique characteristics of MHC class Iheavy chains is the high polymorphism of their a1 and a2domains [1,11]. Crystallographic studies show that the a1and a2 domains of MHC class I heavy chains form a b-sheetplatform, spanned by two a-helices that together form apeptide-binding pocket (Fig. 3). The peptide-binding grooveof MHC class I molecules tethers the amino and carboxyltermini of peptides through a network of hydrogen bonds

between several conserved residues at each end of the grooveand the peptide backbone [13]. By accommodating an eight-to 11-amino acid peptide, the peptide-binding pocket formedby the a1 and a2 domains of MHC class I heterodimersseems to be stabilized [2,5,8]. Since almost all of the poly-morphic amino acids within the family of class I moleculesare clustered around the peptide-binding groove, the shape ofthe groove and the sequence of the bound peptide are uniquefor each MHC class I allele [2,5,11,13]. To efficiently presentappropriate peptides with strictly defined lengths and con-served sequence motifs [2,5,11], polymorphic MHC class Iheterodimers appear to serve as templates for guiding pepti-dases to generate the optimally cleaved peptides in an MHC-dependent manner [14].

Assembly of MHC class I heavy chain-b2M heterodimerswith peptides in the ER is a highly regulated process [2,5].The folding and assembly of MHC class I heavy chains andb2M appear to be orchestrated by the molecular chaperonesof the ER, including BiP, calnexin, calreticulin and ERp57[2,5,8]. Appropriate peptides generated by intracellularproteasome-dependent proteolytic machinery are trans-ported into the ER by TAP complexes and loaded onto MHCclass I heterodimers by MHC class I peptide-loading com-plexes [2,5,8]. The individual components of MHC class Ipeptide-loading complexes, which contain MHC class Iheavy chain-b2M heterodimers, TAP complexes, tapasin,calreticulin and ERp57, appear to interact with each other inmultiple ways, but in a highly cooperative manner [8]. It hasbeen proposed that these interactions facilitate the initialfolding of the MHC class I heavy chain-b2M heterodimersinto a conformation that is able to assemble into the MHCclass I peptide-loading complex [8].Among TAP-transportedpeptides, N-terminal-extended precursor peptides require anaminopeptidase activity in the ER to become optimal MHCclass I-presented peptides [15]. MHC class I heterodimersappear to direct the excision of N-terminal extensions fromlonger precursor peptides in the ER and become the finalrepositories of peptides [16]. Peptide binding, a prerequisitefor stabilizing MHC class I molecules [2,5], promotes therelease of MHC class I molecules. Thereafter, MHC class Ipeptide-loading complexes disassemble, leaving TAP–tapa-sin complexes available for another round of peptide loadingand binding to MHC class I heavy chain-b2M heterodimers[5,8]. The released MHC class I molecules are transported tothe cell surface and present peptides to cytotoxic T lympho-cytes (Fig. 1).

3. MHC class I-presented peptides

The length of MHC class I-presented peptides is usuallyrestricted to eight- to 11-amino acids [2,5,16], because thepeptide-binding pocket of MHC class I molecules is closed atboth ends (Fig. 3). The anchor residues, which are deter-mined by the nature of the side chains in the binding pocket

Fig. 3. Crystal structure of an MHC class I molecule. The a1 (dark blue) anda2 (turquoise) domains of an MHC class I heavy chain form a b-sheetplatform spanned by two a-helices that together form a peptide-bindingpocket and contact both the a3 (green) domain of the MHC class I heavychain and light chain b2M (yellow). The octamer peptide (red) forms acomplex with an MHC class I heterodimer (see [13] for details).

