347 stainless hlau coiled tubing tshuaj tivthaiv, Kev txheeb xyuas cov tshiab interferon-tej tib neeg leukocyte antigen-A (HLA-A) chaperone proteins siv cross-linked mass spectrometry (CLMS)

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Stainless hlau 347L Coil Tubes, Qib Qib: SS347L

Lub interferon signaling system induces ib tug muaj zog cytokine teb rau ntau yam pathogenic thiab intrinsic pathological teeb liab los ntawm ib puag ncig, uas ua rau induction ntawm subsets ntawm interferon-inducible proteins.Peb tau thov DSS-mediated cross-link mass spectrometry (CLMS) txhawm rau txheeb xyuas cov protein-protein kev sib cuam tshuam hauv cov ntsiab lus ntawm interferon-induced proteins.Ntxiv nrog rau qhov xav tau interferon-inducible proteins, peb kuj tau txheeb xyuas cov tshiab intermolecular thiab intramolecular cross-linked adducts ntawm canonical interferon-inducible proteins xws li MX1, USP18, OAS3, thiab STAT1.Peb tsom mus rau orthogonal validation ntawm ib tug tshiab txheej ntawm interferon-inducible protein networks tsim los ntawm HLA-A proteins (H2BFS-HLA-A-HMGA1) siv co-immunoprecipitation thiab lawv kawm ntxiv siv molecular dynamics qauv.Kev ua qauv ntawm cov kev hloov pauv hloov pauv ntawm cov protein complex tau nthuav tawm ntau qhov chaw sib cuam tshuam uas cuam tshuam txog kev sib cuam tshuam hauv kev tshawb pom CLMS.Ua ke, peb nthuav qhia txoj kev tshawb fawb ntawm CLMS txhawm rau txheeb xyuas qhov teeb meem tshiab cuam tshuam los ntawm interferon, thiab tos ntsoov rau kev siv dav dua ntawm CLMS txhawm rau txheeb xyuas cov kev hloov pauv tshiab ntawm cov protein sib cuam tshuam hauv cov qog microenvironment.
Ua ntej yuav pib lub cev tiv thaiv kab mob pib, tus tswv tsev lub cev tiv thaiv kab mob mounts cov tshuaj tiv thaiv kab mob los ntawm ib tsev neeg ntawm secreted alpha-helical cytokines hu ua interferons (IFNs).Hom I IFN chav kawm IFNα thiab IFNβ qhib cov lus teb ntawm tes, suav nrog cov tshuaj tiv thaiv kab mob, proapoptotic, proinflammatory, thiab tshuaj tiv thaiv kab mob.Hauv tib neeg, 13 subtypes ntawm IFNα paub, tag nrho cov pawg ntawm chromosome 91. Kuj ceeb tias, tsuas yog IFNα2 tau kawm rau kev siv tshuaj kho mob.Tsis ntev los no, kev saib xyuas tshwj xeeb tau them rau kev tshawb fawb ntawm lwm yam subtypes ntawm IFNα.Ib txoj kev tshawb fawb tsis ntev los no tau pom tias IFNα14 yog ib qho zoo tshaj plaws isoforms hauv kev txwv HBV2 thiab HIV-13,4 replication piv rau canonical IFNα2 subtype.
Nws tau raug tsim los uas qhib hom I interferon receptor complexes (IFNAR1 thiab IFNAR2) ua rau lub teeb liab hloov pauv cascade mediated los ntawm Janus kinases TYK2 thiab JAK15,6.Cov Janus kinases phosphorylate signal transducers thiab transcriptional protein activators (STAT1 thiab STAT2) ntawm tyrosine residues los pib SH2 domain-mediated heterodimerization6.Tom qab ntawd, IRF9 khi STAT heterodimers los tsim ib qho kev sib txuas ntawm IFN-stimulated factor 3 gene (ISGF3), uas hloov mus rau lub nucleus thiab induces transcription ntawm ntau tshaj 2000 interferon-stimulated noob (ISGs) 5,6,7,8.
ISGs tsim cov pob txha ntawm lub cev tiv thaiv kab mob, tshwj xeeb tshaj yog nyob rau hauv kev teb rau tus kab mob.Raws li thawj kab ntawm kev tiv thaiv kab mob kis tau tus kab mob, cov cell sai sai xa cov kev sib cuam tshuam ntawm cov cellular proteins nrog ntau yam kev ua ub no lom neeg.Cov proteins no suav nrog cov qauv lees paub receptors, taw qhia cov khoom lag luam, cov khoom hloov pauv, thiab cov proteins uas muaj cov tshuaj tiv thaiv kab mob ncaj qha, nrog rau cov tswj tsis zoo ntawm lub cev tiv thaiv kab mob 9.Ntau cov ntaub ntawv ntawm ISG kev ua haujlwm yog los ntawm cov ntxaij vab tshaus ua haujlwm siv cov ntxaij vab tshaus overexpression10,11 lossis gene silencing cov txheej txheem (siRNA, RNAi thiab CRISPR) 12,13 uas tus neeg ISGs tau nthuav tawm lossis cuam tshuam thiab lawv cov haujlwm raug kuaj ntawm ntau yam kab mob.Txawm hais tias cov kev tshawb fawb no tau txiav txim siab cov tshuaj tiv thaiv kab mob ntawm ib tus neeg ISGs, cov txheej txheem molecular ntawm txhua ISG tseem tsis paub ntau.Nws feem ntau lees paub tias ntau cov protein cuam tshuam nrog ib lossis ntau dua cytokines kom ntseeg tau tias muaj kev ua tiav tag nrho, yog li ISGs cuam tshuam ncaj qha lossis lawv cov kev sib cuam tshuam yog kho los ntawm cov cellular proteins.Piv txwv li, kev tshawb fawb photocrosslinked proteomics tsis ntev los no tau txheeb xyuas ATPase VCP/p97 ua tus koom tes loj IFITM3 kev cuam tshuam, uas nws inhibition ua rau muaj qhov tsis xws luag hauv lysosomal sorting, turnover, thiab cotransport ntawm IFITM3 nrog cov kab mob 14.Siv immunoprecipitation, peb tau txheeb xyuas VAPA, ib qho vesicle-associated protein, raws li kev sib koom tes nrog IFITM1/2/3 uas kho cov cholesterol-mediated viral maturation, thiab qhov no tau lees paub los ntawm lwm txoj kev tshawb fawb siv cov poov xab ob-hybrid system.Kev Txhawb Kev Tshawb Fawb 15, 16 .
