Critical Role for the Tapasin-Docking Site of TAP2 in the Functional Integrity of the MHC Class I-Peptide-Loading Complex

Leonhardt, Ralf M.; Keusekotten, Kirstin; Bekpen, Cemalettin; Knittler, Michael R.

doi:10.4049/jimmunol.175.8.5104

Cited by 56 publications

(116 citation statements)

References 43 publications

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confidence: 99%

“…TAP2 is predicted to span the membrane nine times, with the N terminus of the membrane domain in the ER lumen and the C terminus in the cytosol (14, 39). The C-terminal six transmembrane domains of TAP1 and TAP2 combine to form the core unit of the transporter that is responsible for peptide binding and translocation, whereas the N-terminal transmembrane domains of TAP1 and TAP2 interact with tapasin (21,22,28,36).Since the vast majority of peptides presented to CTLs require translocation into the ER by TAP, it is not surprisingly that many viruses interfere with this transport step (3, 8, 12, 17, 18, 23-25, 30, 35, 37, 41). Human cytomegalovirus (HCMV) encodes the protein US6, which is an ER-localized type I integral membrane glycoprotein that inhibits peptide translocation by TAP (2,15,27).…”

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Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

Dugan

Hewitt

2008

J Virol

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The human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.The major histocompatibility (MHC) class I antigen presentation pathway plays a central role in the immune response to viral infection. MHC class I molecules are expressed on the plasma membranes of all nucleated cells and present peptides derived from intracellular proteins to cytotoxic T lymphocytes (CTLs). CTLs monitor these MHC class I molecules for peptides derived from viral proteins and kill the infected cells. The presentation of antigenic peptides to CTLs represents the end point of a complex multistep pathway. The vast majority of peptides presented by MHC class I molecules are generated by the degradation of proteins in the cytosol by the proteasome complex (38). These peptides are then translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) (1). MHC class I molecules are assembled in the ER by a process that involves ER-resident chaperones (5). Peptide binding represents the final step of MHC class I molecule folding and is facilitated by the TAP-associated molecule tapasin, which links the nascent MHC class I molecules to TAP to form the "peptide-loading complex" (5). Only once they have bound peptides can MHC class I molecules dissociate from this complex and exit the ER for transport to the plasma membrane.TAP is a member of the ATP binding cassette (ABC) family of transporters, whose members couple ATP hydrolysis to the translocation of a broad spectrum of different substrates across cellular membranes (1,29,42). All...

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Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

Dugan

Hewitt

2008

J Virol

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“…It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12]. The exact stoichiometry of the PLC has been a matter of debate [13,14]; however, recent findings demonstrate a TAP-tpn ratio of 1:2 [15,16] as proposed previously also by Rufer et al [17].…”

Section: Introductionmentioning

confidence: 49%

“…Since our HLA-B*44:02 stability analysis never showed a complete loss in the presence of TPSN Ex3, the data indicate, that more than one active MHC-I loading site per TAP molecule exist, which has recently been proposed also by others [15,16]. Furthermore, since TPSN Ex3 was not able to displace tpn completely from the TAP complex, it suggests, in a functional sense, that there are at least two differing binding sites for tpn on TAP, which has been observed previously for rat TAP subunits [11]. Since TPSN Ex3 appears to bind to a subset of these binding sites, in future studies TPSN Ex3 might be instrumental to gain further insights into the functional stoichiometry of the PLC.…”

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confidence: 48%

“…In the PLC the MHC-I heterodimer associates with tapasin (tpn), calreticulin, ERp57, and indirectly with TAP; tpn and TAP interact via their transmembrane segments (TMSs), thereby stabilizing the structure and function of TAP [5][6][7][8][9]. It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12]. The exact stoichiometry of the PLC has been a matter of debate [13,14]; however, recent findings demonstrate a TAP-tpn ratio of 1:2 [15,16] as proposed previously also by Rufer et al [17].…”

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A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

et al. 2013

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To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the Exon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the Exon3 variant with ERp57 was strongly impaired. Although the Exon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the Exon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the Exon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the Exon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8 + T-cell responses.Keywords: MHC class I peptide loading r PLC r TAP r Tapasin Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Anne Halenius e-mail: anne.halenius@uniklink-freiburg.de * These authors contributed equally to this work. Eur. J. Immunol. 2013Immunol. . 43: 1459Immunol. -1469 Introduction Presentation of endogenous peptides by MHC class I (MHC-I) to CD8 + T cells is critical to controlling viral infections and tumor growth. Peptides originating from cytosolic proteasomal degradation are translocated into the ER lumen by the heterodimeric peptide transporter TAP1/2 (transporter associated with Ag processing). Optimally TAP transports peptides with a length of 8-16 amino acids [1,2] and these can be further trimmed in the ER to fit the peptide binding groove of MHC class I (MHC-I) molecules [3], yielding a stable peptide-MHC-I complex that is transported through the secretory pathway for cell surface expression. Proper peptide loading of MHC-I is controlled by several chaperones that form the peptide loading complex (PLC) in the ER [4]. In the PLC the MHC-I heterodimer associates with tapasin (tpn), calreticulin, ERp57, and indirectly with TAP; tpn and TAP interact via their transmembrane segments (TMSs), thereby stabilizing the structure and function of TAP [5][6][7][8][9]. It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12]. The exact stoichiometry of the PLC has been a matter of debate [13,14]; however, recent findings demonstrate a TAP-tpn ratio of 1:2 [15,16] as proposed previously also by Rufer et al...

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Functional significance of tapasin membrane association and disulfide linkage to ERp57 in MHC class I presentation

et al. 2009

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Summary Tapasin is disulfide linked to ERp57 within the peptide loading complex. In cell-free assays, a soluble variant of the tapasin-ERp57 dimer recruits MHC class I molecules and promotes peptide binding to them while soluble tapasin alone does not. Here we show that within cells, tapasin conjugation with ERp57 is as critical as its integration into the membrane for efficient MHC class I assembly, surface expression, and antigen presentation to CD8-positive T cells. Elimination of both of these properties severely compromises tapasin function, in keeping with predictions from in vitro studies.

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Critical Role for the Tapasin-Docking Site of TAP2 in the Functional Integrity of the MHC Class I-Peptide-Loading Complex

Cited by 56 publications

References 43 publications

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

Functional significance of tapasin membrane association and disulfide linkage to ERp57 in MHC class I presentation

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