41Y. Yang / Microbes and Infection 5 (2003) 39–47

of a particular MHC class I allele-binding groove, are re-quired for peptide binding [11,13], whereas the N- andC-terminal residues of peptides contribute significantly tobinding affinity [2,5,11,13]. The sum of requirements forallele-specific peptide–MHC class I interactions is believedto define the binding motif for a given MHC class I molecule[5,11,13]. Processing of MHC class I antigens appears tohave adapted a constitutive intracellular proteolytic pathwayfor its use. The majority of peptides presented by MHC classI molecules is most likely derived from cytoplasmic, defec-tive ribosomal products [3], which constitute about 30% ofnewly synthesized proteins in a variety of cell types [3,17].Defective ribosomal products seem to be rapidly processedby proteasome-dependent proteolytic machinery, andthereby, become a major source of antigenic peptides forMHC class I molecules [3,17]. It would make sense to havethe intracellular, proteasome-dependent proteolytic machin-ery in antigen-presenting cells be devoted largely to process-ing newly synthesized proteins rather than long-lived pro-teins, because intracellular infectious pathogens must berapidly detected and eliminated by the immune system [1].Newly synthesized viral glycoprotein antigens could be ret-rograde transported from the ER to the cytoplasm and be-come substrates of proteasome-dependent proteolysis duringinfection [18]. Exogenous particulate antigens, such as bac-teria, introduced by phagocytosis into the cytoplasm or ER ofmacrophages or dendritic cells, could also be processed byproteasome-dependent proteolytic machinery [1,2]. How-ever, these exogenous antigens appear to contribute to a smallsubset of MHC class I-presented peptides. The specificitiesof the proteases in the proteasome-dependent MHC antigen-processing pathway appear to limit the diversity of peptidesavailable for presentation [2,7]. It has been proposed that toovercome this limitation, peptides presented by MHC class Imolecules in professional antigen-presenting cells could bederived from exogenous antigens that are processed via alter-nate mechanisms. Antigen-presenting cells, such as mac-rophages and neutrophils, could process phagocytosed anti-gens by an alternative, vacuolar pathway for processingMHC class I antigens that does not involve intracellularproteasome-dependent proteolysis. Neutrophils could alsosecrete processed peptides that are subsequently presentedby neighboring macrophages or dendritic cells. By present-ing MHC class I antigens directly to effector T lymphocytesat sites of infection or by secreting processed peptides toother antigen-presenting cells that might migrate to lym-phoid organs, these antigen-presenting cells are able to ini-tiate and influence cytotoxic T lymphocyte responses topathogen infection.

4. Intracellular proteasome-dependent proteolysis

The eukaryotic proteasome is an intracellular, multi-catalytic protease complex that is involved in a variety ofcellular functions [7]. The catalytic core of proteasomes is a

cylindrical multi-subunit complex arranged as four axiallystacked heptameric rings (Fig. 4). The two outer rings arecomposed exclusively of seven homologous a subunits,whereas the two inner rings are composed exclusively ofseven homologous b subunits [5,7]. Whereas proteasome asubunits are involved in proteasome structure, b subunitsfunction in catalytic activities [7]. Because the catalytic sitesof proteasomes regulate each other allosterically, protea-somes seem to degrade protein substrates by an ordered,cyclical bite–chew mechanism [5,7]. Crystal structuralanalysis of proteasomes and biochemical studies of mutantproteasomes show that each defined catalytic activity islinked with a specific b subunit [5,7]. In constitutive protea-somes, the three catalytic activities, chymotryptic-like(cleaving after large hydrophobic residues), trypsin-like

Fig. 4. Crystal structure of a proteasome/PA28 complex. Side view ofribbon representation of a proteasome–PA28 complex. The image has beencut away to reveal internal features. PA28 (turquoise); proteasome a subu-nits (purple); proteasome a subunits (blue); residues of the a annulus(green); ordered N-terminal residues of a subunits that do not have counter-parts in a subunits (red); residues of the b annulus (yellow) (see [35] fordetails).


(cleaving after basic residues) and peptidylglutamyl peptidehydrolase-like (cleaving after acidic residues), are contrib-uted by the three b subunits X, Y and Z, respectively, and bytheir corresponding interferon-inducible counterparts in im-munoproteasomes, LMP2, LMP7 and MECL1 [5,7]. Uponinfection, the interferon-inducible LMP2, LMP7 andMECL1 catalytic b subunits replace their catalytic b-subunithomologues, X,Y and Z [5,7], respectively, and form a newlyassembled immunoproteasome that possesses altered cata-lytic specificities [2,5,7]. During the assembly of protea-somes, the interferon-inducible proteasome maturating fac-tor, Ump1p, interacts specifically with proteasome precursorcomplexes and appears to facilitate the maturation and as-sembly of immunoproteasomes [5]. It has been shown thatwhereas constitutive proteasomes are distributed evenlythroughout the cytoplasm and in the nucleus, immunoprotea-somes are concentrated around the ER [5,19]. In comparisonto constitutive proteasomes, immunoproteasomes seem todegrade protein substrates with hydrophobic and basic aminoacids more rapidly than substrates with acidic amino acids[5,7,20,21]. It has been reported that among the peptidesgenerated by proteasomes, 50% are not made by immuno-proteasomes, and 30% are identical to those made by immu-noproteasomes [21]. By generating a partially overlappingbut different repertoire of peptides from proteasomes, immu-noproteasomes could profoundly influence cytotoxic T lym-phocyte responses during infection. Cytotoxic Tlymphocyte-mediated immune responses seem to be mainlydirected to immunoproteasome-dependent T-cell epitopesduring the peak phase of viral and bacterial elimination,because infection with pathogens, such as the lymphocyticchoriomeningitis virus and Listeria monocytogenes, leads toan almost complete replacement of constitutive proteasomesby immunoproteasomes [22]. As a result of an increasedintracellular level of immunoproteasomes with a concomi-tant decreased amount of 26S proteasomes [19,21], infectionseems to limit the generation of self-peptides and optimizesthe access of non-self-peptides to MHC class I heterodimers.Because of its ER localization [5,19], altered catalytic speci-ficities [5,7,20] and increased expression induced by inter-feron [5,7,19,21], immunoproteasome-dependent processingof MHC class I antigens appears to efficiently generate pep-tides that are optimal for translocation by TAP complexesand for binding to MHC class I heterodimers.

