CHAPTER 26 – ASSOCIATED WITH ANTIGEN PROCESSING AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE

CHAPTER 26 – ASSOCIATED WITH ANTIGEN PROCESSING AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE CHAPTER 26 – ASSOCIATED WITH ANTIGEN PROCESSING AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE CHAPTER 26 – ASSOCIATED WITH ANTIGEN PROCESSING AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE CHAPTER 26 – ASSOCIATED WITH ANTIGEN PROCESSING AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE CHAPTER 26 – ASSOCIATED WITH ANTIGEN PROCESSING AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE

533 THE TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING PHYLOGENETIC AND (TAP): A PEPTIDE TRANSPORT FUNCTIONAL CLASSIFICATION OF AND L OADINGCASSETTE COMPLEX) ABC (ATPBINDING ESSENTIAL FOR CSYSTEMS ELLULAR * IMMUNEER ESPONSE LIE DASSA BRIGITTE LANKAT-BUTTGEREIT AND ROBERT TAMPÉ OVERVIEW OF THE MHC CLASS I ANTIGEN PROCESSING During evolution, the adaptive immune system has developed to protect the organism against pathogens This system consists of three interrelated branches of defense, depending on where the first step of elimination of foreign antigens occurs The humoral system is responsible for recognition and elimination of intact pathogens such as viruses or bacteria in the extracellular space via antibodies These are produced by B-lymphocytes and, subsequently, they activate the complement system The cellular immune system is subdivided into two components In the first, endogenous proteins are degraded in the cytosol by the proteasome and the resulting peptides are transported into the endoplasmic reticulum (ER), where they bind to major histocompatibility complex (MHC) class I molecules In the second, exogenous proteins are degraded after internalization in specialized late endosomal or pre-lysosomal compartments which contain MHC class II molecules The antigenic peptides are directly loaded onto the MHC class II complexes Both peptide-loaded MHC class I and class II ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 26 CHAPTER CHAPTER molecules are transported on the cell surface and recognized by cytotoxic and helper Tlymphocytes, respectively The focus of this chapter is on the MHC class I pathway In the non-infected state, MHC class I complexes are presented on the cell surface by binding peptides derived from normal cellular proteins Cytotoxic T-lymphocytes (CTLs) are not activated by this chronic presentation of self-peptides, because T-cells with the ability to respond to these molecules are eliminated during thymus development The pathways leading to the generation of peptides, their binding to MHC molecules, and their subsequent expression on the cell surface are called antigen processing and presentation (Figure 26.1A) During viral infection or malignant transformation, a set of ‘non-self’ peptides of the target cell is presented to the CTLs, which recognize the MHC class I molecule as a self-component loaded with a peptide derived from non-self proteins and which eliminate these cells (for review, see Ljunggren et al., 1990; Townsend et al., 1989) Interference with this antigen presentation pathway is an effective method for pathogens to dodge the immune response MHC class I molecules are composed of a polymorphic heavy chain (␣-chain), which is encoded within the MHC locus, and an invariant Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 534 ABC PROTEINS: FROM BACTERIA TO MAN CTL A MHC class I molecule PM proteasome Golgi protein peptides tapasin ERp57 β m TAP B α1 calreticulin ER MHC heavy chain calnexin translocon ribosome peptide α2 α1 α2 β2m heavy chain α3 α3 Figure 26.1 A, Antigen processing pathway via MHC class I In the ER, the MHC class I heavy chain associates with the chaperone calnexin After assembly with ␤2-microglobulin (␤2m) and the thiol reductase ERp57, calnexin is replaced by calreticulin Subsequently, tapasin mediates the association with TAP, forming a macromolecular transport and loading complex for antigenic peptides Peptides are generated within the ubiquitin–proteasome pathway in the cytosol After peptide loading, MHC class I molecules are released from the TAP complex and traffic via the Golgi apparatus and the trans-Golgi network to the cell surface There CTLs recognize the antigenic peptides in complex with MHC class I molecules B, Structure of the peptide–MHC complex Side and top view of the MHC class I molecules HLA-A2 with bound antigenic peptide derived from tax protein of human T-cell lymphotropic virus (Madden et al., 1993) The peptide epitope (LLFGYPVYV, yellow, residues are numbered) binds in a groove of the heavy chain of HLA-A2 (blue), which is formed by two ␣-helices on the rim and eight ␤-strands at the bottom of the ␣1 and ␣2 domain Peptide, heavy chain and ␤2-microglobulin (green) form a stable complex with a half-life of several days The groove fits peptides with a length of eight to ten amino acids Peptides are fixed via their free amino and carboxy termini as well as via side-chain interactions at position two or three and the C-terminus Side-chains, which point out of the groove, are monitored by the T-cell receptor non-MHC encoded subunit, ␤2-microglobulin (for review, see Ljunggren et al., 1990; Townsend et al., 1989) The assembly of the different subunits proceeds in the ER by a folding process that is synchronized in time and space by various chaperones After co-translational translocation, the ␣-chain of the MHC class I molecule associates with the chaperone protein, THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE calnexin, and forms a dimer with ␤2-microglobulin (for review, see Pamer and Cresswell, 1998) ␤2-Microglobulin interacts extensively with domains of the ␣-chain and, consequently, the correct folding of the ␣-chain is dependent on the dimerization with ␤2-microglobulin Calnexin is then replaced by another chaperone, calreticulin, and the thiol reductase ERp57 then associates with the complex For the binding of peptides to the MHC class I heterodimer, a macromolecular loading complex, together with tapasin and an ATP-binding cassette (ABC) heterodimeric protein known as the transporter associated with antigen processing (TAP) (ABCB2/ABCB3), is formed Tapasin is an ERresident type I glycoprotein that mediates the efficient interaction of TAP and class I molecules (Sadasivan et al., 1996) The peptide loading onto the MHC class I heterodimer stabilizes the molecule and it is released from the assembly complex for transport to the plasma membrane via the Golgi apparatus and the trans-Golgi network (Figure 26.1B) Normal cellular proteins, as well as viral proteins or proteins that are artificially introduced into the cytosol (Moore et al., 1988; Yewdell et al., 1988), are degraded by an extralysosomal pathway (Morrison et al., 1986) The major sources for antigenic peptides are proteins cleaved by a proteasomebased mechanism, which degrades unfolded or ubiquitinated proteins in the cytoplasmic compartment (Rock and Goldberg, 1999; York et al., 1999) One form of the proteasome is a 20S (700 kDa) cylindrical particle, consisting of 28 subunits arranged in four heptameric rings The outer rings are composed of seven ␣ subunits with regulatory and structural functions, while the inner rings consist of seven ␤ subunits containing the catalytic sites (Baumeister et al., 1998) The 26S proteasome (1500 kDa) is associated with additional subunits, which have a regulatory function Approximately one-third of newly synthesized proteins are degraded by proteasomes into peptides with a size distribution of 3–30 amino acid residues (Kisselev et al., 1999; Schubert et al., 2000; Turner and Varshavsky, 2000) The optimal size is 6–11 residues, which overlaps with the size of antigenic peptides (8–11 residues) bound to MHC class I molecules (Kisselev et al., 1999) The transport of the peptides generated in the cytosol into the ER lumen is executed by TAP (ABCB2/ABCB3) It has been established that any defect in TAP severely impairs antigen presentation Since peptide binding is necessary for stabilization of the MHC complex, a reduced or abolished transport activity of TAP results in reduced cell surface expression of MHC class I molecules Thus, TAP function is essential for antigen presentation and, consequently, inhibition of TAP function is an effective strategy for pathogens to avoid immune surveillance, leading to chronic or latent infections During the last few years, the understanding of the function of TAP has increased significantly Disturbance of peptide delivery to the MHC class I complex is associated with various human diseases from tumor development to infections In this chapter, the current knowledge of the mechanisms enabling transport of peptides from the cytosol into the lumen of the ER for antigen presentation is