Ib qho tseem ceeb ntawm cov txheej txheem lom neeg koom nrog hauv kev tawm tsam ntawm kev kis kab mob thiab kev hloov pauv tsis zoo yog kev nthuav qhia antigen, uas yog kho los ntawm cov tshuaj histocompatibility complex (MHC) molecules.Peptides (8-12 amino acids ntev) los ntawm cleaved, prematurely txiav los yog misfolded proteins yog loaded rau hauv MHC-I heterodimer (muaj MHC-I hnyav thiab lub teeb chains, hu ua β-2-microglobulin; β2M) 17,18 .Qhov ua tau ruaj khov MHC-I trimers raug thauj mus rau ntawm lub xov tooj ntawm tes, qhov chaw uas lawv nthuav tawm intracellular peptides rau CD8 + T hlwb (cytotoxic T cells)17.T hlwb paub thiab rhuav tshem cov kab mob no thiab cov hlwb uas nqa cov qog tshwj xeeb antigen.Yog li ntawd, cov kab mob thiab cov qog hlwb feem ntau cuam tshuam cov txheej txheem kev nthuav qhia antigen kom tsis txhob muaj kev tiv thaiv kab mob.Tsis tas li ntawd, MHC-I yog downregulated nyob rau hauv 40-90% ntawm tib neeg qog nqaij hlav thiab feem ntau txuam nrog ib tug tsis zoo prognosis19.
Cov noob koom nrog hauv kev teb rau cov kab mob yuav tsum hloov pauv sai sai ntawm lub xeev ntawm so thiab lub xeev ntawm kev ua haujlwm.Yog li ntawd, ntau lub cellular proteins yog hypothesized los koom tes nyob rau hauv cov lus teb rau siab IFN thov nyob rau lub sij hawm luv luv ntawm lub sij hawm, nrog rau kev hloov kho thiab kev hloov kho ntawm promoting chromatin 20,21.Feem ntau cov kev tshawb fawb tau tsom mus rau kev txheeb xyuas tus neeg ISG cov neeg koom tes protein nyob rau hauv lub xub ntiag ntawm IFN.Ntau qhov kev tshawb fawb proteomic thiab transcriptomic hauv cov qauv ntawm tes tau piav qhia cov txiaj ntsig ntawm IFN ntawm thaj chaw cellular.Txawm li cas los xij, txawm hais tias muaj kev nkag siab ntau ntxiv ntawm cov kev cuam tshuam los ntawm interferons, peb tseem paub me ntsis txog kev koom tes ntawm ISGs.Thaum xav txog qhov nyuaj thiab lub sij hawm-dependent dynamics ntawm interferon signaling, ob lo lus nug tshwm sim: (i) nws puas muaj peev xwm mus stabilize thiab cuab cov multiprotein complexes koom nyob rau hauv ceev ceev signaling, thiab (ii) cov kev sib cuam tshuam yuav mapped rau hauv 3D qhov chaw?
Txhawm rau daws cov teeb meem no, peb tau siv disuccinimide suberate-mediated chemical cross-linking (DSS) ua ke nrog huab hwm coj spectrometry (CLMS) los kawm txog IFNα-induced protein sib cuam tshuam network thiab nws cov dynamics.DSS ntxiv covalent bonds ntawm proximal residues ntawm cov proteins thiab/los yog protein complexes hauv vivo.Tom qab MS kev tshuaj xyuas qhia pom cov chaw sib txuas sib txuas uas cuam tshuam txog qhov sib thooj ntawm thaj chaw hauv ib qho protein, hu ua kev sib txuas sab hauv, lossis subunits hauv protein complexes, hu ua kev sib txheeb.Siv txoj hauv kev no, peb tau txheeb xyuas ntau yam tshiab protein-protein complexes nrog rau interferon-induced multiprotein kev sib koom tes tes hauj lwm.Los ntawm kev sim txuas ntxiv ntawm cov kev sib cuam tshuam tshiab no, peb pom tau tias H2BFS (H2B histone-type FS; hereinafter hu ua H2B) thiab MDN1 ua raws li kev sib koom tes rau HLA-A.
Flo-1 hlwb yog ib qho zoo tshaj plaws paub hauv vitro qauv ntawm esophageal adenocarcinoma raws li lawv ua raws li cov yam ntxwv tseem ceeb ntawm cov qog nqaij hlav esophageal 22,23.Txawm li cas los xij, tsis yog txhua tus qog nqaij hlav yog immunogenic, thiab txhawm rau txiav txim siab tias Flo-1 hlwb teb rau kev kho interferon, peb kho Flo-1 hlwb nrog 10 ng / ml IFNα rau 72 teev.Flo-1 hlwb pom thaum ntxov induction ntawm pSTAT1 thiab IRF1, pib 2 teev tom qab kev kho mob thiab txuas ntxiv mus rau 72 teev, nrog rau lub sij hawm-dependent txo nyob rau hauv cov theem ntawm IRF1 (Daim duab 1A).ISGs (MX1, IFITM1, OAS1/2, thiab ISG15) tau pom tias muaj kev cuam tshuam loj heev tom qab 6 teev, ua raws li cov lus teb hauv nruab nrab thiab lig rau IFNα (Daim duab 1A).Ua ke, cov ntaub ntawv no qhia tias tus qauv cellular no tuaj yeem siv los kawm cov lus teb interferon.
Qhov sib txawv ntawm cov protein qhia cov lus teb hauv Flo-1 hlwb tom qab kev kho IFNα.(A) Protein qhia hauv Flo-1 hlwb kho nrog 10 ng / ml IFNα rau 2, 6, 24, 48 thiab 72 teev tau soj ntsuam los ntawm immunoblot siv cov tshuaj tiv thaiv ISG.(B) Coomassie xiav stained SDS-PAGE gels ntawm tag nrho cov cell rho tawm tom qab kev sib txuas nrog DSS rau lub sijhawm qhia thiab qhov ntau.(C) Tus neeg sawv cev immunoblot tshuaj xyuas nrog p53 (DO-1) antibody los ntawm tib cov qauv los ntsuas cov qib ntawm cov protein sib txuas.
Txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhim kho cov kab mob, peb siv DSS.Lub sij hawm luv luv ntawm cov tshuaj tiv thaiv pab tiv thaiv kev tsim ntawm cov kab sib txuas loj ntawm cov protein sib txuas, yog li tswj kev ruaj ntseg ntawm crosslinker.Txhawm rau txiav txim siab qhov zoo tshaj plaws DSS concentration thiab tsis txhob hla dhau, peb xub nthuav tawm cov hlwb rau 5, 2.5, thiab 1 mM DSS rau 5, 10, 5, thiab 30 feeb, raws li, thiab tshuaj xyuas cov lysates los ntawm Coomassie-stained SDS-PAGE (cov ntaub ntawv tsis qhia).Cell lysates zoo li muaj kev sib txuas ntawm qhov qis tshaj plaws thiab lub sijhawm luv tshaj plaws.Yog li, DSS tau titrated rau 1, 0.5, thiab 0.1 mM dhau 5 feeb (Daim duab 1B).Qhov zoo tshaj plaws crosslinking tau pom nrog 0.5 mM DSS rau 5 feeb, thiab cov xwm txheej no tau raug xaiv rau cov hlwb kho nrog IFNα.Tsis tas li ntawd, daim duab 1C qhia tau hais tias Western blot tau ua los ntawm p53 (DO-1) antibody los ntsuas cov qib ntawm cov protein sib txuas.