It has been suggested that proteasomes are the only en-zymes that generate carboxyl termini of protein substrates,yielding a mixture of proteolytic intermediates rather thansingle, precisely cleaved peptide epitopes [5,7,21]. Some ofthese proteolytic intermediates could be further degraded bycytoplasmic proteases, such as the tricorn protease, whichhas been reported to functionally and physically interact withproteasomes [23]. The intracellular proteasome-dependentproteolytic machinery is capable of generating not only finalproducts of MHC class I-presented peptides but alsoN-terminal-extended precursor peptides [5,7,20,21,24]. Inthe cytoplasm or ER, a subset of these precursor peptides

could be further cleaved of their N-terminal extensions andultimately presented by MHC class I molecules [14–16,24].Though the requirements for N-terminal trimming of precur-sor peptides remain to be determined, trimming ofN-terminal extensions might involve several peptidases, suchas tripeptidyl peptidase II, ERAAP (see below), puromycin-sensitive aminopeptidase, interferon-inducible Gp96 andbleomycin hydrolase [24–28]. The likely complementarityand redundancy of the MHC class I antigen-processing sys-tem acting downstream of proteasomes appear to be neces-sary for generating MHC class I-presented peptides reliablyand for reducing the chance of immuno-evasion throughinterference with antigen processing by pathogens. As anabundant, interferon-inducible ER-resident chaperone, Gp96has been shown to bind to peptides transported by TAPcomplexes and might deliver the bound peptides to MHCclass I heterodimers [27]. Though the aminopeptidase activi-ties reported for Gp96 have been shown to be caused byco-purifying proteins [28], rather than Gp96 itself, the Gp96-associated aminopeptidase activities have been reported toeffectively cleave amino acids from the amino terminus of aprecursor T-cell epitope [27]. It is conceivable that duringinfection, increased expression of Gp96 and/or increasedgp96-associated aminopeptidase activities might enhancepresentation of MHC class I antigens by protecting anddelivering peptides to MHC class I molecules and by trim-ming precursor peptides in the ER. A previously identifiedpuromycin-insensitive leucyl-specific aminopeptidase thathas recently been renamed ER aminopeptidase associatedwith antigen processing (ERAAP), appears to be one of themissing links between the N-terminal extended peptides ofcytosolic processing and the final peptide products presentedby MHC class I molecules on the cell surface. ERAAP is theonly known lumenal protease with a broad substrate specific-ity in the ER, and its expression is strongly upregulated byinterferon [24]. ERAAP seems to be required for MHC classI antigen processing, because reducing the expression ofERAAP prevents the N-terminal trimming of certain precur-sor peptides in the ER and reduces the expression of MHCclass I molecules on the cell surface.

Several peptidases could purportedly participate inN-terminal trimming of precursor peptides in the cytoplasm.Among them, leucine aminopeptidase is most likely to play amajor role in the processing of MHC class I antigens, be-cause expression of leucine aminopeptidase is upregulatedby interferon [29]. Although trimming of precursor peptidescould contribute to generating an optimal repertoire of pep-tides for MHC class I molecules, trimming of precursorpeptides could also take part in destroying some epitopes,thus inhibiting presentation of some immuno-potent epitopes[15]. It is worth noting that HLA-E molecules preferablybind peptides derived from amino acid residues three to 11 ofthe signal sequences of most HLA-A, -B, -C and -G mol-ecules. At least two specific peptidases are needed to gener-ate HLA-E-presented peptides: one signal peptidase for gen-erating signal peptides from pre-proteins of MHC class I


heavy chains and one signal peptide peptidase for furtherprocessing. These signal peptide-derived peptides are foundto be subsequently transported into the lumen of the ER viathe TAP complexes and bound to HLA-E molecules. Conse-quently, surface expression of HLA-E protects HLA-E-expressing cells from natural killer cell-mediated killing [1].