summarized In addition, TAP serves as an important model system to aid in understanding the topic of multisubstrate specificity, transport mechanism and inhibition of function by natural inhibitors THE TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING, TAP (ABCB2/ABCB3) GENOMIC ORGANIZATION AND REGULATION OF TAP Some years ago, it was observed that some tumor cell lines exhibit a low cell surface expression of MHC class I molecules and are deficient in antigen presentation However, at low temperatures, the expression of the MHC class I ␣-chain and ␤2-microglobulin could be restored to normal levels (Ljunggren et al., 1990; Townsend et al., 1989) The defect was located in the MHC locus and it was concluded that a gene or genes were involved in peptide loading of the class I molecules In the following years, four groups independently discovered candidate genes for proteins that were implicated in the transport of peptides from the cytosol into the lumen of the ER (Deverson et al., 1990; Monaco et al., 1990; Spies et al., 1990; Trowsdale et al., 1990) Since then, these human, 535 536 ABC PROTEINS: FROM BACTERIA TO MAN mouse and rat genes have been renamed as the transporter associated with antigen processing, TAP (TAP1/ABCB2 for RING4, PSF1, mtp1 and HAM1; TAP2/ABCB3 for RING11, PSF2, mtp2 and HAM2) Transfection of defective cell lines with TAP1 (ABCB2) and/or TAP2 (ABCB3) cDNAs restores MHC class I surface expression and antigen presentation These findings indicate that MHC class I molecules are stabilized by binding peptides and that the majority of peptides are transported by TAP from the cytosol into the ER lumen The human TAP genes are located on chromosome band p21.3 in the MHC II locus They are 8–12 kb in size and consist of 11 exons each (Hanson and Trowsdale, 1991) The TAP1 2.5 kb mRNA encodes a protein of 748 amino acids, while the TAP2 2.8 kb mRNA encodes a protein of 686 amino acids Sequence alignments of the coding region from human to horned shark, the most distant vertebrate class displaying an adaptive immune system, exhibit the expected phylogenetic differences For example, human TAP1 shares 98.8% amino acid homology with the gorilla TAP1 protein, 69.2% with the hamster protein, and approximately 43% with the horned shark protein The homology between TAP1 and TAP2 is approximately 35% in all species examined thus far and the two proteins share a similar predicted membrane topology It is likely that these genes evolved from a common ancestral gene by gene duplication prior to the development of the adaptive immune system in jawed vertebrates The human TAP genes contain putative GC-rich elements (Sp1-binding sites) in their 5Ј-flanking sequences, but no TATA box motifs (Beck et al., 1992) It was shown by mutagenesis that the Sp1-binding sites are necessary for the basal promoter activity of TAP1 (Wright et al., 1995) In addition, several other motifs induce TAP1 promoter activity such as interferon (IFN)-␥- and p53-responsive elements (Zhu et al., 1999) Interestingly, the TAP1 gene is coordinately regulated by a bi-directional promoter with the divergently transcribed LMP2 gene (Israel et al., 1989) LMP2 encodes the alternative ␤-type proteasomal subunit, which is important for differential processing of epitopes by constitutive and immunoproteasomes (Gaczynska et al., 1994; Toes et al., 2001) Both genes are stimulated by tumor necrosis factor (TNF)-␣ The induced expression of TAP1 and LMP2 concordantly with upregulated MHC class I genes suggests a link between generation of peptides and expression levels of the transporter Expression of the MHC class I molecules correlates with CTL function and can be increased by cytokines such as IFNs (for review, see Früh and Yang, 1999) TAP1 and TAP2 mRNA and protein levels are rapidly upregulated by IFN-␥, whereas MHC class I ␣-chains and cell surface expression increase more slowly (Ma et al., 1997) A similar enhancement of TAP1 was observed by in vitro treatment of tumor samples with TNF-␣ (Nagy et al., 1998) In contrast to these cytokines, interleukin-10 has a reverse effect on TAP expression and reduces TAP1 and TAP2 levels (Salazar-Onfray et al., 1997) In addition to the interference of TAP function by cytokines, some other mechanisms are known to regulate TAP activity In certain breast cancer cell lines, TAP expression was observed to be dependent on the cell cycle, and the overall amounts of TAP mRNAs were lower than in normal breast epithelial cells (Alpan et al., 1996) Tumor cells may evade host tumor surveillance by mutations that inhibit TAP function Because more than 50% of human tumors have no functional p53, the influence of p53 on TAP1 levels was examined (Zhu et al., 1999) Overexpression of p53 increased TAP1 mRNA and protein levels and, subsequently, MHC class I cell surface expression The authors suggested that a non-functional p53 cannot induce TAP following genotoxic stress Thus, p53 may act as a tumor suppressor by inducing TAP and thereby tumor surveillance STRUCTURAL ORGANIZATION OF TAP Like MDR1 (ABCB1) and MDR3 (ABCB4), TAP1 and TAP2 belong to subfamily B of the ABC superfamily Each TAP protein consists of one ATP-binding domain (nucleotide-binding domain: NBD) and one hydrophobic region of 10 (TAP1) or (TAP2) transmembrane (TM) helices The homology with other ‘half-size’ transporters indicated that a functional TAP complex consisted of either a homodimer of TAP1 or TAP2, or a TAP1/TAP2 heterodimer Heterologous coexpression of TAP1 and/or TAP2 in yeast and insect cells demonstrated that TAP is active as a heterodimer and that no additional factors of the adaptive immune system are needed for TAP function (Meyer et al., 1994; Urlinger et al., 1997) Moreover, immunoprecipitation with antibodies directed against TAP1 co-precipitate THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE TAP1 and TAP2 (Kelly et al., 1992) The TAP complex is located in the ER as shown by immunoelectron and immunofluorescence microscopy (Kleijmeer et al., 1992; Meyer et al., 1994) Both proteins lack an NH2-terminal signal sequence for ER targeting The complex is retarded in the ER by an internal signal sequence Recent studies with truncated proteins indicate that ER retention of both TAP1 and TAP2 is achieved by multiple signals in the transmembrane regions (Vos et al., 1999) TAP1 has three predicted glycosylation sites, two facing the cytosol and one placed in an ER loop, which is likely to be too short for glycosylation Consistent with these predictions, it was found that both proteins are predominantly non-glycosylated (Meyer et al., 1994) A minor subpopulation of TAP has been reported to be N-glycosylated, but this may consist of misfolded protein (Russ et al., 1995) Hydrophobicity analysis predicts that each TAP protein contains 6–10 TM helices depending on the algorithm used (Figure 26.2A) (Elliott, 1997; Gileadi and Higgins, 1997; Nijenhuis and Hämmerling, 1996; Tampé et al., 1997) A core domain of six TM-spanning helices, which is found in all other ABC transporters, may possibly serve to align the translocation pore, and the sequence similarity increases from TM1 through to TM6 By sequence alignments, the first 175 and the first 140 NH2-terminal amino acid residues of TAP1 and TAP2, respectively, which are encoded by exon 1, show no corresponding domains in related ABC transporters It is assumed that these hydrophobic regions contain an additional four and three TM helices, respectively, as extensions and might be necessary for specialization or assembly of the TAP complex (Tampé et al., 1997) To clarify the topology of the TAP complex, it may be useful to construct cysteine-less mutants of TAP1 and TAP2, which are functionally active Single cysteines can then be reintroduced in predicted loop regions and the accessibility checked by thiol-specific reagents Linked to the hydrophobic domains are the NBDs containing the conserved Walker A and B motifs and the ‘C’ transport family signature sequence located in the cytosol According to this model, the complex contains large cytosolic loops, but only a small part passes into the lumen of the ER (Tampé et al., 1997) It was proposed that these TM-spanning domains are arranged in a head–head/tail–tail orientation (Vos et al., 2000), but this alignment contradicts established models of other transporters such A TMD ER N N N C C B B ATP ATP NBD cytosol NBD A C A C peptides TAP1 TAP2 TM1 TM4 B TM5 TM2 TM3 TM6 peptidebinding site TM6 TM5 TM4 TM3 TM2 TM1 Figure 26.2 A, Structural organization of the TAP transporter Membrane topology was predicted based on sequence alignments with other ABC transporters including MDR1 and hydrophobicity analysis The translocation pore is framed by ϫ transmembrane helices from TAP1 and TAP2 (blue cylinders) N-terminal regions of TAP1 and TAP2 have no counterparts in other ABC proteins