Flo-1 hlwb raug kho nrog 10 ng / ml IFNα rau 24 teev ua ntej ntxiv cov crosslinker.Cov hlwb sib txuas tau tom qab lysed los ntawm ob-kauj ruam proteolysis thiab cov proteins tau ua tiav los ntawm FASP (Fig. 2)24,25.Hla-linked tryptic peptides tau soj ntsuam los ntawm huab hwm coj spectrometry (Fig. 2).Lub MS / MS spectra yog tom qab ntawd sib npaug rau cov protein ntau thiab suav nrog MaxQuant26,27.Hla-txuas peptides tau txheeb xyuas los ntawm qhov tau txais spectra siv SIM-XL qhov kev pab cuam, thiab cov tib neeg sib txuas tau muab tso ua ke rau hauv ib qho kev sib txuas uas siv xQuest28 thiab SIM-XL29 qhib qhov chaw suav cov kav dej (Fig. 2).SIM-XL qhia txog kev sib cuam tshuam ntawm cov protein-protein, cov saw hlau sab hauv thiab cov saw hlau ib leeg hauv cov protein sib xyaw yooj yim lossis nyuaj thiab muab cov ntawv sau rau kev pom kev sib cuam tshuam hauv cov qauv protein.Tsis tas li ntawd, nws nyob rau txhua qhov sib txawv raws li tus qhab nia ID raws li MS / MS29 spectrum zoo.Ob peb qhov kev sib raug zoo ntawm cov protein-protein kev sib cuam tshuam thiab cov complexes tau raug txheeb xyuas, thiab cov txheej txheem tshiab ntawm kev sib cuam tshuam tau raug tshawb xyuas ntxiv los ntawm kev siv co-immunoprecipitation thiab conformational hloov ntawm complexes siv molecular dynamics (MD) qauv (Fig. 2) 30, 31.
Schematic txheej txheem cej luam ntawm CLMS txoj kev.Flo-1 hlwb tau kho nrog 10 ng / ml IFNα rau 24 teev tom qab los ntawm kev sib txuas ntawm cov protein sib txuas siv DSS ua raws li cov cell lysis thiab trypsinization.Cov qauv sib txuas sib txuas tau txheeb xyuas siv lub Orbitrap mass spectrometer thiab ua piv txwv ntxiv rau kev tawg ntawm peptide precursors thaum LC-MS / MS.Ob qhov sib txuas peptides tau txheeb xyuas los ntawm qhov tau txais spectrum siv Spectrum Recognition Machine ntawm Crosslinked Peptides (SIM-XL) program, thiab tag nrho cov tebchaw tau muab tso rau hauv ib qho kev sib txuas uas siv cov kav dej sib txuas.Lim tawm qhov kev ntseeg siab tsis tshua muaj kev cuam tshuam raws li cov qhab nia tsis zoo (FDR).Ntau qhov kev sib raug zoo ntawm cov protein-protein tshiab tau raug lees paub ntxiv los ntawm kev siv co-immunoprecipitation, thiab kev hloov pauv hauv cov complexes raug tshuaj xyuas siv cov qauv molecular dynamics (MD).
Tag nrho ntawm ~ 30,500 thiab ~ 28,500 peptides tau kuaj pom siv MaxQuant hauv cov qauv uas tsis muaj zog thiab txhawb nqa IFNα, raws li (Sab Ntxiv Table S1, Fig. 3A).Qhov ntev peptide faib nyob rau hauv ob qho tib si pom muaj ntau dua ntawm cov peptides loj, qhia tias muaj kev sib txuas ntawm peptides (Fig. 3B, C).Tsis tas li ntawd, qhov feem ntau ntawm cov peptides loj dua muaj nyob rau hauv 40-55 ntau yam hauv IFNα-kho cov qauv (Daim duab 3C).Protein mapping tiv thaiv log2 siv tau pom tias classic interferon-stimulated proteins yog ntau tshaj piv rau cov qauv tsis kho, xws li MX1, IFIT1/3, OAS2/3, DDX58, thiab HLA-F (Daim duab 3D).Kev soj ntsuam ntawm txoj hauv kev rau cov protein ntau tshaj li peb-fold enriched nyob rau hauv cov lus teb rau IFNα kev kho mob siv lub Reactome pathway database qhia tias MHC-I-mediated antigen kev nthuav qhia thiab kev ua yog txoj kev tseem ceeb tshaj plaws (Daim duab 3E).Raws li cov ntaub ntawv tshaj tawm ua ntej, cov lus teb tshuaj tiv thaiv kab mob sib kho los ntawm OAS thiab ISG15 nrog rau IFNα / β thiab cytokine signaling yog ntawm txoj hauv kev qhib.Tsis tas li ntawd, lysine- thiab serine-specific protein cross-links tau txheeb xyuas los ntawm qhov pib tau txais MS / MS spectra siv SIM-XL.Ib txoj kev tshawb fawb tsis ntev los no tau tshaj tawm txog 104 ISGs hla 20 tus kab mob los ntawm 9 chav kawm kab mob los ntawm kev tshuaj ntsuam meta ntawm ib tus neeg ISG overexpression kev tshawb fawb hauv 5 cell type9.Txawm li cas los xij, txhawm rau kov yeej cov kev txwv kev suav ntawm kev tshuaj xyuas cov ntaub ntawv loj, peb pib nrog cov ntaub ntawv me dua los tshawb txog kev sib cuam tshuam ntawm cov npe ntawm IRDS cov noob qhia los ntawm Padaria li al., feem ntau yog ISGs.
Kev txheeb xyuas qhov sib txawv ntawm cov kab sib txuas sib txuas hauv cov lus teb rau IFNα (cov ntaub ntawv tau los ntawm MaxQuant).(A) Venn daim duab sawv cev rau tus naj npawb ntawm cov peptides ntau thiab tshwj xeeb uas tau txheeb xyuas hauv IFNα14 kho thiab tsis kho Flo-1 kuaj.Peptide ntev tis ntawm untreated (B) thiab IFNα kho (C) crosslinked kuaj.(D) Daim ntawv qhia tshav kub uas sawv cev rau lub log2 (LFQ siv) ntawm cov tsis kho thiab IFNα14 kho Flo-1 hlwb.Lub vaj huam sib luag sab laug qhia tau hais tias cov proteins uas nquag nquag nquag ua haujlwm nyob rau hauv lub xub ntiag ntawm IFNα.(E) Histogram sawv cev rau 20 txoj hauv kev txhawb nqa loj tom qab kho IFNα.Cov ntaub ntawv Reactome pathway tau soj ntsuam ntau dua plaub zaug hloov pauv hauv cov txheej txheem IFNα-teb cov proteins.