5. Proteasome regulatory complexes

The intrinsic proteolytic activities of proteasomes appearto be determined by the accessibility of protein substrates,because proteasomes are molecular-gated proteases with anaxial-pore diameter of ~1.3 nm [5,7]. To open the gate and toallow substrates to reach the catalytic chamber of protea-somes, several proteasome regulatory complexes appear tohave evolved to associate and modulate proteasome function[5,7,30,31]. The association between proteasomes and theirregulatory complexes appears to be reversible and is prob-ably regulated by phosphorylation [5,19]. Among the protea-some regulatory complexes in processing MHC class I anti-gens, PI31 [30], ATPase complex [7] and proteasomeregulator PA28 [5] are relatively well characterized, but func-tions of other regulatory complexes, such as PA200 [31],remain to be defined. PI31, which acts in vitro as an inhibitorof proteasome activity, appears to function as an inhibitor ofimmunoproteasome formation and of immunoproteasome-mediated MHC class I antigen processing.

ATPase complex consists of 15–20 subunits and is able tobind to one or both of the terminal a rings of proteasomes inan ATP-dependent, cooperative manner [7]. The associationof proteasomes with ATPase complexes forms 26S protea-somes [5,7], which appear to be responsible for degrading awide variety of cellular proteins that are tagged with poly-ubiquitin chains by the cellular ubiquitination machinery.Specific subunits of ATPase complex, such as S5a, have beenshown to bind to polyubiquitin chains [5,7]. It has beenreported that ATPase complex is capable of recognizing,unfolding and de-ubiquitinating ubiquitinated protein sub-strates [7]. In addition, ATPase complex appears to modulatethe function of proteasomes by overcoming their structuralrestrictions for accessing substrates and, therefore, can beconsidered as a proteasome activator [5,7]. It has been pro-posed that ubiquitination machinery is functionally con-nected to proteasomes [32]. Some of the E3 ubiquitin ligasesof the ubiquitination machinery are found to be directlylinked to proteasomes via physical interactions with PLIC, afamily of proteins that contain ubiquitin-like domains [32].Thus, ATPase complexes appear not to only recognize andcatalyze unfolding of ubiqutinated protein substrates but alsoact to open the molecular gate of proteasomes.

The gene family of proteasome regulator PA28 containsthree members, a, b and c, which share approximately 50%amino acid identity [5,7,33]. Orthologous genes for PA28 donot exist in the yeast genome. PA28a and b subunits form aheteropolymer, whereas the PA28c subunit forms a ho-

mopolymer [5,7,33,34]. Since none of the PA28 subunits isan ATPase, activation of proteasomes by PA28 complexesseems to be restricted to peptide substrates [5,7]. The notionthat the PA28ab complex is involved in proteasome-dependent MHC class I antigen processing is supported bythe main localization of PA28a and b on the ER with immu-noproteasomes [5,19] and by the upregulated expression ofPA28a and b subunits, but not PA28c, by interferon andduring infection [5,33,34]. Studies on PA28b-deficient miceshow that in the absence of PA28b function, presentation ofMHC class I antigens is severely altered [34]. By bindingdirectly to the ends of proteasomes, PA28 complexes appearto induce conformational changes on the catalytic sites ofproteasomes and activate selectively the peptidase activitiesof proteasomes. Moreover, binding of PA28 complexes toproteasomes seems to open the molecular gate of protea-somes [35]. The mechanisms by which PA28 and ATPasecomplexes open the proteasome gate probably differ becauseof the distinct structural differences between PA28 and AT-Pase complexes. Crystal structures of proteasomes and pro-teasome–PA28 complexes reveal that by imposing a topo-logical closure on proteasomes in their latent state, a-subunittails of proteasomes prevent the exit or entry of substrates[35]. However, binding of PA28 complexes forces protea-somes to selectively shift the positions of their a-subunittails, allowing them to protrude into the cavity of PA28complexes and open the proteasome gates (Fig. 4). BecauseMHC class I molecules are found to preferentially bindpeptides that are longer than the average-sized peptides thatare generated by proteasomes [2,5,7], these peptide specifici-ties and preferences appear to have evolved from the changesin catalytic activity of the immunoproteasomes and from theselectively increased rate of peptide–substrate dissociationthat results from opening the gates of immunoproteasomesby PA28ab complexes. Preferred binding of PA28ab com-plexes to immunoproteasomes would thus favor the genera-tion of longer peptides, which are optimal for transport byTAP complexes and for binding to MHC class I molecules. Ithas been proposed that immunoproteasomes with an ATPasecomplex at one end and a PA28ab complex at the other endare ideal protease complexes for generating MHC classI-presented peptides [5]. While the ATPase complex recruitsand unfolds ubiquitinated protein substrates, the PA28abcomplex opens the exit gate at the end of the immunoprotea-some that is opposite from the ATPase complex. Most impor-tantly, immunoproteasome and PA28ab complexes havebeen reported to be physically associated with MHC classI—TAP complexes on the ER [5]. It is conceivable that directinteractions between PA28ab complexes, immunoprotea-somes, ATPase complexes, TAP complexes and MHC class Imolecules provide a direct functional link between the sitesof peptide generation by immunoproteasomes, peptide trans-location by TAP complexes and peptide loading onto MHCclass I heterodimers by MHC class I peptide-loading com-plexes.