They putatively contain four and three transmembrane helices, respectively (orange cylinders) The yellow circle encompasses the highly conserved NBD with the Walker A (P loop) and B motifs (red bars) and the C-loop (blue bar) The cytosolic loops following TM6 and within TM5 and TM6 of both subunits delineate the potential peptide-binding site B, Arrangement of the transmembrane helices The transmembrane helices (light blue for TAP1 and dark blue for TAP2) are organized according to the model for MDR1 (Loo and Clarke, 2001a) as P-glycoprotein (ABCB1) (Loo and Clarke, 1995, 2001a) The peptide-binding site is shared by both subunits as was shown by peptide photo-crosslinking and binding experiments (Androlewicz and Cresswell, 1994; Androlewicz et al., 1993; van Endert et al., 1994) Digestion of TAP after photo-crosslinking and subsequent immunoprecipitation with antibodies directed against different epitopes of TAP 537 538 ABC PROTEINS: FROM BACTERIA TO MAN provided more detailed insight into the peptidebinding region of this transporter (Nijenhuis and Hämmerling, 1996) The cytosolic loops between TM4 and TM5 and a COOH-terminal stretch of approximately 15 amino acids following TM6 of TAP1 and TAP2 participate in peptide binding (Figure 26.2A and B) Deletion of some of these potential binding sites of TAP1 resulted in loss of transporter function (Ritz et al., 2001) According to one topological model of TAP (Abele and Tampé, 1999; Tampé et al., 1997), these regions should all be located in the cytosol A different model was proposed by Vos et al (1999), which is in contrast to the established membrane topology of other ABC transporters (Loo and Clarke, 1995) These authors could find no evidence for membrane integration of the two hydrophobic regions adjacent to the NBDs This could be a misleading experimental result derived from singularly expressed, non-functional deletion constructs of TAP1 and TAP2 HOMOLOGUES OF TAP As mentioned previously, sequence alignments show that both TAP proteins belong to subfamily B of the ABC transporter superfamily Members of this subfamily may be ‘full’ transporters like P-glycoprotein/MDR1 (ABCB1) and MDR3 (ABCB4), or half-size transporters like TAP1 (ABCB2), TAP2 (ABCB3) and ABCB9 These transporters translocate a variety of molecules across different biological membranes, e.g steroids and hydrophobic compounds by P-glycoprotein, phosphatidylcholine by MDR3/ ABCB4 (see Chapter 22), possibly iron/glutathione complexes by ABCB6 and ABCB7 (Chapter 25), and monovalent bile salts by BSEP (ABCB11, sPgp) For other members of this subfamily, the substrates are unknown at present Sequence alignments of the NBDs and a phylogenetic tree of the members of subfamily B reveals that TAP1 and TAP2 are most related to ABCB9, a half-size transporter of unknown function (Zhang et al., 2000) (Figure 26.3) The three genes may have arisen by duplication from an ancestral gene Because of the close relationship with TAP1 and TAP2, it is likely that ABCB9 may act as a peptide transporter but this remains to be established At the moment, no partner protein for ABCB9 is known; therefore, the functional complex may be a homo- or a heterodimer The next closest relatives to the TAP proteins are ABCB8 and ABCB10, two MDR subfamily (drugs, lipids) sPgp(N) ABCB5 MDR3(N) MDR1(C) MDR3(C) sPgp(C) MDR1(N) 0.1 ABCB6 MTABC3 MDL1 ABCB7 ABCB10 M-ABC2 MDL1 subfamily (peptides) ABCB8 M-ABC1 ATM1 subfamily (Me-glutathione, peptides) TAP1 ABCB9 TAP2 TAP subfamily (peptides) Figure 26.3 Phylogenetic analysis of TAP homologues The NBDs of all members of the subfamily B of human ABC transporters including the TAP homologue MDL1 in yeast (S cerevisiae) are aligned by ClustalW The member of this family most closely related to TAP is ABCB9 Also, the putative peptide transporters ABCB6, ABCB7, ABCB8 and ABCB10, all located in the inner membranes of mitochondria, are close relatives The yeast mitochondrial peptide transporter MDL1 is included for comparison (gray) The other members of subfamily B transport hydrophobic drugs and lipids (indicated in red) (N) and (C) refer to NBDs of the N- or C-terminal half or full-size transporters mitochondrial ABC transporters which, due to their relation to the yeast transporter MDL1, putatively transport peptides (see Chapter 25) SUBSTRATE SELECTION AND SPECIFICITY OF TAP The first data concerning the character of peptides that are transported by the TAP complex was obtained by trapping peptides in the ER via glycosylation or by binding to MHC class I molecules (Androlewicz et al., 1993; Neefjes et al., 1993) Peptides with a length of 8–16 amino acids were found to have equal affinity for TAP (van Endert et al., 1994), but are most efficiently translocated into the ER when they are 8–12 residues long (Koopmann et al., 1996) Moreover, free NH2- and COOH-termini are prerequisites for transport (Momburg et al., 1994; Schumacher et al., 1994a; Uebel et al., 1997) By screening combinatorial peptide libraries, the contribution of each amino acid to the THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE Diversity Specificity Amino acid Specificity Specificity A D E F G H I K L M N P Q R S T V W Y Affinity for TAP G kJ kJ Ϫ8 kJ Position H H Promiscuity H ϩ N in sequence and length C Contact sites (TAP1/2) T-cell recognition HLA restricted Figure 26.4 Specificity of TAP By using combinatorial peptide libraries and statistical analysis, human TAP was found to be most specific for the three N-terminal and C-terminal residues (Uebel et al., 1997) Favored amino acids (K, N and R in the first, R in the second and W and Y in the third position) are shown in blue (negative ⌬ ⌬G values) in the middle panel At the C-terminus F, L, R or Y are preferred for binding Disfavored amino acids are shown in red (positive ⌬ ⌬G values) In the lower panel a model of the peptide-binding site to TAP is shown with the variable region of the peptide labeled in yellow stabilization of peptide binding to TAP was analyzed (Uebel et al., 1997) A randomized peptide mixture with one defined residue was compared with a totally randomized peptide library, and the influence of each amino acid on the affinity for TAP was determined The peptide with the best binding characteristics showed a 45-fold higher affinity for TAP than a totally randomized peptide mixture The effect of each amino acid was found to be critically dependent on its position in the peptide (Figure 26.4) Thus, the first three NH2terminal residues and the COOH-terminal amino acid were most important for substrate specificity TAP displayed preferences for peptides with Lys, Asn and Arg in the first, Arg in the second, and Trp and Tyr in the third position at the NH2-terminus The most profound differences were observed for peptide residues at the COOH-terminus The highest affinity for TAP binding was found for peptides with hydrophobic or basic amino acids (Phe, Leu, Tyr or Arg) in this position It is interesting that these residues at the COOH-terminus are also advantageous for binding to MHC class I molecules Moreover, none of the disfavored amino acids served as a preferred anchor for MHC class I binding Thus, it is speculated that the recognition and binding principles of TAP and MHC class I molecules coevolved The contribution of the peptide backbone to the substrate specificity of TAP was determined by using peptides of different length by exchanging each residue with its D-enantiomer D-Amino acids in positions 1–3 and the COOHterminal position resulted in a markedly reduced affinity for TAP Thus, contact between a peptide and TAP seems to occur via the peptide backbone, the amino acid side-chains and the free NH2- and COOH-termini, which is fixed by hydrogen bonding (Uebel et al., 1997) The residues in the center of the peptide between positions 1–3 and the COOH-terminal amino acid seem to have only a little or no effect on the substrate specificity of TAP This binding property can explain how larger peptides can bulge out of the binding pocket and how large amino acid side-chains and even fluorescence labels can be accommodated (Neumann and Tampé, 1999; Uebel et al., 1995) Interestingly, these residues are responsible for the detection of the MHC class I-bound peptide by the T-cell receptor Therefore, by binding at the termini, TAP transports peptides with maximal diversity in the center of the peptide (positions 5–8), where T-cell recognition occurs Therefore, a coevolution of the genes involved in antigen presentation seems likely to have taken place in order to optimize the antigen processing and recognition machinery (Uebel and Tampé, 1999) Polymorphisms in TAP have been found in human, mouse and rat by sequence analysis and restriction length polymorphism analysis (Daniel et al., 1997; Momburg et al., 1994; Powis et al., 1992; Schumacher et al., 1994a, 1994b) Although