Interferon-mediated ISG stimulation tau sau tseg zoo, tab sis ntawm qib molecular nws tsis nkag siab tias cov proteins no ua rau muaj ntau yam kev ua haujlwm lom neeg.Peb tau tshawb xyuas cov protein sib cuam tshuam nrog kev ntseeg siab ntawm ISGs paub.Interestingly, peb tau txheeb xyuas lub network xws li MX1, USP18, ROBO1, OAS3, thiab STAT1 cov proteins uas tsim cov txheej txheem loj hauv kev teb rau IFNα kev kho mob (Daim duab 4, Table S2) 32,33,34.Qhov tseem ceeb tshaj plaws, cov kev sib cuam tshuam no tau pom nyob rau hauv tag nrho cov triplicates kho nrog IFNα thiab tsis pom nyob rau hauv cov qauv tsis kho, qhia tias lawv tau tsim tshwj xeeb hauv kev teb rau IFNα kev kho mob.Nws paub tias STAT1 transcriptionally tswj cov kev qhia ntawm cov ISGs, tab sis nws kev cuam tshuam nrog ISGs ntawm cov protein tsis tau kawm.Cov qauv siv lead ua ntawm STAT1 tau pom tias nws cov helical domain (CCD) tsis koom nrog kev cuam tshuam nrog DNA lossis protomers thaum tsim cov dimers35.Cov α-helices tsim cov qauv helical helix uas muab thaj chaw hydrophilic feem ntau rau kev sib cuam tshuam tshwm sim 35 .Hauv peb cov ntaub ntawv CLMS, peb tau pom tias feem ntau ntawm kev sib cuam tshuam nrog STAT1 tshwm sim hauv SH2 sau ua ntej CCD, qhov txuas txuas, lossis C-terminal tail (residues 700-708) (Daim duab 4A).Ib txoj kev tshawb fawb yav dhau los tau tshaj tawm tias USP18 khi rau CCD thiab DNA-binding domain (DBD) ntawm STAT2 thiab raug xaiv mus rau lub subunit ntawm hom I interferon receptor IFNAR2 los kho qhov inhibition ntawm hom I interferon signaling 24 .Peb cov ntaub ntawv kuj tau qhia tias USP18 catalytic domain cuam tshuam nrog STAT1 DBD (Daim duab 4A, D), qhia tias ob qho tib si STAT1 thiab STAT2 tuaj yeem ua lub luag haujlwm hauv kev nyiam USP18 rau IFNAR2.
Protein-protein ISG network txheeb xyuas nyob rau hauv cov kab txuas txuas txuas nrog IFNα.(A) 2D kev sib cuam tshuam zaj duab xis uas qhia txog kev sib cuam tshuam ntawm cov protein-protein (tsim nyob rau hauv SIM-XL qhov kev pab cuam), nrog cov kab uas sawv cev rau kev sib cuam tshuam (crosslink cutoff teem rau 3.5).Cov npe sib txawv yog cim los ntawm lawv cov xim 32: MX1 sau, Dynamin_N (73–249), Dynamin_M (259–547), thiab GED (569–660).OAS3 domains: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), thiab OAS1_C (903-108).Domain ROBO1, Ig_3 (67–151), I-set (170–258), I-set (262–347), Ig_3 (350–432), Ig_3 (454–529), fn3 (562–646), fn3 (678–758) thiab fn3 (777–864).STAT1 teb: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), thiab STAT1_TAZ2bind (715–739).(B) Cov neeg saib lub voj voog ntawm cov kab sib txuas sib txuas (MX1, UBP18, OAS3, ROBO1, thiab STAT1) nrog kev sib cuam tshuam thiab kev sib cuam tshuam uas sau ua xim xiav thiab liab, raws li.Qhov kev sib txuas sib txuas tau teeb tsa ntawm 3.5.Dot plots qhia STAT1 qhov chaw sib cuam tshuam nrog MX1 (C), USP18 (D), ROBO1 (E), thiab OAS3 (F), nrog rau K lossis S qhov chaw sib cuam tshuam ntawm ob lub peptides.Hauv daim duab, tus qhab-nees sib txuas ntawm qhov pib yog teem rau 3.0.(G) Ntau qhov chaw sib cuam tshuam ntawm STAT1 thiab OAS3 DI domains superimposed ntawm lawv cov qauv protein hauv PyMol (PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) and OAS3 (pdb id: 4s3n34).) program.
Ob lub isoforms ntawm USP18 tau piav nyob rau hauv tib neeg, ib tug tag nrho-ntev protein uas yog feem ntau nyob rau hauv lub nucleus, thiab ib tug isoform tsis muaj ib tug N-terminal domain, USP18-sf, uas yog sib npaug zos faib nyob rau hauv lub cytoplasm thiab nucleus 36.Tsis tas li ntawd, N-terminus tau kwv yees tias yuav tsis muaj teeb meem thiab tsis tas yuav muaj kev ua haujlwm isopeptidase lossis ISG1537 khi.Feem ntau ntawm kev sib cuam tshuam hauv peb txoj kev tshawb fawb tau nyob ntawm N-qhov kawg ntawm cov protein, tawm tswv yim tias cov kev sib cuam tshuam no cuam tshuam nrog USP18 tag nrho (Daim duab 4A, D) thiab yog li yuav tshwm sim hauv cov nucleus.Ntxiv mus, peb cov ntaub ntawv kuj qhia tau hais tias N-terminus yog tshwj xeeb rau cov protein-rau-protein kev sib cuam tshuam.Lub IFNAR2 khi qhov chaw nyob nruab nrab ntawm cov seem 312-368, thiab qhov tseem ceeb, tsis muaj cov proteins nyob rau hauv txoj hlua khi rau thaj tsam no (Fig. 4A) 37,38 .Cov ntaub ntawv no tau coj los ua ke qhia tias IFNAR2 binding domain tsuas yog siv los ntawm receptor protein.Tsis tas li ntawd, tsuas yog OAS3 thiab ROBO1 tau pom tias muaj feem cuam tshuam nrog cov thawj coj ntawm N-terminus thiab IFNAR2 khi qhov chaw (Daim duab 4A).
ROBO1 belongs rau immunoglobulin (Ig) superfamily ntawm transmembrane signaling molecules thiab muaj tsib Ig domains thiab peb fibronectin (Fn) domains hauv cheeb tsam extracellular.Cov extracellular domains yog ua raws li los ntawm ib cheeb tsam membrane-proximal thiab ib qho transmembrane helix 39. Ib cheeb tsam intracellular unstructured nyob rau ntawm C-terminus thiab muaj conserved sequence motifs uas mediate effector protein binding39.Cov cheeb tsam txuas ntxiv los ntawm cov amino acids ~ 1100 txog 1600 feem ntau tsis zoo.Peb pom tias MX1 cuam tshuam nrog ROBO1 los ntawm Ig, Fn, thiab intracellular domains, thaum feem ntau kev cuam tshuam nrog STAT1 tshwm sim ntawm nws CCD, linker domain, thiab C-terminus ntawm ROBO1 (Fig. 4A, E).Ntawm qhov tod tes, kev sib cuam tshuam nrog DI, DIII, thiab OAS3 txuas cov cheeb tsam tau muab faib thoob plaws hauv ROBO1 protein (Fig. 4A).
