Error bars show -/+SD.****p0.0001, ***p0.001 using unpaired two-tailed t-tests. == Physique 2figure supplement 1. provide A 77-01 novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity. Research organism:Human == Introduction == Major histocompatibility complex class I (MHC I) molecules play a critical role in immmunosurveillance, particularly in the context of viral infections and cancer, by presenting antigenic peptides to CD8 +T cells. Prior to their cell surface export, MHC I molecules undergo peptide editing, a process that involves the exchange of A 77-01 low-affinity peptides for those of higher affinity. In addition to ensuring that only stable peptide:MHC I complexes are released to the plasma membrane, peptide editing ultimately controls the peptide repertoire that is displayed for immune detection. For over two decades, tapasin was the only known peptide editor for MHC I, facilitating peptide selection within the confines of the peptide loading complex (PLC) (Williams et al., 2002;Howarth et al., 2004;Chen and Bouvier, 2007;Wearsch and Cresswell, 2007). However, it is now well-recognised that this tapasin-related protein TAPBPR is usually a second impartial peptide editor that performs peptide exchange outside the PLC (Boyle et al., 2013;Hermann et al., 2013;Hermann et al., 2015;Morozov et al., 2016). Furthermore, TAPBPR can work in cooperation with UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) to reglucosylate MHC I, causing recycling of MHC molecules to the PLC (Neerincx et al., 2017). Although TAPBPR usually functions as an intracellular peptide editor, we have recently made the fascinating discovery that when given access to surface expressed MHC I molecules, TAPBPR retains its function as a peptide exchange catalyst and can be utilised to display immunogenic peptides of choice directly onto the surface of cells (Ilca et al., 2018). We have therefore identified that manipulation of TAPBPR function may be utilised as a potential immunotherapeutic that facilitates the presentation of both neoantigens and viral-derived peptides, thereby overriding the endogenous A 77-01 cellular antigen processing pathway. Moreover, we have also developed two novel functional assays that enable detailed interrogation of TAPBPR-mediated peptide exchange on MHC I (Ilca et al., 2018). Precisely how tapasin and TAPBPR function at the molecular level remains largely enigmatic. The recently reported crystal structures of human TAPBPR in complex with mouse MHC I captured the endpoint of peptide editing, thereby suggesting that TAPBPR facilitates peptide exchange by widening the peptide binding groove of MHC I at the 21 region (Jiang et al., 2017;Thomas and Tamp, 2017). However, there remains an incomplete understanding of the step-by-step processes by which the two peptide editors, TAPBPR and tapasin, recognise peptide-loaded MHC I molecules and actively facilitate peptide dissociation to result in the conformations observed in the crystal structures. Indeed, McShan et al. have recently used NMR in an attempt to further delineate the dynamic process of peptide exchange on MHC I by TAPBPR (McShan et al., 2018). Intriguingly, the structure reported by Thomas and Tampe identified a loop of TAPBPR FCRL5 that was proposed to interact with the peptide binding groove of MHC I, where the C-terminus of bound peptide usually resides (Thomas and Tamp, 2017). While this study exhibited the localisation of the loop, to date, there is no experimental evidence to support the notion that this loop mediates peptide exchange on MHC I. In contrast, Jianget al.failed to capture the loop in proximity to the peptide-binding groove (Jiang et al., 2017), further questioning the relevance and importance of this loop in TAPBPR-mediated peptide exchange. Given the discordance between the data reported for the captured structures and the lack of functional evidence to support any role for this loop, it is vital to reconcile these discrepancies to understand whether the TAPBPR loop is usually involved in peptide exchange. Here, we A 77-01 investigate the functional importance of the K22-D35 loop using two newly developed assays in combination with immunopeptidomic analysis. Our data demonstrates that this loop is critical for peptide dissociation from MHC I. Furthermore, we spotlight key molecular features governing TAPBPR:MHC I A 77-01 conversation and provide insight into the mechanism(s) of peptide selection on MHC I molecules. == Results == == The TAPBPR K22-D35.