6. Viral interference of proteasomes

The relationship between infectious pathogens and hostsis a dynamic process [18]. While hosts attempt to suppressand eliminate infections, infectious pathogens try to coexistwith their hosts by evading host immune surveillance [1,18].Because presentation of MHC class I antigens initiates thedetection and clearance of pathogen-infected cells by cyto-toxic T lymphocytes [1], interference with the proteasome-dependent processing of MHC class I antigens appears to beone of the main strategies that pathogens have evolved forevading detection and destruction by the immune system[5,18]. Mutation of viral epitopes during the course of infec-tion represents an effective means for a virus to escaperecognition by cytotoxic T lymphocytes [36]. Structuralvariation in peptides could result in interferences withproteasome-dependent processing of MHC antigens, subse-quent translocation of peptides by TAP complexes, peptideloading and binding to MHC class I molecules and intracel-lular transport of MHC class I molecules from the ER to thesurface of antigen-presenting cells. Structural variation couldalso affect direct recognition by cytotoxic T lymphocytes[18,36]. Moreover, by acting as T-cell receptor antagonists,peptides with mutated amino acids could even prevent acytotoxic T lymphocyte-mediated immune response [37,38].Under the pressure of immune responses, viruses that containmutated amino acid residues in epitopes that are recognizedby cytotoxic T lymphocytes are most likely to be selected andthus establish in their hosts life long infections with few, ifany, clinical manifestations. Epstein–Barr virus, a herpesvirus, appears to establish a long-lasting latent infection of Blymphocytes and nasopharynx epithelial cells by interferingwith the function of proteasomes after primary infection[18]. The internal glycine–alanine repeats of Epstein–Barrnuclear antigen-1 generate a cis-acting inhibitory signal thatblocks its own translocation into proteasomes, thereby pre-venting antigen processing by proteasome-dependent pro-teolysis [39]. Human cytomegalovirus suppresses MHCclass I antigen presentation by a multi-step process thatinvolves the matrix protein kinase pp65 [18,40] and a familyof unique, short region-encoded glycoproteins US2–11[18,41]. By phosphorylating the immediate–early viral tran-scription factor pp72, pp65 inhibits proteasome-dependentgeneration of immuno-potent epitopes derived from pp72[18,40]. US2 and US11 promote retrograde transport ofMHC class I heavy chains from the ER to the cytoplasm viaSec61 translocon complexes for proteasome-dependent deg-radation [18,41,42], thus decreasing surface expression ofMHC class I molecules. Moreover, several types of virusesare capable of transcriptionally downregulating the expres-sion levels of immunological genes, such as MHC class Iheavy chains, LMP2, LMP7, PA28 and TAP[18], conse-quently decreasing the presentation of MHC class I antigens.