Cov tsev neeg oligoadenylate synthase (OAS) protein txais thiab khi intracellular ob-stranded RNA (dsRNA), ua rau muaj kev hloov pauv, thiab ua ke 2′,5′-txuas oligoadenylates (2-5 As) 40 .Nws tau pom tias ntawm peb OASs, OAS3 nthuav tawm qhov kev sib raug zoo siab tshaj plaws rau dsRNA thiab ua kom muaj qhov tsawg kawg nkaus ntawm 2-5 As, uas tuaj yeem qhib RNase L thiab yog li txwv tsis pub kis kab mob 41.OAS tsev neeg muaj cov polymerase beta (pol-β) zoo li nucleotide transferase domains.Cov kev tshawb fawb yav dhau los tau pom tias cov kev ua haujlwm catalytic ntawm C-terminal domain (DIII) yog nyob ntawm dsRNA-binding domain (DI), uas yuav tsum tau ua kom OAS342.Peb pom tias DI thiab DII ntawm OAS3 cuam tshuam nrog CCD thiab thaj tsam me me ntawm SH2 thiab STAT1 TAD (Daim duab 4A, F).Overlaying sib txawv crosslinking qhov chaw ntawm cov qauv protein tau qhia txog kev sib cuam tshuam ntawm β-daim ntawv thiab DBD STAT1 voj thiab ib lub hnab qhib lossis kab noj hniav tsim los ntawm cov seem 60–75 hauv DI domain ntawm OAS3 (Fig. 4G).Kev taw qhia ntawm cov proteins hauv cov complex kuj tau qhia tias tsis muaj kev cuam tshuam nrog OAS3 cuam tshuam nrog DNA-binding peev xwm ntawm nws cov DI domain (Fig. S1A).Tsis tas li ntawd, N-terminal domains ntawm GTPase MX1 cuam tshuam nrog DI thiab DIII domains ntawm OAS3 (Fig. 4A).Peb kuj tau soj ntsuam kev sib cuam tshuam ntawm OAS1 thiab MX1 hauv tag nrho peb IFNα-kho rov ua dua, qhov twg ib qho OAS1 domain (kuj catalytically active) cuam tshuam nrog tag nrho peb MX1 domains (Daim duab S2A, B).
MX proteins yog ib feem ntawm tsev neeg loj ntawm dynein-zoo li GTPases uas muaj N-terminal GTPase domain uas khi thiab hydrolyzes GTP, ib qho chaw nruab nrab uas kho nws tus kheej, thiab C-terminal leucine zipper uas ua raws li GTPase (LZ. ).domain effector domain25,43.MX1 khi rau subunits ntawm viral polymerases los thaiv kev hloov pauv ntawm tus kab mob gene43.Ib qho yav dhau los qhia cov poov xab ob-hybrid tshuaj ntsuam pom tau tias PIAS1-koom nrog MX1 inhibits STAT1-mediated gene activation los ntawm thaiv DNA-binding kev ua si thiab kuj muaj SUMO E344,45 ligase kev ua.Ntawm no, peb pom tias MX1 khi rau STAT1 (Daim duab 4C, D), txawm li cas los xij, qhov kev cuam tshuam no cuam tshuam li cas rau STAT1-mediated gene activation hauv teb rau IFNα xav tau kev kawm ntxiv.Tsis tas li ntawd, peb kuj pom tias MX1 cuam tshuam nrog IFIT3 thiab DDX60 hauv tag nrho peb IFNα-kho rov ua dua (Fig. S2C).
DDX60 yog IFN-induced cytoplasmic helicase uas yav dhau los tau tshaj tawm los ua lub luag haujlwm hauv RIG-I-kev ywj pheej degradation ntawm tus kab mob RNA46.Nws cuam tshuam nrog RIG-I thiab ua kom nws cov teeb liab nyob rau hauv ib qho ligand-specific yam 46. DDX60 muaj DEXD / H-Box helicase domain thiab C-terminal helicase domain uas khi tus kab mob RNA thiab DNA47.Feem ntau ntawm nws cov kev cuam tshuam nrog MX1 thiab IFIT3 tshwm sim nyob rau hauv ntev N- thiab C-terminal cheeb tsam tsis muaj canonical domains lossis motifs (Fig. S2E, F).Txawm li cas los xij, MX1 kuj tseem cuam tshuam nrog DEXD / H-Box helicase domain (Fig. S2E).Proteins ntawm tsev neeg IFIT muaj tandem luam ntawm qhov txawv helix-tig-helix motif hu ua tetrapeptide repeat (TPR).IFIT3 tau pom tias yog ib qho zoo modulator ntawm RIG-I signaling thiab yog li ib feem ntawm MAVS complex.Ua ke, peb cov ntaub ntawv qhia tias IFIT3 thiab DDX60 cuam tshuam feem ntau hauv cheeb tsam ntawm TPR 3-6 ntawm IFIT3 thiab tuaj yeem ua lub luag haujlwm hauv RIG-I / MAVS signaling (Fig. S2F).
Muab hais tias kev tshuaj xyuas tag nrho cov proteome yog suav nrog, peb mam li tshuaj xyuas tag nrho tib neeg UniProt database rau muaj ib qho ntawm IFNα-kho rov ua dua.Hauv qhov kev hloov pauv no, peb pom ntau qhov kev sib cuam tshuam zoo heev rau HLA-A.Kev soj ntsuam ntawm txoj hauv kev protein txheeb xyuas los ntawm MS / MS spectra tau pom tias MHC-I-based antigen ua thiab kev nthuav qhia yog txoj hauv kev tseem ceeb ntawm kev cuam tshuam los ntawm interferon (Fig. 3D).Yog li ntawd, peb tau tsom mus rau kev kawm txog kev sib cuam tshuam ntawm cov protein ntawm MHC-I molecules nrog kev ntseeg siab hauv txhua qhov kev sib txuas sib txuas.HLA muaj xws li α1, α2 thiab α3 domains thiab lub teeb chains, thiab microglobulin β2 (β2m) yog ib qho tsis tu ncua chaperone protein49.Thaum sib sau ua ke hauv endoplasmic reticulum, HLA tsis ruaj khov thaum tsis muaj peptide ligands50.Lub peptide-binding zawj yog tsim los ntawm cov polymorphic heev thiab tsis muaj qauv α1 thiab α2 domains nyob rau hauv non-peptide daim ntawv thiab qhov tsawg dua polymorphic α351 domains.Nyob rau hauv lub xub ntiag ntawm IFNα, peb tau kuaj pom ob lub HLA-A complexes: ib qho cuam tshuam nrog HMGA1 thiab H2B (Daim duab 5, Table S3) thiab lwm yam cuam tshuam nrog MDN1, LRCH4 thiab H2B (Daim duab 6).
