{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073419/ZNFX1_XLMS_Results_insol_5.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073419/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073419/FL_20220418_Dan_SV_20220314_ZNFX1_XL_5.raw"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["s.s.virdee@dundee.ac.uk"],"submitter":["Satpal Virdee"],"technology_type":["Mass Spectrometry","Chemical cross-linking coupled with mass spectrometry proteomics"],"software":["Not available"],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD073419"],"sample_protocol":["Purified full-length human ZNFX1 protein (35 μg) was incubated with biotinylated UBE2D2~Ub activity-based probe (175 μg; eightfold molar excess) in 50 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl in a final volume of 250 μL. Labelling reactions were performed for 4 hours at 30 °C. Probe labelling efficiency was assessed by SDS-PAGE and Coomassie staining.  Labelled and unlabelled ZNFX1 species were separated from excess probe by gel filtration chromatography using a Superdex 200 Increase 10/300 GL column. Protein-containing fractions were pooled and concentrated by centrifugal filtration, followed by incubation with pre-equilibrated streptavidin resin for 3 hours at room temperature to capture probe-labelled species.  Beads were washed extensively prior to reduction and alkylation. Resin-bound proteins were digested overnight with GluC and trypsin proteases. Resulting peptides were desalted, freeze-dried, and resuspended in 5% formic acid prior to LC–MS/MS analysis.  Approximately 2 μg of peptides were injected onto an UltiMate 3000 RSLC nano system coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Peptides were trapped on an Acclaim PepMap trap column and separated on a PepMap RSLC C18 analytical column using either 60- or 120-minute linear gradients from 3–60% solvent B (0.08% formic acid in 80:20 acetonitrile:water). Data were acquired in data-dependent acquisition mode using a TOP-3s method."],"repository":["Pride"],"modification":[""],"quantification_method":[""],"data_protocol":["Raw LC–MS/MS data were searched for cross-linked peptides using pLink software. Searches were performed against a custom sequence database containing human ZNFX1 (UniProt Q9P2E3), UBE2D2* (UniProt P62837; harbouring C21S, C107S, and C111S mutations), and ubiquitin (UniProt P0CG47; residues 1–73 only). A reverse decoy database was generated for false discovery rate estimation.  Cross-linked peptides were filtered to a 1% false discovery rate. A crosslinker mass of 306.181 Da with elemental composition C14H22N6O2 was specified manually. Variable modifications included carbamidomethylation of cysteine, methionine oxidation and dioxidation, and asparagine and glutamine deamidation. Minimum peptide length was set to six residues, with a maximum peptide mass of 10 kDa and up to three missed cleavages permitted. Peptide and fragment mass tolerances were set to +/-10 ppm.  Candidate cross-linked spectra were manually inspected and validated using pLabel to assess fragment ion coverage and crosslink assignment. Data interpretation was qualitative in nature and no statistical hypothesis testing was applied."],"omics_type":["Proteomics"],"labhead":["Satpal Virdee"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, UK"],"species":["Homo Sapiens (human)"],"publication":["41861786 Squair DR, Rivers E, Sowar H, Balci A, Harmo R, Wright DJ, Beniwal G, Soetens M, Mathur S, Tollervey A, Wood NT, Pao KC, Stanton C, Fletcher AJ, Virdee S. ZNFX1 uses two-component ubiquitin circuitry to quarantine viral RNA. Mol Cell. 2026 86(6):1164-1181.e12 10.1016/j.molcel.2026.02.015"],"submitter_mail":["s.s.virdee@dundee.ac.uk"],"submitter_affiliation":["University of Dundee"],"submitter_country":["United Kingdom"],"pubmed_abstract":["The detection of viral RNA inside cells triggers a diverse range of antiviral responses, including global translation inhibition, interferon secretion, and RNA sequestration. Mutations in the gene zinc-finger NFX1-type containing 1 (ZNFX1) cause severe pediatric immunodeficiencies, including chronic viral infection and autoinflammation. Here, we show that ZNFX1 is an RNA helicase with cryptic and unusual bifurcating E3 ubiquitin ligase activity. Nucleotide-dependent RNA binding stimulates ZNFX1 to generate complex ubiquitin chains via a two-component ubiquitin circuit wired in parallel, with ubiquitin flux occurring via two competing paths. One route produces K63-linked polyubiquitin that drives RNA entrapment within self-propagating ZNFX1 aggregates, and the other route produces K48-linked polyubiquitin that drives ZNFX1 turnover. RNA entrapment restricts RNA virus replication and is reversible by deubiquitination. Pathogenic ZNFX1 variants are defective for viral restriction, linking RNA entrapment to antiviral immunity in vivo."],"pubmed_title":["ZNFX1 uses two-component ubiquitin circuitry to quarantine viral RNA."],"pubmed_authors":["Squair Daniel R DR, Rivers Eilidh E, Sowar Hanna H, Balci Arda A, Harmo Roosa R, Wright David J DJ, Beniwal Gaurav G, Soetens Mathieu M, Mathur Sunil S, Tollervey Aidan A, Wood Nicola T NT, Pao Kuan-Chuan KC, Stanton Callum C, Fletcher Adam J AJ, Virdee Satpal S"],"additional_accession":[]},"is_claimable":false,"name":"ZNFX1 activity-based probe crosslinking mass spectrometry","description":"The aim of this experiment was to identify the catalytic cysteine in the antiviral E3 ligase ZNFX1 using activity-based probe (ABP)–enabled cross-linking mass spectrometry (XL-MS). A biotinylated E2~ubiquitin activity-based probe containing a cysteine-electrophile trap was used to covalently capture ubiquitin a transfer intermediate between UBE2D2~Ub and full-length human ZNFX1. Cross-linked peptide mapping was performed to identify probe-labelled residues and putative catalytic site.","dates":{"publication":"2026-05-22","submission":"2026-01-22"},"accession":"PXD073419","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:184922","NEWT:6703","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2102","NEWT:2042546","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:5722","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","NEWT:1211601","NEWT:876138","NEWT:3654","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:1096976","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NCBITaxon:4896","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:28903","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:913645","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:84645","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:5888","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:197","NEWT:1390363","NEWT:288705","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:374990","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:286","NEWT:391619","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:1148","NEWT:580240","NEWT:294128","NEWT:11676","NEWT:55571","NEWT:35500","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1390","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:3712","NEWT:105023","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:375146","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:243277","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:460519","NEWT:1187947","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:9483","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:432359","NEWT:46835","NEWT:1182263","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:253","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:93061","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:89920","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:108458","NEWT:272623","NEWT:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:32644","NEWT:225117","NEWT:499175","NEWT:109779","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:357","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NEWT:28038","NEWT:1663","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:327159","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:108931","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:303","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:115104","NEWT:1121114","NEWT:663","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:941442","NEWT:220668","NEWT:13076","NEWT:1249668","NEWT:7108","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:9644","NEWT:415540","NEWT:550","NEWT:675060","NEWT:4081","NEWT:334542","NEWT:554","NEWT:27592","NEWT:98334","NEWT:426428","NEWT:9639","NEWT:7574","NEWT:1715256","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:7460","NEWT:507601","NEWT:643680","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:102169","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NEWT:1908","NEWT:13164","NEWT:216129","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:353152","NEWT:2829","NEWT:366581","NEWT:216599","NEWT:216595","NEWT:1194669","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:8479","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:630","NEWT:334747","NEWT:61235","NEWT:15368","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:170187","NEWT:469008","NEWT:260707","NEWT:2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