polymorphisms can contribute to immune diversity, no effect of the amino acid changes on the substrate specificity for human and mouse TAP was observed However, a rat TAP polymorphism has a significant influence 539 540 ABC PROTEINS: FROM BACTERIA TO MAN on peptide selectivity (Powis et al., 1992) Human TAP and rat TAP from the RT1a strain were found to prefer peptides with hydrophobic or basic residues at the COOH-terminus, while mouse TAP and rat TAP from the strain RT1u favored peptides with hydrophobic COOHterminal residues The TAP complexes from RT1a and RT1u strains differ by the exchange of 25 amino acids in TAP2: 23 in the transmembrane domain (TMD) and two in the NBD In addition to gene polymorphisms, altered substrate specificity can be achieved by alternative splicing Recently, a variant of the human TAP2 protein, called TAP2iso, was described (Yan et al., 1999) This splice variant lacks exon 11 comprising a part of the coding region and the original 3Ј-untranslated sequence Instead, it contains a previously unidentified exon 12 Expression of TAP2iso mRNA was found to be coincident with TAP2 mRNA in several cell lines Interestingly, heterodimers of TAP1 and TAP2iso more resemble mouse and rat RT1u TAP, with preferences for peptides with hydrophobic COOH-termini Thus, alternative splicing may be a strategy for the organism to acquire broadened epitope diversity How the variable COOH-terminus of the TAP2 NBD affects TAP substrate specificity remains an open and intriguing question TRANSPORT MECHANISM OF TAP The transport mechanism of TAP is the subject of intensive investigation because of its important role in immune recognition Translocation into the lumen of the ER is a multistep process, consisting of binding of the peptide to TAP, isomerization of the complex, and transport (Figure 26.5) (review by Abele and Tampé, 1999) Peptide interaction with the binding site shared by both TAP subunits is ATP-independent and follows a monophasic 1:1 Langmuir adsorption model (Uebel et al., 1995) In direct binding or competition assays, no evidence for a second interaction site was found However, it cannot be excluded that a second binding site with a very low affinity, or with a similar affinity, exists Real-time kinetic analysis of the peptide binding with environmentally sensitive fluorescence labeled peptides revealed that this process could be subdivided into two steps (Neumann and Tampé, 1999) The peptide binding occurs in a fast bimolecular association step and determines the specificity of TAP; subsequently, a slow isomerization of the Peptide ATP ADP TAP TAP ATP ADP TAP TAP ADP ϩ Pi ADP ϩ Pi ADP ATP TAP TAP ADP TAP TAP Figure 26.5 Working model of the translocation mechanism by TAP In the ground state, ATP interacts primarily with the TAP1 subunit, whereas TAP2 most probably contains pre-bound ADP in an occluded state (red) (Alberts et al., 2001; Karttunen et al 2001; Lapinski et al., 2001; Saveanu et al., 2001) High-affinity peptide binding occurs in a fast reaction followed by a slow isomerization of the TAP complex, promoting allosteric coupling between the two NBDs (Neumann and Tampé, 1999) Peptide binding to TAP triggers ATP hydrolysis and subsequent translocation of the solute (Gorbulev et al., 2001) ATP hydrolysis and peptide binding are tightly coupled The release of inorganic phosphate and subsequently ADP at TAP1 might catalyze nucleotide exchange at TAP2 ATP hydrolysis at TAP2 finally closes the transport cycle by restoration of the high-affinity peptide-binding pocket The maximal turnover rate of the transport cycle was determined to be around ATP per second (Gorbulev et al., 2001) TAP complex takes place It is proposed that the conformational change of the molecule triggers ATP hydrolysis and, thereby, peptide transport into the lumen of the ER The isomerization of the complex also affects its lateral mobility as analyzed by fluorescence recovery after photobleaching (Reits et al., 2000) This increases when TAP is inactive and decreases during peptide translocation, as was shown in studies with TAP1 tagged with green fluorescent protein (GFP) at its cytosolic COOHterminus However, owing to the presence of endogenous TAP, the activity of the GFPtagged complex could not be unequivocally established THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE The transport of peptides from the cytosol to the ER lumen strictly requires hydrolysis and not merely binding of ATP, UTP, CTP or GTP (Androlewicz et al., 1993) Non-hydrolyzable analogues of ATP, nucleotide depletion by apyrase, or competition with ADP, completely abrogate peptide translocation (Meyer et al., 1994; Neefjes et al., 1993; Shepherd et al., 1993) Evidence of the binding of nucleotides to the NBDs was obtained by crosslinking experiments with 8-azido-ATP (Müller et al., 1994; Russ et al., 1995) Nucleoside tri- and diphosphates can compete for binding and have similar affinities for TAP Thus, for example, ADP inhibits peptide translocation By developing an enrichment and reconstitution protocol for TAP, it was possible to restore the function in proteoliposomes and to examine the specific ATPase activity (Gorbulev et al., 2001) Nucleotide hydrolysis was found to be strictly dependent on binding of peptides and on crosstalk with the peptide-binding and the translocation sites The strict correlation between peptide binding and stimulation of ATP hydrolysis may be a strategy to avoid ‘wasting’ ATP without transport of peptides A further indication of the tight coupling between ATP hydrolysis and peptide transport is the observation that sterically restricted peptides, which cannot be transported by TAP, not induce ATP hydrolysis Maximal ATPase activity of TAP was found to be independent of substrate affinity, because peptides with different KD values for the transporter exhibited the same Vmax values (Gorbulev et al., 2001) The two NBDs of the functional TAP complex can both interact with ATP, even if TAP1 or TAP2 are expressed separately (Müller et al., 1994; Wang et al., 1994) However, alone, the NBDs are unable to hydrolyze ATP Thus, communication between the NBDs and TMDs leading to a conformational change of the NBDs by peptide binding to TAP seems to be a requirement to activate ATPase function Furthermore, TAP function is dependent on the presence of both NBDs since disruption of one NBD leads to loss of transport (Chen et al., 1996) Thus, it has been speculated that hydrolysis at one NBD is necessary for the beginning of the transport, whereas hydrolysis at the second NBD completes the cycle and may promote the reconversion of the peptide binding site to the initial state (Abele and Tampé, 1999) But one question remains: are the two NBDs equal in function or they have distinct functional properties? To address this point, several groups have introduced mutations in the Walker A and/or B motifs in the NBDs of TAP1 and TAP2 and examined the effects on transport function Lysine mutations in the Walker A sequences affecting nucleotide binding/hydrolysis by TAP1 or TAP2 suggest that each NBD plays a distinct functional role (Karttunen et al., 2001; Lapinski et al., 2001; Saveanu et al., 2001) Even if the data concerning nucleotide and peptide binding from the different groups are in part contradictory, it seems that nucleotide binding to TAP2 maintains a peptide-receptive TAP conformation, and that TAP2-mediated ATP hydrolysis is essential for translocation The functional consequences of mutations in the Walker A motifs of TAP1 and TAP2 have led to speculations that ATP hydrolysis at TAP1 initiates the transport cycle and is a requirement for binding of ATP to the NBD of TAP2 (Alberts et al., 2001) Hydrolysis by TAP2 might then complete the cycle by restoring the peptidebinding site The use of chimeric proteins consisting of the TAP1 membrane-spanning domain and the TAP2 NBD, and vice versa, indicate that both membrane-spanning domains of TAP1 and TAP2 are necessary, but that neither NBD encompasses signals unique for peptide binding (Arora et al., 2001) These observations are in agreement with earlier data demonstrating that the TM domains of both TAP1 and TAP2 are needed to form the peptide-binding pocket The NBD-switched complexes are all transport competent (Arora et al., 2001) Even TAP complexes with two identical NBDs are able to translocate peptides, although with a lower efficiency The two NBDs of TAP1 and TAP2 appear to possess different functional properties Further studies will elucidate the exact roles of each NBD within the transport cycle TAP AS A CENTRAL PART OF A MACROMOLECULAR PEPTIDE TRANSPORT AND CHAPERONE COMPLEX TAP not only delivers peptides necessary for antigen presentation into the ER lumen, but is also part of a large macromolecular loading complex which is critical for MHC class I 541 ABC PROTEINS: FROM BACTERIA TO