IFNα induces HLA-A kev sib koom tes nrog H2B (H2BFS) thiab HMGA1.(A) 2D zaj duab xis (tsim hauv SIM-XL software) qhia txog ntau hom kev sib cuam tshuam hauv H2B-HLA-A-HMGA1 complex: interlink (xiav), interlink (liab) thiab ib qhov txuas (dub)..Cov npe ntawm cov cim sib txawv yog xim coded32: H2B (histone; 2–102) thiab MHC-I (MHC_1; 25–203, pawg C1; 210–290 thiab MHC_I_C; 337–364).Qhov kev sib txuas sib txuas tau teeb tsa ntawm 3.5.Dot plots qhia HLA-A qhov chaw sib cuam tshuam nrog H2B (B) thiab HMGA1 (C), nrog rau K lossis S qhov chaw sib cuam tshuam ntawm ob lub peptides.Hauv daim duab, tus qhab-nees sib txuas ntawm qhov pib yog teem rau 3.0.(D) Kev sib raug zoo ntawm cov protein uas pom nyob rau hauv cov qauv ntawm H2B, HLA-A, thiab HMGA1 cov proteins hauv PyMOL program.Cov qauv no tau ua qauv siv Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2) thiab cov qauv qauv rau H2B, HLA-A thiab HMGA1 proteins yog 1kx552, 1kj349 thiab 2eze55, raws li.
IFNα induces HLA-A kev sib koom tes nrog H2B (H2BFS), MDN1 thiab LRCH4.(A) Intramolecular (liab) thiab intermolecular (xiav) crosslinks nthuav tawm ntawm 2D daim ntawv qhia kev sib tham (tsim hauv SIM-XL software) nrog MDN1 sawv cev ua lub voj voog.Qhov kev sib txuas sib txuas tau teeb tsa ntawm 3.5.Cov npe ntawm cov cim sib txawv yog xim coded32: H2B (histone; 2–102), MHC-I (MHC_1; 25–203, pawg C1; 210–290 thiab MHC_I_C; 337–364) thiab LRCH4 (LRR_8 (68–126), LRR_8 (137–194) thiab CH (535–641)).(B) Kev sib raug zoo ntawm cov proteins uas pom hauv cov qauv ntawm H2B, HLA-A, LRCH4, thiab MDN1 cov proteins hauv PyMOL program.Cov qauv no tau ua qauv siv Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2) nrog cov qauv qauv 1kx552, 1kj349, 6hlu62 thiab 6i2665 rau H2B, HLA-A, LRCH4 thiab MDN1 proteins, raws.Dot cov phiaj xwm qhia K lossis S qhov chaw sib cuam tshuam rau HLA-A nrog H2B (C), LRCH4 (D), thiab MDN1 (E).Rau cov phiaj xwm, qhov sib txuas sib txuas tau raug teeb tsa rau 3.0.
Ntxiv nrog rau kev tswj hwm kev ncaj ncees ntawm cov genome, histone H2B kuj tseem koom nrog hauv kev tswj hwm ntawm kev sau ntawv.H2B protein muaj ib tug central histone domain (HFD) tsim los ntawm peb α-helices sib cais los ntawm loops thiab ib tug C-terminal tail 41,52.Feem ntau ntawm kev cuam tshuam nrog H2B tshwm sim hauv α1 helix, uas muab trimerization nrog HFD heterodimer (Fig. 5A, B).Txawm hais tias lysins koom nrog DNA khi, qee qhov lysins kuj yog lwm qhov acetylation lossis methylation sites.Piv txwv li, residues K43, K46, thiab K57 los ntawm H2B tsis koom nrog kev khi ncaj qha DNA, tab sis yog lub hom phiaj ntawm ntau yam kev hloov pauv tom qab kev hloov pauv53.Ib yam li ntawd, residues K44, K47, thiab K57 hauv H2B tuaj yeem ua lub luag haujlwm lwm txoj hauv kev muaj IFNα, suav nrog kev cuam tshuam nrog lwm cov proteins (Fig. 5A, B).Tsis tas li ntawd, lub extrachromosomal histone H2B activates lub cev tiv thaiv kab mob nyob rau hauv ntau hom ntawm tes, ua raws li ib tug cytosolic sensor mus ntes ob-stranded DNA (dsDNA) fragments muab tau los ntawm cov kab mob los yog puas hlwb54.Thaum muaj cov kab mob DNA, H2B depletion inhibited IFN-β ntau lawm thiab STAT154 phosphorylation.H2B tseem paub tias txav mus rau hauv thiab tawm ntawm lub nucleus sai dua li lwm cov tub ntxhais histones54.Kev sib cuam tshuam H2B nrog MDN1 thiab LRCH4 kuj tau pom nyob rau hauv cov qauv xaiv tsis kho.Peb pom tias HLA-A cuam tshuam nrog H2B hauv tag nrho peb qhov IFNα-kho cov qauv thiab hauv ib qho piv txwv uas tsis tau kho dua.Cov ntaub ntawv no qhia txog lub luag haujlwm ntawm H2B hauv lwm txoj haujlwm physiological tsis muaj kev tswj hwm kev hloov pauv.
HMGA1 (High Mobility Group AT-Hook 1), ib tug me me nucleoprotein nplua nuj nyob rau hauv cov kab mob txhawb cov amino acids, tau raug txheeb xyuas nyob rau hauv koom nrog HLA-A.Nws muaj acidic C-terminal tail thiab peb qhov sib txawv DBDs hu ua AT hooks vim lawv khi rau qhov me me ntawm thaj tsam AT-nplua nuj hauv dsDNA55,56.Qhov kev khi no ua rau DNA khoov lossis ncaj, tso cai rau cov ntaub ntawv hloov pauv hloov mus rau nws qhov kev pom zoo ua ntu zus.C-terminal tus Tsov tus tw yog ntseeg tau koom nrog hauv kev sib cuam tshuam ntawm cov protein-protein thiab kev nrhiav neeg ua haujlwm ntawm kev hloov pauv, txij li C-terminal deletion mutants tsis tuaj yeem pib transcription57.Ntxiv mus, lub npe no muaj ntau qhov chaw khaws cia phosphorylation uas paub cov substrates rau kinases 58 .Peb tau soj ntsuam HLA-A thiab H2B kev sib cuam tshuam nrog HMGA1 sab nraum C-terminal sau, qhia tias C-terminal domain feem ntau yog siv rau kev sib txuas lus (daim duab 5A, C).HMGA proteins sib tw nrog histone H1 rau kev khi rau DNA adapter, yog li nce kev nkag mus tau57.Ib yam li ntawd, nws zoo li tias HMGA cuam tshuam nrog histone H2B raws li qhov txuas DNA hauv kev sib tw nrog histone H1.HMGB1 induces kev qhia ntawm HLA-A, -B, thiab -C nyob rau hauv dendritic hlwb, ua rau lawv activation59, tab sis ib tug kev sib raug zoo ntawm HMG thiab HLA tsis tau yav tas los qhia.Peb pom tias HMGA1 cuam tshuam nrog α1 thiab α3 qhov ntawm HLA-A, nrog rau feem ntau ntawm kev sib cuam tshuam sab nraud ntawm nws 3 DBD (Daim duab 5A, C).Nyob rau hauv peb txhais tes, HLA-A tau pom nyob rau hauv lub nucleus (cov ntaub ntawv tsis qhia), thiab muab hais tias H2B thiab HMGA1 kuj muaj nyob rau hauv lub nucleus, qhov kev sib cuam tshuam yuav tshwm sim nyob rau hauv lub nucleus.Cov adducts tshwj xeeb ntsuas ntawm H2B, HLA-A, thiab HMGA1 yog qhia hauv daim duab 5D.