At least four types of viruses, hepatitis B virus, humanimmunodeficiency virus type I, human papillomavirus andhuman T-cell leukemia virus type 1, have gene products that

appear to have evolved to physically bind to proteasomes anddirectly interfere with proteasome function [18]. Hepatitis Bvirus X protein, essential for establishing hepatitis B virusinfection in vivo, has been shown to associate with twosubunits of the 26S proteasome, PSMA7 and PSMC1 [43].As one of the ATPase complex subunits, PSMC1 interactswith hepatitis B virus X protein and one of the seven homolo-gous a subunits of proteasome, PSMA7. Because expressionof hepatitis B virus X protein causes a decrease in protea-somes’ chymotrypsin- and trypsin-like activities and hy-drolysis of ubiquitinated substrates, hepatitis B virus X pro-tein seems to act as a negative regulator of proteasomes [5].Hepatitis B virus X protein also interacts with the C-terminalportion of the proteasome a subunit, XAPC7. However, thebiological significance of this interaction in modulating pro-teasome function and in processing MHC class I antigensremains to be investigated. As a viral transcriptional transac-tivator of human immunodeficiency virus type I, Tat not onlyis essential for viral replication but also functions as a repres-sor for a variety of cellular genes. It has been reported thatbesides interacting with three ATPases, MSS1, TBP1 andTBP7, which are highly homologous to the ATPases presentin the 26S proteasome, Tat is able to bind proteasomes andcompetes with PA28 for binding to proteasomes [44]. Be-cause expression of Tat inhibits the chymotrypsin-like activ-ity of proteasomes, Tat interferes with the processing ofMHC class I antigens by preventing the formation ofPA28–proteasome complexes and by altering the catalyticactivities of proteasomes. Infection of human papillomavirusdecreases the expression levels of LMP2 and LMP7 andaffects the presentation of a human papillomavirus-derivedepitope to cytotoxic T lymphocytes. By interacting with theS4 ATPase present in the ATPase complex [45], the E7oncoprotein of human papillomavirus is able to stimulate theATPase activity of 26S proteasomes. Tax of human T-cellleukemia virus type 1 specifically interacts with the b subunitHsN3 and the a subunit HC9 of proteasomes [46]. Thoughthe roles of some of these viral gene products in processingMHC class I antigens remain to be determined, it is conceiv-able that viruses have exploited the intracellular proteasome-dependent proteolytic machinery to evade host immune sur-veillance.

7. Concluding remarks

Elucidating the underlying mechanisms of MHC class Iantigen processing and presentation holds the key to achiev-ing a better understanding of the molecular basis of MHC-associated immunological disorders and infectious diseases.Clearly, many lessons concerning generation of MHC anti-gens and immune responses to infection are yet to be learned.Because availability of pathogen-derived peptides can varygreatly depending on how the functions of proteasomes andaminopeptidases are regulated, further studies are needed todetermine whether additional aminopeptidases are involved


in generating and destroying peptides and whether ami-nopeptidases are constitutively or temporally expressed in acell-, tissue- or development-specific fashion. It will be par-ticularly interesting to study how variable processing ofMHC class I antigens influences the states of infection andvice versa. What is the relationship between infection andcytotoxic T lymphocyte-mediated immune responses withinthe context of MHC antigen processing? How is generationof MHC antigens modulated in states of infection? What arethe possible consequences of disturbances during infectionon expression of the genes that encode MHC antigen pro-cessing machinery? The co-evolution of infection and theMHC antigen processing machinery must still be addressed.The development and clinical severity of multiple neurode-generative diseases are greatly influenced by the function ofMHC class I antigen processing and presentation [47] as wellas by the extent of abnormal, intracellular deposition ofaggregated and ubiquitinated antigens [48,49], which can beinduced by inflammatory immune responses. Because in-flammation is most likely triggered by infection, studying theresponse of the intracellular proteasome-dependent process-ing of MHC antigens to infection and the contribution ofinfection-altered proteasomes to the deposition of aggre-gated and ubiquitinated antigens will help to define the exactcauses of neurodegenerative diseases [5,47]. We have al-ready translated our knowledge of MHC antigen presentationto many clinical applications [5], and one of our great chal-lenges for the future is to fully exploit generation of MHCclass I antigens for advancing immunotherapy-based strate-gies. With rapidly progressing studies of the biochemistryand immunology of MHC antigen processing, we anticipatethat future studies will lead to therapeutic strategies in whichthe processing and presentation of MHC antigens will bemanipulated to treat a variety of neurodegenerative diseases,immunological disorders and infectious diseases.

Acknowledgements

I thank J.G. Luz and I.A. Wilson of the Scripps ResearchInstitute, California, for kindly providing me with Figs. 2 and3, and F.G. Whitby and C.P. Hill of the University of Utah,Utah, for kindly providing me with Fig. 4. My apologies tothose whose work could only be cited indirectly owing tospace limitations.

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generation of major histocompatibility complex class i antigens

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