MAN VIRAL STRATEGIES OF IMMUNE EVASION In non-infected cells, MHC class I molecules are stably expressed on the cell surface presenting peptides derived from intracellular proteins herpes simplex virus human cytomegalovirus TAP2 TAP1 US6 TAP2 maturation A number of proteins have been identified which play an important role in this complex (for review, see Cresswell et al., 1999) At least three proteins are involved in the assembly of peptide-loaded TAP and MHC class I heterodimers: the chaperone calreticulin, the thiol reductase ERp57, and the glycoprotein tapasin (TAP-associated glycoprotein) Calreticulin acts as a chaperone to ensure proper folding (Sadasivan et al., 1996), whereas ERp57 probably supports the correct formation of disulfide bridges (Lindquist et al., 1998), and tapasin mediates the efficient interaction of TAP and the MHC class I molecules and stabilizes the loading complex (Ayalon et al., 1998; Ortmann et al., 1997) The stoichiometry of this complex was determined to be four MHC class I heterodimers associated with four tapasins to one TAP heterodimer (Ortmann et al., 1997) In the absence of tapasin, the assembly in the ER is impaired and MHC class I antigen presentation decreases However, in a tapasin-deficient cell line, the class I cell surface expression and function is restored by a truncated soluble tapasin lacking the transmembrane region, even if the remaining cytosolic tail is not linked to TAP (Lehner et al., 1998) The physical association of TAP and class I molecules leads to the assumption that the peptides are directly loaded from TAP onto the MHC class I complex However, application of anti-peptide antibodies inhibits peptide binding to class I molecules (Hilton et al., 2001) Therefore, these authors suggested that most TAP-transported peptides diffuse through the ER lumen before being loaded onto MHC class I molecules The binding of the peptides is necessary for the dissociation of TAP–MHC class I complexes and is dependent on conformational signals from TAP in an ATP-dependent manner (Cresswell et al., 1999; Knittler et al., 1999) Recent observations point to a more pronounced conformational role of TAP1 in the dynamic activity of the loading complex (Alberts et al., 2001) The release of the MHC class I molecules for transport to the cell surface is synchronized with peptide binding and peptide translocation by TAP TAP1 542 ER cytosol ICP47 Figure 26.6 Inhibition of TAP function by viral proteins The herpes simplex virus-encoded protein ICP47 blocks the TAP-mediated peptide transport by binding to the cytosolic part of the TAP complex (left side), whereas the human cytomegalovirus protein US6 inhibits TAP function by binding to the ER-luminal side (right side) The association of TAP with tapasin and MHC class I molecules seems to be unaffected by both proteins Presenting peptides from viral proteins enables T-cells to recognize and eliminate infected cells To propagate in the presence of an active immune system, the virus must develop a strategy to avoid an immune response One mechanism is to interfere with the antigen presentation pathway at different stages (for review see Ploegh, 1998) Many steps are susceptible to viral disturbances, such as the generation of peptides (Gilbert et al., 1996; Levitskaya et al., 1997), the export of class I molecules to the cell surface (Früh et al., 1999; Hengel et al., 1999), and the transport of peptides by TAP Here, we will focus on the latter point (Figure 26.6) Human cytomegalovirus (HCMV) encodes several proteins inhibiting cell surface expression of MHC class I molecules (for review, see Hengel and Koszinowski, 1997) One of these proteins is known to interfere with intracellular peptide transport: gpUS6 gpUS6 is an ER-resident glycoprotein that probably binds to the ER luminal part of the TAP complex (Ahn et al., 1997; Hengel et al., 1997; Lehner et al., 1997) The association of TAP with tapasin, calreticulin and MHC class I molecules seems to be unaffected by gpUS6, as is the binding of peptides to TAP Recent data point to a binding of gpUS6 to TAP, which results in stabilization of a TAP1 conformation that is unable to bind ATP (Hewitt et al., 2001; Kyritsis et al., 2001) Consequently, the energy for peptide transport is lacking It is possible to override this inhibition by overexpression of TAP, for example by induction with IFN-␥ Herpes simplex virus type I (HSV-1) has evolved a completely different strategy to avoid immune recognition This virus encodes the THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE immediate early protein ICP47 of 88 amino acids The expression of ICP47 leads to a downregulation of MHC class I antigen presentation in human fibroblasts (York et al., 1994) ICP47 inhibits TAP-mediated peptide transport into the ER lumen by binding with high affinity to the cytosolic side of the TAP complex (Früh et al., 1995; Hill et al., 1995), thereby preventing the binding of peptides and translocation (Ahn et al., 1996; Tomazin et al., 1996) Moreover, it was found that ICP47 has a 100-fold higher affinity for human TAP than for murine TAP and thus it acts in a species-specific manner The amino acids strictly required for ICP47 function include residues 3–34 (Neumann et al., 1997) This active domain seems to be unstructured in aqueous solution, but in a lipid-like environment it adopts an ␣-helical structure with two helical regions from amino acid 4–15 and 22–32 (Beinert et al., 1997; Pfänder et al., 1999) The two helices are linked by a flexible loop (helixloop-helix motif) and the authors propose that one helix binds to TAP, while the other one is attached to the membrane The elucidation of the ICP47 structure can serve as a template for new specific therapeutic agents, either for use as immune suppressors or for vaccination strategies against HSV Viruses from other families can also interfere with immune recognition via TAP impairment Two of these viruses are the adenovirus and the human papilloma virus (HPV) (Bennett et al., 1999; Vambutas et al., 2000) Adenoviruses express E3/19K, a protein which serves two purposes On the one hand, it directly binds to MHC class I molecules, trapping them in the ER On the other hand, it seems to bind to TAP and consequently inhibit TAP–tapasin association and thereby the assembly of the macromolecular loading complex (Bennett et al., 1999) HPV interferes with immune recognition via another mechanism This virus causes different clinical courses with recurrent papillomatosis and can lead to significant morbidity Although the precise mechanism is unclear at the moment, it was found that in laryngeal papilloma tissue biopsies and in cell culture of primary explants, TAP levels are markedly downregulated and correlate inversely with the frequency of disease recurrence (Vambutas et al., 2000) The expression of TAP1 is even considered a measure for disease severity By decreasing the TAP levels and thereby MHC class I levels, HPV may evade immune recognition, which in turn leads to persistent infection of the host cells For other malignancies induced by HPV, such as HPV-16 and HPV-18 infected carcinomas of the cervix, a similar downregulation of TAP1 and MHC class I molecules has been described (Cromme et al., 1994) One mechanism by which a decrease in TAP can occur is known from studies of the Epstein–Barr virus (EBV) (Zeidler et al., 1997) EBV expresses an interleukin-10like protein, causing the downregulation of TAP1 and subsequent MHC class I cell surface expression This strategy may promote persistent EBV infections TUMOR ESCAPE STRATEGIES Although defects in the TAP complex are associated with a subgroup of individuals with a rare genetic disease (bare lymphocyte syndrome type 1) (de la Salle et al., 1994, 1999; Teisserenc et al., 1997), TAP may play a more important role in tumor development Tumors often avoid immune recognition by ineffective display of the antigen presentation pathway In some cancers, such as melanomas, it was demonstrated that MHC class I molecules on the cell surface were reduced (Seliger et al., 1997b; Sherman et al., 1998) This effect can be due to low levels of TAP1 and/or TAP2 as was shown for cell lines from human small cell lung cancer (Seliger et al., 1997b) and breast cancer (Alimonti et al., 2000) In various cases, the TAP levels could be restored by treatment of the cells with IFN-␥ Also, TAP1 is downregulated in other human tumors, but the mechanism or mutation responsible is unknown (Chen et al., 1996; Seliger et al., 1997a) A defective or non-existent TAP complex causes a decrease in, or even loss of, MHC class I expression on the cell surface One way of reducing TAP levels may be via cytokines, such as interleukin-10, which are known to downregulate TAP (Zeidler et al., 1997) A large number of tumors secrete interleukin-10; accordingly, this fact may be