Feem ntau kev sib cuam tshuam ntawm HLA-A nrog lwm cov proteins tshwm sim nyob rau hauv nws cov α1 thiab α2 domains thiab qhov tsis sib haum xeeb C-terminal domain (Fig. 6).Hauv ib qho piv txwv no, peb pom tias HLA-A cuam tshuam nrog qhov tsis sib haum xeeb N-terminal tail ntawm LRCH4 (Daim duab 6A,D).LRCH4 tswj TLR4 ua kom thiab LPS cytokine induction, yog li hloov kho lub cev tiv thaiv kab mob 60,61.Nws yog ib qho membrane protein nrog cuaj leucine-nplua nuj repeats (LRRs) thiab ib qho calmodulin (CH) homology motif nyob rau hauv nws ectodomain, ua raws li los ntawm ib tug transmembrane domain (TMD) 60 , 62 .CH domains tau tshaj tawm los kho cov kev sib tshuam ntawm cov protein-protein 60 .Kev ncab ntawm txog 300 amino acids ntawm LRR thiab CH domains yog qhov nkag tau yooj yim tab sis tsis meej.Raws li kev ua haujlwm ntawm cov cheeb tsam tsis sib haum xeeb raws li cov neeg kho kom haum xeeb ntawm cov protein-protein networks thiab kev thauj mus los ntawm vesicular 63, peb pom tias feem ntau cov protein sib cuam tshuam tshwm sim hauv cov cheeb tsam tsis sib haum xeeb.Kev cuam tshuam nrog MDN1 tau muab faib thoob plaws qhov ntev ntawm cov protein, suav nrog LRR1, LRR6, CH domains, thiab random regions, thaum H2B feem ntau khi rau CH domains (Fig. 6A, B).Qhov tseem ceeb, tsis muaj ib qho kev cuam tshuam nrog TMJ, qhia txog qhov tshwj xeeb ntawm CLMS mus kom ze (Daim duab 6A, B).
MDN1 kuj tau raug txheeb xyuas tias yog ib feem ntawm HLA-A protein network (Daim duab 6A).Nws belongs rau AAA tsev neeg cov proteins (ATPases txuam nrog ntau yam dej num).Qhov no yog tib lub N-terminal AAA domain uas npaj rau hauv lub nplhaib hexameric thiab tshem tawm cov khoom sib dhos los ntawm 60S 64 ribosomal subunit.zoo li dynein64,65,66.Tsis tas li ntawd, thaj av Asp / Glu nplua nuj yog ua raws los ntawm MIDAS domain (hlau ion nyob ntawm qhov chaw).Vim qhov loj me me ntawm MDN1 (kwv yees li 5600 amino acids) thiab nws cov kev txwv homology nrog cov proteins uas tau kawm zoo, me ntsis paub txog nws cov qauv thiab kev ua haujlwm hauv tib neeg.Peb tau txheeb xyuas HLA-A, H2B, thiab LRCH4 ua MDN1 cov neeg koom tes thiab qhia lawv txoj kev taw qhia raws li cov protein ntau hauv PyMol (Fig. 6A, B).Peb cov proteins no cuam tshuam nrog AAA domain, dynein-like linker domain, thiab tejzaum nws yog MIDAS MDN1 domain.Nyob rau hauv tsab ntawv tshaj tawm dhau los, affinity purification of bait proteins identified MDN1 as a protein related with histone H2B67.Tsis tas li ntawd, ib txoj kev tshawb fawb tsis ntev los no kuj tau tshaj tawm txog kev sib cuam tshuam ntawm MDN thiab HLA-B hauv HCT116 hlwb siv affinity-purified mass spectrometry, txhawb peb qhov kev tshawb pom68.Kev txheeb xyuas qhov nyuaj no hauv IFNα-kho cov qauv qhia txog lub luag haujlwm rau MDN1 hauv interferon signaling.
Vim HLA noob yog polymorphic heev, peb rho tawm sequencing nyeem daim ntawv qhia HLA-A, -B, thiab -C los ntawm RNA sequencing cov ntaub ntawv ntawm Flo-1 hlwb (cov ntaub ntawv tsis qhia).Peptide sequences ua raws li kev nyeem ntawv sib txuas qhia qhov sib txawv tseem ceeb ntawm HLA-A, -B, thiab -C hauv thaj chaw uas cov peptides sib txuas nyob hauv HLA-A (Daim duab S3).Tsis tas li ntawd, peb tsis tau soj ntsuam cov protein-to-protein cross-linking ntawm HLA-B/C molecules nrog H2B/HMGA1/MDN1/LRCH4 proteins.Qhov no qhia tau hais tias cov protein sib cuam tshuam ntawm HLA-A, MDN1, LRCH1 thiab HMGA1 yog HLA-A tshwj xeeb.Tsis tas li ntawd, kev soj ntsuam proteomic ntawm cov qauv tsis sib txuas (Table S4) tau pom tias HLA-A muaj kev pab cuam ntau dua piv rau HLA-B lossis HLA-C.Cov peptides tau txheeb xyuas rau HLA-A tau siv ntau hauv IFNα-kho thiab tsis kho cov qauv.
