Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/db.protein-peptides.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/protein-peptides.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-131-D4.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-131-D4.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-129-D4.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/23-0922-MUT-191-MR1-TD-D6.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-131-D6.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-129-D6.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/23-0922-MUT-191-MR1-TD-D4.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-131-D6.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/23-0922-MUT-191-MR1-TD-D6.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-129-D6.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/22-1102-MR1_CHAP_42_TD-MUT-129-D4.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063725/23-0922-MUT-191-MR1-TD-D4.wiffprimaryOK200william-hildebrand@ouhsc.eduJason KrawicData-dependent acquisitionMass SpectrometryNot availableLcmsMr1https://www.ebi.ac.uk/pride/archive/projects/PXD063725Epithelial CellSamples were resuspended in 100 μl of PBS and sent for on-bead digestion. Tryptic digests were conducted using Thermo Scientific’s Pierce In-Solution Tryptic Digestion and Guanidation Kit (Cat # 89895) directly on magnetic beads at 5x volume. Sample cleanup was completed using either Thermo Scientific’s Pierce C18 Spin Columns (Cat #89870), or 10 μl Spin Tips (Cat #87782). Samples were dried via speedvac and resuspended in 6 μl of a solvent composed of 10% acetic acid, 2% acetonitrile, and indexed Retention Time (iRT) peptides (Biognosys, Schlieren, Switzerland) at a 1:50 dilution in HPLC grade water. Samples were then injected using an Eksigent NanoLC (nanoscale liquid chromatography) 415 reversed-phase HPLC. A trap-elution method was used with a 5-mm-long, 350-μm-internal diameter ChromXP C18 trap column by Optimize Technologies with 3-μm particles and 120-Å pores. After desalting, samples were then loaded onto a 150-mm-long, 75-μm-internal diameter ChromXP Eksigent C18 reversed-phase separation column with 3-μm particles and 120-Å pores. An acetonitrile gradient was run at pH 3.0 using two solvents. Solvent A was 0.1% formic acid in HPLC-grade water, and solvent B was 0.1% formic acid in 95% acetonitrile in HPLC-grade water. After column equilibration with 2% solvent B, samples were loaded at 5 μl over 10 min onto the trap column and injected through the separation column at 300 nl/min with a gradient of 10–40% B for 70 min, followed by a clearance gradient of 40–80% B for 7 min. Sample passing through the separation column was ionized using the PicoView ion source by New Objective, through an 11-cm, 20-μm-inner diameter pulled glass emitter that tapered to 10 μm at the tip. The ionized sample was injected into an AB Sciex TripleTOF 5600 quadrupole time of flight (TOF) mass spectrometer in positive ion mode with parameters set at the manufacturer’s default tryptic digest settings.PridePeptide sequences were determined using PEAKS Studio X+ software (Bioinformatics Solutions, Waterloo, Canada). A database composed of SwissProt Homo sapiens (Proteome ID UP000005640, Taxon ID 9606), MR1-GFP construct sequence, and iRT peptide sequences was used as the reference for database search. The following variable post-translational modifications were included in the database search: acetylation, deamination, pyroglutamate formation, oxidation, sodium adducts, phosphorylation, cysteinylation, and carbamidomethylation. Identified peptides were further refined using a false discovery rate of 1%, requiring at least two unique, significant peptides for protein identification. Peptide intensity values for each protein ID were normalized by dividing by the corresponding protein's molecular weight, adjusting for protein size. This normalization was applied across all biological replicates prior to performing t-tests.ProteomicsWilliam HildebrandUniversity of Oklahoma Health Sciences Center, Department of Microbiology & ImmunologyPARTIALHomo Sapiens (human)jason-krawic@ouhsc.edu41759736 Kulicke CA, Lemon C, Krawic JR, Nieto Ramirez LM, Kim SJ, Narayanan G, Tafesse FG, Hildebrand WH, Dobos KM, Lewinsohn DM. Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens. J Biol Chem. 2026 302(4):111317 10.1016/j.jbc.2026.111317University of Oklahoma Health Sciences CenterUnited StatesThe antigen presenting molecule major histocompatibility complex class I-related protein 1 (MR1) binds small molecule metabolites derived from the microbial riboflavin biosynthetic pathway and presents them at the cell surface for surveillance by MR1-restricted mucosal-associated invariant T cells (MAIT cells). MR1 ligands can originate in the extracellular space or in endosomal compartments that contain microbial pathogens. Distinct, complementary antigen processing and presentation pathways enable MR1 to survey diverse intracellular locations and present both exogenous and intracellular antigens. Here, we generated a panel of BEAS-2B MR1 KO cells reconstituted with MR1 proteins mutated at amino acids 9 to 16. The overexpressed mutated MR1 molecules differentially translocated to the cell surface in response to 6-formylpterin and differed in their ability to present mycobacterial antigens to MAIT cell clones. While they barely presented Mycobacterium smegmatis supernatant and other exogenous MAIT cell antigens, their ability to present antigens derived from mycobacterial infection and a 5-amino-6-D-ribitylaminouracil prodrug that requires endosomal processing remained largely intact. Protein coimmunoprecipitation and mass spectrometry-based proteomic analysis showed that mutated MR1 differentially associated with calnexin and β₂-microglobulin (B2M). Knock down of B2M in cells overexpressing wildtype MR1 phenocopied the loss of exogenous antigen presentation but did not impact presentation of intracellular antigens. Thus, the MR1-mediated presentation of exogenous antigen appears to be limited by binding to B2M whereas a lower sensitivity to B2M deficiency suggests that MAIT cell activation via the endosomal antigen presentation pathway may be limited by the availability of MR1 itself.Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens.Kulicke Corinna A CA, Lemon Chance C, Krawic Jason R JR, Nieto Ramirez Luisa Maria LM, Kim Se-Jin SJ, Narayanan Gitanjali G, Tafesse Fikadu G FG, Hildebrand William H WH, Dobos Karen M KM, Lewinsohn David M DMfalseCo-immunoprecipitation and proteomic analysis of Major Histocompatibility Complex Class I-related (MR1) mutantsIdentification of cellular proteins mediating presentation of bacterial antigens by the MHC I-related protein MR1. MR1 mutant proteins that differ in their capacity to present antigens from different sources were immunoprecipitated and their interacting proteins were compared to identify candidate proteins involved in the different antigen presentation pathways.2026-04-062025-05-07PXD063725NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NEWT:1129NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:383379NEWT:10029NEWT:913645NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:4442NEWT:192875NEWT:59729NEWT:2164133NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:77133NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:55571NEWT:35500NEWT:1140NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:263NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:2850NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:536231NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:571256NEWT:40483NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:9778NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:6494NEWT:6491NEWT:507601NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:27448NEWT:1194669NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:6063NEWT:49240NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:206411NCBITaxon:6191NEWT:596153NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:51515NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NEWT:8839NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:430615NEWT:326423NEWT:452467NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:70448NEWT:698936NEWT:9940NEWT:521001NEWT:192011NEWT:1813887NEWT:224911NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:4513NEWT:1247NEWT:6938NCBITaxon:10407NEWT:1736309NEWT:10360NEWT:47664NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:466585NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:34305NCBITaxon:183674NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:431241NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:288705NEWT:29176NEWT:4787NEWT:3218NEWT:556182NEWT:1270NEWT:374990NEWT:1432059NEWT:156471NEWT:4784NEWT:360106NEWT:156476NEWT:4543NEWT:360104NEWT:10117NEWT:10116NEWT:1280NEWT:10358NEWT:1735272NEWT:83334NEWT:83332NEWT:317513NEWT:580240NEWT:294128NEWT:11676NEWT:100226NEWT:4530NEWT:75058NEWT:13616NEWT:296543NEWT:173NEWT:316435NEWT:42157NEWT:1895NEWT:1034304NEWT:105023NEWT:8666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