of clinical importance and needs further investigation In addition to downregulation of TAP expression, mutations in the TAP genes can also contribute to tumor escape from immune recognition In a small cell lung cancer cell line, a mutation was found which introduces an amino acid exchange at position 659 of TAP1 (Chen et al., 1996) This mutation is located between the ‘C’ motif and the Walker B motif and might interfere with ATP binding or hydrolysis The resulting TAP complex is non-functional It is 543 544 ABC PROTEINS: FROM BACTERIA TO MAN important to stress the fact that mutations in TAP1 occur frequently in a variety of human tumors, such as primary breast cancers, and that the expression of TAP1 can be linked with tumor grading and reduced survival (Kaklamanis et al., 1995; Vitale et al., 1998) In contrast to high-grade breast carcinoma lesions, low-grade lesions exhibited normal TAP levels (Vitale et al., 1998) Based on these findings, the therapeutic potential of exogenous TAP was tested in a TAP-deficient small cell lung carcinoma cell (Alimonti et al., 2000) A clear regression of tumors in mice was shown after infection with vaccinia virus containing the TAP1 gene Therefore, this method could increase the immune response against tumors and should be kept in mind for implementation in cancer therapies Some studies show a correlation between deficiencies in MHC class I antigen presentation and tumor progression (Kaklamanis et al., 1995; Seliger et al., 1997b; Vitale et al., 1998), but the significance for tumor development is still uncertain To gain a better insight into the problem, matched panels of TAP1-positive and TAP1-negative tumor cell lines were established and inoculated into mice (Johnsen et al., 1999) The TAP-negative cells resulted in large and persistent tumors, but the TAP-positive cells did not produce lasting tumors As a control, both cell lines were shown to generate tumors in athymic mice, thereby confirming that the tumorigenicity can be attributed to the T-cell immune response Because of the different types of tumors, the clinical significance of reduced MHC class I cell surface expression varies markedly and needs further studies to explore potential diagnostic and therapeutic applications CONCLUSIONS The transport of peptides from the cytosol into the ER lumen is a key step in the MHC class I antigen presentation pathway This translocation is performed by the TAP complex, a heterodimer of TAP1 and TAP2 It is known that disruption of peptide transport severely interferes with the T-cell-mediated immune response The TAP transporter may be involved in several (patho) physiological processes; the elucidation of the molecular structure, topology and function of TAP will enhance our knowledge of the underlying mechanisms of various diseases and, thereby, provide the basis for the development of new therapeutic strategies It is conceivable that new vaccines may be developed by understanding how some viruses escape the immune response Knowledge about every step in TAP action may even improve the treatment of some cancers, because some studies have shown that loss of TAP function occurs frequently in metastatic tumors One possible therapeutic strategy may be to restore the immune response against tumor cells which evade immune recognition by disruption of TAP function However, many questions remain unanswered at the moment For example, what are the conformational changes upon peptide binding? And how the molecules involved in the macromolecular peptide transport and loading complex interact? Further investigations will provide new insights about the significance of the loss of TAP function, and about the mechanisms by which viruses and some malignant tumor cells escape immune recognition REFERENCES Abele, R and Tampé, R (1999) Function of the transport complex TAP in cellular immune recognition Biochim Biophys Acta 1461, 405–419 Ahn, K., Meyer, T.H., Uebel, S., Sempé, P., Djaballah, H., Yang, Y., Peterson, P.A., Früh, K and Tampé, R (1996) Molecular mechanism and species-specificity of TAP inhibition by Herpes-Simplex virus protein ICP47 EMBO J 15, 3247–3255 Ahn, K., Gruhler, A., Galocha, B., Jones, T.R., Wiertz, E.J.H.J., Ploegh, H.L., Peterson, P.A., Yang, Y and Früh, K (1997) The ER-luminal domain of the HCMV glycoprotein US6 inhibits peptide translocation by TAP Immunity 6, 613–621 Alberts, P., Daumke, O., Deverson, E.V., Howard, J.C and Knittler, M.R (2001) Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle Curr Biol 11, 242–251 Alimonti, J., Zhang, Q.J., Gabathuler, R., Reid, G., Chen, S.S and Jefferies, W.A (2000) TAP expression provides a general method for improving the recognition of malignant cells in vivo Nat Biotechnol 18, 515–520 Alpan, R.S., Zhang, M and Pardee, A.B (1996) Cell cycle-dependent expression of TAP1, TAP2, and HLA-B27 messenger RNAs in a human breast cancer cell line Cancer Res 56, 4358–4361 THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE Androlewicz, M.J and Cresswell, P (1994) Human transporters associated with antigen processing possess a promiscuous peptide binding site Immunity 1, 7–14 Androlewicz, M.J., Anderson, K.S and Cresswell, P (1993) Evidence that transporter associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner Proc Natl Acad Sci USA 90, 9130–9134 Arora, S., Lapinski, P.E and Raghavan, M (2001) Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation Proc Natl Acad Sci USA 98, 7241–7246 Ayalon, O., Hughes, E.A., Cresswell, P., Lee, J., Odonnell, L., Pardi, R and Bender, J.R (1998) Induction of transporter associated with antigen-processing by interferongamma confers endothelial-cell cytoprotection against natural killer-mediated lysis Proc Natl Acad Sci USA 95, 2435–2440 Baumeister, W., Walz, J., Zühl, F and Seemüller, E (1998) The proteasome – paradigm of a self-compartmentalizing protease Cell 92, 367–380 Beck, S., Kelly, A., Radley, E., Khurshid, F., Alderton, R.P and Trowsdale, J (1992) DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing J Mol Biol 228, 433–441 Beinert, D., Neumann, L., Uebel, S and Tampé, R (1997) Structure of the Viral TAPinhibitior ICP47 induced by membrane association Biochemistry 36, 4694–4700 Bennett, E.M., Bennink, J.R., Yewdell, J.W and Brodsky, F.M (1999) Cutting edge: adenovirus E19 has two mechanisms for affecting class I MHC expression J Immunol 162, 5049–5052 Chen, H.L., Gabrilovich, D., Tampé, R., Girgis, K.R., Nadaf, S and Carbone, D.P (1996) A functionally defective allele of TAP1 results in loss of MHC class I antigen presentation in a human lung cancer Nat Genet 13, 210–213 Cresswell, P., Bangia, N., Dick, T and Diedrich, G (1999) The nature of the MHC class I peptide loading complex Immunol Rev 172, 21–28 Cromme, F.V., Airey, J., Heemels, M.T., Ploegh, H.L., Keating, P.J., Stern, P.L., Meijer, J.C.L.M and Walboomers, C.M.M (1994) Loss of transporter protein, encoded by the TAP-1 gene, is highly correlated with loss of HLA expression in cervical carcinomas J Exp Med 179, 335–340 Daniel, S., Caillatzucman, S., Hammer, J., Bach, J.F and Van Endert, P.M (1997) Absence of functional relevance of human transporter associated with antigen processing polymorphism for peptide selection J Immunol 159, 2350–2357 de la Salle, H., Hanau, D., Fricker, D., Urlacher, A., Kelly, A., Salamero, J., Powis, S.H., Donato, L., Bausinger, H and Laforet, M (1994) Homozygous human TAP peptide transporter mutation in hla class-I deficiency Science 265, 237–241 de la Salle, H., Zimmer, J., Fricker, D., Angenieux, C., Cazenave, J.P., Okubo, M., et al (1999) HLA class I deficiencies due to mutations in subunit of the peptide transporter TAP1 J Clin Invest 103, R9–R13 Deverson, E.V., Gow, I.R., Coadwell, W.J., Monaco, J.J., Butcher, G.W and Howard, J.C (1990) MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters Nature 348, 738–741 Elliott, T (1997) Transporter associated with antigen processing Adv Immunol 65, 47–109 Früh, K and Yang, Y (1999) Antigen presentation by MHC class I and its regulation by interferon gamma Curr Opin Immunol 11, 76–81 Früh, K., Ahn, K., Djaballah, H., Sempé, P., van Endert, P.M., Tampé, R., Peterson, P.A and Yang, Y (1995) A viral inhibitor of peptide transporters for antigen presentation Nature 375, 415–418 Früh, K., Gruhler, A., Krishna, R.M and Schoenhals, G.J (1999) A comparison of viral immune escape strategies targeting the MHC class I assembly pathway Immunol Rev 168, 157–166 Gaczynska, M., Rock, K.L., Spies, T and Goldberg, A.L (1994) Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complexencoded genes for lmp2 and lmp7 Proc Natl Acad Sci USA 91, 9213–9217 Gilbert, M.J., Riddell, S.R., Plachter, B and Greenberg, P.D (1996) Cytomegalovirus selectively blocks antigen-processing and presentation of its immediate-early geneproduct Nature 383, 720–722 Gileadi, U and Higgins, C.F (1997) Membrane topology of the ATP-binding cassette 545 546 ABC PROTEINS: FROM BACTERIA TO MAN transporter associated with antigen presentation (TAP1) expressed in Escherichia coli J Biol Chem 272, 11103–11108 Gorbulev, S., Abele, R and Tampe, R (2001) Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP Proc Natl Acad Sci USA 98, 3732–3737 Hanson, I.M and Trowsdale, J (1991) Colinearity of novel genes in the class II regions of the MHC in mouse and human Immunogenetics 34, 5–11 Hengel, H and Koszinowski, U.H (1997) Interference with antigen-processing by viruses Curr Opin Immunol 9, 470–476 Hengel, H., Koopmann, J.O., Flohr, T., Muranyi, W., Goulmy, E., Hämmerling, G.J., Koszinowski, U.H and Momburg, F (1997) A viral ER-resident glycoprotein inactivates the MHC-encoded peptide transporter Immunity 6, 623–632 Hengel, H., Reusch, U., Gutermann, A., Ziegler, H., Jonjic, S., Lucin, P and Koszinowski, U.H (1999) Cytomegaloviral control of MHC class I function in the mouse Immunol Rev 168, 167–176 Hewitt, E.W., Gupta, S.S and Lehner, P.J (2001) The human cytomegalovirus gene product US6 inhibits ATP binding by TAP EMBO J 20, 387–396 Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H and Johnson, D (1995) Herpes simplex virus turns off the TAP to evade host immunity Nature 375, 411–415 Hilton, C.J., Dahl, A.M and Rock, K.L (2001) Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum J Immunol 166, 3952–3956 Israel, A., Le Bail, O., Hatat, D., Piette, J., Kieran, M., Logeat, F., Wallach, D., Fellous, M and Kourilsky, P (1989) TNF stimulates expression of mouse MHC class I genes by inducing an NF kappa B-like enhancer binding activity which displaces constitutive factors EMBO J 8, 3793–3800 Johnsen, A.K., Templeton, D.J., Sy, M and Harding, C.V (1999) Deficiency of transporter for antigen presentation (TAP) in tumor cells allows evasion of immune surveillance and increases tumorigenesis J Immunol 163, 4224–4231 Kaklamanis, L., Leek, R., Koukourakis, M., Gatter, K.C and Harris, A.L (1995) Loss of transporter in antigen-processing-1 transport protein and major histocompatibility complex class-i molecules in metastatic versus primary breast-cancer Cancer Res 55, 5191–5194 Karttunen, J.T., Lehner, P.J., Gupta, S.S., Hewitt, E.W and Cresswell, P (2001) Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP) Proc Natl Acad Sci USA 98, 7431–7436 Kelly, A.P., Powis, S.H., Kerr, L.-A., Mockridge, I., Elliott, T., Bastin, J., UchanskaZiegler, B., Ziegler, A., Trowsdale, J and Townsend, A (1992) Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex Nature 355, 641–644 Kisselev, A.F., Akopian, T.N., Woo, K.M and Goldberg, A.L (1999) The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes – implications for understanding the degradative mechanism and antigen presentation J Biol Chem 274, 3363–3371 Kleijmeer, M., Kelly, A., Geuze, H.J., Slot, J.W., Townsend, A and Trowsdale, J (1992) Location of MHC-encoded transporters in the endoplasmic reticulum and cis-golgi Nature 357, 342–344 Knittler, M.R., Alberts, P., Deverson, E.V and Howard, J.C (1999) Nucleotide binding by TAP mediates association with peptide and release of assembled MHC class I molecules Curr Biol 9, 999–1008 Koopmann, J.O., Post, M., Neefjes, J.J., Hämmerling, G.J and Momburg, F (1996) Translocation of long peptides by transporters associated with antigen processing (TAP) Eur J Immunol 26, 1720–1728 Kyritsis, C., Gorbulev, S., Hutschenreiter, S., Pawlitschko, K., Abele, R and Tampé, R (2001) Molecular mechanism and structural aspects of transporter associated with antigen processing inhibition by the cytomegalovirus protein US6 J Biol Chem 276, 48031–48039 Lapinski, P.E., Neubig, R.R and Raghavan, M (2001) Walker a lysine mutations of TAP1 and TAP2 interfere with peptide translocation but not peptide binding J Biol Chem 276, 7526–7533 Lehner, P.J., Karttunen, J.T., Wilkinson, G.W and Cresswell, P (1997) The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation Proc Natl Acad Sci USA 94, 6904–6909 THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE Lehner, P.J., Surman, M.J and Cresswell, P (1998) Soluble tapasin restores MHC class I expression and function in the tapasinnegative cell line 220 Immunity 8, 221–231 Levitskaya, J., Sharipo, A., Leonchiks, A., Ciechanover, A and Masucci, M.G (1997) Inhibition of ubiquitin/proteasomedependent protein degradation by the GlyAla repeat domain of the Epstein-Barr virus nuclear antigen Proc Natl Acad Sci USA 94, 12616–12621 Lindquist, J.A., Jensen, O.N., Mann, M and Hammerling, G.J (1998) ER-60, a chaperone with thiol-dependent reductase activity involved in MHC class I assembly EMBO J 17, 2186–2195 Ljunggren, H.G., Stam, N.J., Ohlen, C., Neefjes, J.J., Hoglund, P., Heemels, M.T., Bastin, J., Schumacher, T.N., Townsend, A., Karre, K., et al (1990) Empty MHC class I molecules come out in the cold Nature 346, 476–480 Loo, T.W and Clarke, D.M (1995) Membrane topology of a cysteine-less mutant of human P-glycoprotein J Biol Chem 270, 843–848 Loo, T.W and Clarke, D.M (2001a) Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil J Biol Chem 276, 14972– 14979 Loo, T.W and Clarke, D.M (2001b) Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers J Biol Chem 276, 36877–36880 Ma, W.L., Lehner, P.J., Cresswell, P., Pober, J.S and Johnson, D.R (1997) Interferon-gamma rapidly increases peptide transporter (TAP) subunit expression and peptide-transport capacity in endothelial-cells J Biol Chem 272, 16585–16590 Madden, D.R., Garboczi, D.N and Wiley, D.C (1993) The antigenic identity of peptide-MHC complexes – a comparison of the conformations of viral peptides presented by HLAA2 Cell 75, 693–708 Meyer, T.H., van Endert, P.M., Uebel, S., Ehring, B and Tampé, R (1994) Functional expression and purification of the ABC transporter complex-associated with antigenprocessing (TAP) in insect cells FEBS Lett 351, 443–447 Momburg, F., Roelse, J., Howard, J.C., Butcher, G.W., Hämmerling, G.J and Neefjes, J.J (1994) Selectivity of MHCencoded peptide transporters from human, mouse and rat Nature 367, 648–651 Monaco, J.J., Cho, S and Attaya, M (1990) Transport protein genes in the murine MHC: possible implications for antigen processing Science 250, 1723–1726 Moore, M.W., Carbone, F.R and Bevan, M.J (1988) Introduction of soluble protein into the class I pathway of antigen processing and presentation Cell 54, 777 Morrison, L.A., Lukacher, A.E., Braciale, V.L., Fan, D.P and Braciale, T.J (1986) Differences in antigen presentation to MHC class I- and class II-restricted influenza virusspecific cytolytic T lymphocyte clones J Exp Med 163, 903–921 Müller, K.M., Ebensperger, C and Tampé, R (1994) Nucleotide binding to the hydrophilic C-terminal domain of the transporter associated with antigen processing (TAP) J Biol Chem 269, 14032–14037 Nagy, N., Vanky, F and Klein, E (1998) Tumor surveillance: expression of the transporter associated with antigen processing (TAP-1) in ex-vivo human tumor samples and its elevation by in vitro treatment with IFNgamma and TNF-alpha Immunol Lett 64, 153–160 Neefjes, J.J., Momburg, F and Hämmerling, G.J (1993) Selective and ATP-dependent translocation of peptides by the MHC-encoded transporter Science 261, 769–771 Neumann, L and Tampé, R (1999) Kinetic analysis of peptide binding of the TAP transporter complex: evidence for structural rearrangements induced by substrate binding J Mol Biol 294, 1203–1213 Neumann, L., Kraas, W., Uebel, S., Jung, G and Tampé, R (1997) The active domain of the herpes-simplex virus protein ICP47 – a potent inhibitor of the transporter associated with antigen-processing (TAP) J Mol Biol 272, 484–492 Nijenhuis, M and Hämmerling, G.J (1996) Multiple regions of the transporter associated with antigen processing (TAP) contribute to its peptide binding site J Immunol 157, 5467–5477 Ortmann, B., Copeman, J., Lehner, P.J., Sadasivan, B., Herberg, J.A., Grandea, A.G., Riddell, S.R., Tampé, R., Spies, T and Trowsdale, J (1997) A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes Science 277, 1306–1309 547 548 ABC PROTEINS: FROM BACTERIA TO MAN Pamer, E and Cresswell, P (1998) Mechanisms of MHC class I-restricted antigen processing Annu Rev Immunol 16, 323–358 Pfänder, R., Neumann, L., Zweckstetter, M., Seger, C., Holak, T.A and Tampé, R (1999) Structure of the active domain of the herpes simplex virus protein ICP47 in water/ sodium dodecyl sulfate solution determined by nuclear magnetic resonance spectroscopy Biochemistry 38, 13692–13698 Ploegh, H.L (1998) Viral strategies of immune evasion Science 280, 248–253 Powis, S.J., Deverson, E.V., Coadwell, W.J., Ciruela, A., Huskisson, N.S., Smith, H., Butcher, G.W and Howard, J.C (1992) Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule Nature 357, 211–215 Reits, E.A.J., Vos, J.C., Grommé, M and Neefjes, J (2000) The major substrates for TAP in vivo are derived from newly synthesized proteins Nature 404, 774–778 Ritz, U., Momburg, F., Pircher, H.P., Strand, D., Huber, C and Seliger, B (2001) Identification of sequences in the human peptide transporter subunit TAP1 required for transporter associated with antigen processing (TAP) function Int Immunol 13, 31–41 Rock, K.L and Goldberg, A.L (1999) Degradation of cell proteins and the generation of MHC class I-presented peptides Annu Rev Immunol 17, 739–779 Russ, G., Esquivel, F., Yewdell, J.W., Cresswell, P., Spies, T and Bennick, J.R (1995) Assembly, intracellular localization, and nucleotide binding properties of the human peptide transporters TAP1 and TAP2 expressed by recombinant vaccinia viruses J Biol Chem 270, 21312–21318 Sadasivan, B., Lehner, P.J., Ortmann, B., Spies, T and Cresswell, P (1996) Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP Immunity 5, 103–114 Salazar-Onfray, F., Charo, J., Petersson, M., Freland, S., Noffz, G., Qin, Z.H., Blankenstein, T., Ljunggren, H.G and Kiessling, R (1997) Down-regulation of the expression and function of the transporter associated with antigen-processing in murine tumor-cell lines expressing IL-10 J Immunol 159, 3195–3202 Saveanu, L., Daniel, S and van Endert, P.M (2001) Distinct functions of the ATP binding cassettes of transporters associated with antigen processing: a mutational analysis of Walker A and B sequences J Biol Chem 276, 22107–22113 Schmitt, L and Tampé, R (2000) Affinity, specificity, diversity: a challenge for the ABC transporter TAP in cellular immunity ChemBioChem 1, 16–35 Schubert, U., Anton, L.C., Gibbs, J., Norbury, C.C., Yewdell, J.W and Bennink, J.R (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes Nature 404, 770–774 Schumacher, T.N., Kantesaria, D.V., Heemels, M.T., Ashton-Rickardt, P.G., Shepherd, J.C., Früh, K., Yang, Y., Peterson, P.A., Tonegawa, S and Ploegh, H.L (1994a) Peptide length and sequence specificity of the mouse TAP1/TAP2 translocator J Exp Med 179, 533–540 Schumacher, T.N.M., Kantesaria, D.V., Serreze, D.V., Roopenian, D.C and Ploegh, H.L (1994b) Transporters from H-2(b), H-2(d), H-2(s), H-2(k), and H-2(g7) (NOD/lt) haplotype translocate similar sets of peptides Proc Natl Acad Sci USA 91, 13004–13008 Seliger, B., Hohne, A., Jung, D., Kallfelz, M., Knuth, A., Jaeger, E., Bernhard, H., Momburg, F., Tampé, R and Huber, C (1997a) Expression and function of the peptide transporters in escape variants of human renal-cell carcinomas Exp Hematol 25, 608–614 Seliger, B., Maeurer, M.J and Ferrone, S (1997b) TAP off-tumors on Immunol Today 18, 292–299 Shepherd, J.C., Schumacher, T.N., AshtonRickardt, P.G., Imaeda, S., Ploegh, H.L., Janeway, C.A.J and Tonegawa, S (1993) TAP1-dependent peptide translocation in vitro is ATP dependent and peptide selective Cell 74, 577–584 Sherman, L.A., Theobald, M., Morgan, D., Hernandez, J., Bacik, I., Yewdell, J., Bennink, J and Biggs, J (1998) Strategies for tumor elimination by cytotoxic T lymphocytes Crit Rev Immunol 18, 47–54 Spies, T., Bresnahan, M., Bahram, S., Arnold, D., Blanck, G., Mellins, E., Pious, D and DeMars, R (1990) A gene in the human major histocompatibility complex class II region controlling the class I antigen presentation pathway Nature 348, 744–747 Tampé, R., Urlinger, S., Pawlitschko, K and Uebel, S (1997) The transporters associated with antigen processing (TAP) In: Unusual Secretory Pathways: From Bacteria to Man THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE (ed K Kuchler, A Rubartelli, and B Holland), pp 115–136 New York: Springer Teisserenc, H., Schmitt, W., Blake, N., Dunbar, R., Gadola, S., Gross, W.L., Exley, A and Cerundolo, V (1997) A case of primary immunodeficiency due to a defect of the major histocompatibility gene complex class I processing and presentation pathway Immunol Lett 57, 183–187 Toes, R.E., Nussbaum, A.K., Degermann, S., Schirle, M., Emmerich, N.P., Kraft, M., et al (2001) Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products J Exp Med 194, 1–12 Tomazin, R., Hill, A.B., Jugovic, P., York, I., van Endert, P., Ploegh, H.L., Andrews, D.W and Johnson, D.C (1996) Stable binding of the Herpes Simplex virus ICP47 protein to the peptide binding-site of TAP EMBO J 15, 3256–3266 Townsend, A., Ohlen, C., Foster, L., Bastin, J., Ljunggren, H.G and Karre, K (1989) A mutant cell in which association of class I heavy and light chains is induced by viral peptides Cold Spring Harb Symp Quant Biol 54, 299–308 Trowsdale, J., Hanson, I., Mockridge, I., Beck, S., Townsend, A and Kelly, A (1990) Sequences encoded in the class II region of the MHC related to the ‘ABC’ superfamily of transporters Nature 348, 741–744 Turner, G.C and Varshavsky, A (2000) Detecting and measuring cotranslational protein degradation in vivo Science 289, 2117–2120 Uebel, S and Tampé, R (1999) Specificity of the proteasome and the TAP transporter Curr Opin Immunol 11, 203–208 Uebel, S., Meyer, T.H., Kraas, W., Kienle, S., Jung, G., Wiesmüller, K.H and Tampé, R (1995) Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries J Biol Chem 270, 18512–18516 Uebel, S., Kraas, W., Kienle, S., Wiesmüller, K.-H., Jung, G and Tampé, R (1997) Recognition principle of the TAPtransporter disclosed by combinatorial peptide libraries Proc Natl Acad Sci USA 94, 8976–8981 Urlinger, S., Kuchler, K., Meyer, T.H., Uebel, S and Tampé, R (1997) Intracellular location, complex-formation, and function of the transporter associated with antigen processing in yeast Eur J Biochem 245, 266–272 Vambutas, A., Bonagura, V.R and Steinberg, B.M (2000) Altered expression of TAP-1 and major histocompatibility complex class I in laryngeal papillomatosis: correlation of TAP-1 with disease Clin Diagn Lab Immunol 7, 79–85 van Endert, P.M., Tampé, R., Meyer, T.H., Tisch, R., Bach, J.F and McDevitt, H.O (1994) A sequential model for peptide binding and transport by the transporters associated with antigen processing Immunity 1, 491–500 Vitale, M., Rezzani, R., Rodella, L., Zauli, G., Grigolato, P., Cadei, M., Hicklin, D.J and Ferrone, S (1998) Hla class-i antigen and transporter associated with antigenprocessing (tap1 and tap2) down-regulation in high-grade primary breast-carcinoma lesions Cancer Res 58, 737–742 Vos, J.C., Spee, P., Momburg, F and Neefjes, J (1999) Membrane topology and dimerization of the two subunits of the transporter associated with antigen processing reveal a three-domain structure J Immunol 163, 6679–6685 Vos, J.C., Reits, E.A., Wojcik-Jacobs, E and Neefjes, J (2000) Head-head/tail-tail relative orientation of the pore-forming domains of the heterodimeric ABC transporter TAP Curr Biol 10, 1–7 Wang, K.N., Früh, K., Peterson, P.A and Yang, Y (1994) Nucleotide binding of the C-terminal domains of the major histocompatibility complex-encoded transporter expressed in Drosophila melanogaster cells FEBS Lett 350, 337–341 Wright, K.L., White, L.C., Kelly, A., Beck, S., Trowsdale, J and Ting, J.P.Y (1995) Coordinate regulation of the human tap1 and lmp2 genes from a shared bidirectional promoter J Exp Med 181, 1459–1471 Yan, G., Shi, L.J and Faustman, D (1999) Novel splicing of the human MHC-encoded peptide transporter confers unique properties J Immunol 162, 852–859 Yewdell, J.W., Bennink, J.R and Hosaka, Y (1988) Cells process exogenous proteins for recognition by cytotoxic T lymphocytes Science 239, 637–640 York, I.A., Roop, C., Andrews, D.W., Riddell, S.R., Graham, F.L and Johnson, D.C (1994) A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8ϩ T lymphocytes Cell 77, 525–535 York, I.A., Goldberg, A.L., Mo, X.Y and Rock, K.L (1999) Proteolysis and class I 549 550 ABC PROTEINS: FROM BACTERIA TO MAN major histocompatibility complex antigen presentation Immunol Rev 172, 49–66 Zeidler, R., Eissner, G., Meissner, P., Uebel, S., Tampé, R., Lazis, S and Hammerschmidt, W (1997) Downregulation of TAP1 in B lymphocytes by cellular and Epstein–Barr virusencoded interleukin-10 Blood 90, 2390–2397 Zhang, F., Zhang, W., Liu, L., Fisher, C.L., Hui, D., Childs, S., Dorovini-Zis, K and Ling, V (2000) Characterization of ABCB9, an ATP binding cassette protein associated with lysosomes J Biol Chem 275, 23287– 23294 Zhu, K., Wang, J., Zhu, J., Jiang, J., Shou, J and Chen, X (1999) p53 induces TAP1 and enhances the transport of MHC class I peptides Oncogene 18, 7740–7747  ... associates with the chaperone protein, THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE calnexin, and forms a dimer with ␤2-microglobulin (for review, see Pamer and. .. TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE Androlewicz, M.J and Cresswell, P (1994) Human transporters associated with antigen processing possess a promiscuous... transporter associated with antigen processing- dependent peptide translocation Proc Natl Acad Sci USA 94, 690 4–6 909 THE TAP: A PEPTIDE TRANSPORT AND LOADING COMPLEX ESSENTIAL FOR CELLULAR IMMUNE RESPONSE