Txhawm rau kom ntseeg tau tias qhov kev sib cuam tshuam uas tau txheeb pom ntawm no tsis yog vim qhov tsis sib xws ntawm kev sib txuas ntawm ob lub proteins nyob rau hauv qhov chaw sib ze, peb tau lees paub ntxiv ob qho tshiab HLA-A cuam tshuam los ntawm kev ua cov tshuaj tiv thaiv kab mob sib kis.HLA-A kev cuam tshuam nrog endogenous MDN1 thiab H2B tau kuaj pom hauv ob qho tib si IFNα-kho thiab tsis kho Flo-1 hlwb (Daim duab 7, Daim duab S4).Peb tau lees paub tias HLA-A raug ntes los ntawm H2B hauv immunoprecipitates thiab tias lub koom haum no yog vim kev kho IFNα txij li HLA-A tsis nyob hauv cov qauv immunoprecipitate los ntawm cov hlwb tsis kho (Daim duab 7A).Txawm li cas los xij, peb cov ntaub ntawv qhia tias IFNα sib txawv tswj HLA-A khi rau H2B thiab MDN1.IFNα induces koom haum ntawm H2B thiab HLA-A, tab sis txo nws cov koom haum nrog MDN1.Peb pom tias MDN1 cuam tshuam nrog HLA-A hauv kev tswj hwm, thiab qhov sib ntxiv ntawm IFNα txo qhov kev sib cuam tshuam ntawm MDN1 induction los ntawm IFNα (Daim duab 7B,C).Tsis tas li ntawd, HLA-A immunoprecipitation ntes H2B hauv A549 hlwb (Fig. S4), qhia tias qhov kev sib cuam tshuam no yog ywj siab ntawm hom cell.Ua ke, cov txiaj ntsig no txhawb kev sib cuam tshuam interferon ntawm HLA-A nrog H2B thiab MDN1.
HLA-A co-purifies H2B thiab MDN1.Cov neeg sawv cev endogenous H2B (A) thiab MDN1 (B) immunoblots tau immunoprecipitated los ntawm IFNα-kho Flo-1 hlwb thiab soj ntsuam rau cov tshuaj tiv thaiv.Nas thiab luav IgG tau siv los ua kev tswj tsis zoo.(C) Cov txheeb ze (cov tswv yim) ntawm cov antigens sib txawv tau piav qhia los ntawm immunoblots soj ntsuam tiv thaiv cov tshuaj tiv thaiv, β-actin tau siv los tswj kev thauj khoom.
Cov yam ntxwv ntawm ib qho ntawm cov interferon-induced siab txhim khu kev qha cross-linked tes hauj lwm, H2B-HLA-A-HMGA1, raug soj ntsuam.Peb siv cov qauv molecular dynamics ua ib txoj hauv kev kom nkag siab txog kev sib haum xeeb ntawm cov proteins koom nrog hauv qhov complex (Daim duab 8).Cov ntaub ntawv pov thawj los ntawm CLMS cov ntaub ntawv qhia txog qhov muaj peev xwm sib txawv ntawm H2B, HLA-A, thiab HMGA1 cov proteins.Yog li ntawd, cov nram qab no muaj peev xwm complexes tau ua qauv nyob rau hauv ib tug hnyav nruab nrab: H2B-HLA-A, HMGA1-HLA-A, thiab H2B-HLA-A-HMGA1.Ib qho kev tshuaj ntsuam protein-protein docking thawj zaug uas siv MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) pob qhia txog kev ua tau zoo uas txawv ntawm cov protein no (Fig. 8A).Kev pom ntawm docking protein complex qhia ntau yam kev sib cuam tshuam thiab ua tau zoo (Daim duab 5A, 8).Yog li, ib qho ua tau raws li tau pom nyob rau hauv daim duab 8A (nrog rau cov ntawv hla kev sib txuas) thiab nws tau raug soj ntsuam ntxiv los ntawm kev siv MD qauv kav dej.Tsis tas li ntawd, kev khi ntawm H2B lossis HMGA1 rau HLA-A qhia txog kev sib raug zoo dua ntawm H2B rau HLA-A (Fig. 8A).
Conformational dynamics ntawm tau tes hauj lwm ntawm H2B (H2BFS)-HLA-A, HMGA1-HLA-A, thiab H2B-HLA-A-HMGA1 complexes.(A) Sab laug vaj huam sib luag yog 2D daim ntawv qhia (tsim nyob rau hauv SIM-XL software) ntawm intramolecular (liab) thiab intermolecular (xiav) crosslinks (crosslink cutoff teem rau 3.5).Tsis tas li ntawd, cov kab sib txuas sib txuas tau txheeb xyuas tau sau rau ntawm cov qauv ntawm H2B, HLA-A, thiab HMGA1 proteins.Cov kev sib raug zoo ntawm cov proteins no tau muab rho tawm los ntawm kev siv cov docking pipeline siv hauv pob MOE.Lub vaj huam sib luag sab laug qhia tau hais tias muaj ntau yam ua tau raws li H2B-HLA-A thiab HMGA1-HLA-A complexes nrog sib txawv protein-protein binding affinities (GBVI / WSA dG; kcal / mol).(B) Tus qauv sib txawv (RMSD) ntawm atomic txoj haujlwm (tsis suav nrog hydrogen atoms) rau txhua tus qauv protein.(C) Intermolecular protein-protein hydrogen bond kev sib cuam tshuam los ntawm ntau yam simulated complexes xav txog kev sib cuam tshuam ntawm lub sijhawm ≥ 10 ns.H-bond pub-acceptor cutoff nrug tau teem rau 3.5 Å, thiab tus pub-H-acceptor txiav lub kaum sab xis yog teem rau ≥ 160 ° -180 °.(D) Cov ntaub ntawv pov thawj uas tsim HLA-A protein-protein kev sib cuam tshuam nrog lawv cov neeg koom tes, ncua ≥ 20 ns, muab rho tawm los ntawm dummy HLA-A-H2B thiab HLA-A-HMGA1 complexes.Protein structures sawv cev rau tus qauv nruab nrab ntawm 100 ns MDS.(E) Kev sib cuam tshuam ntawm HLA-A-H2B thiab HLA-A-HMGA1 complexes piv rau kev sib cuam tshuam los ntawm H2B-HLA simulation tshaj 100 ns raws li K lossis S kev sib cuam tshuam ntawm ob lub peptides.Complexes /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Tus nqi pib rau kev ntsuam xyuas ntawm kev sib txuas sib txuas tau teeb tsa rau 3.0, thiab kev sib cuam tshuam tshwj xeeb los ntawm MDS noj ≥ 10 ns tau muab coj los rau hauv tus account.Cov qauv Protein pom tau siv BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) thiab Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada) pob.
Kev ruaj ntseg ntawm HLA-A molecules nyob rau lub sijhawm (tus qauv sib txawv; RMSD lossis tus qauv sib txawv; RMSF) qhia tias muaj H2B lossis HMGA1 cov proteins hauv cov complexes stabilized HLA-A (Daim duab 8B, Daim duab S5).HMGA1 protein khi nruj rau B2M qhov chaw ntawm HLA-A, ua rau muaj kev ruaj ntseg ntawm HLA-A amino acids hauv HLA-A-HMGA1 lossis H2B-HLA-A-HMGA1 complex (Daim duab 8B, Daim duab S5).tshwj xeeb, HLA residues ~ 60-90 thiab ~ 180-210 tau pom tias tsis hloov pauv thaum muaj H2B (FIG. 8B).H2B thiab HMGA1 tau pom zoo khi rau HLA-A hauv H2B-HLA-A-HMGA1 complex piv rau HLA-A khi rau H2B lossis HMGA1 ib leeg (Daim duab 8C, D; Table S5).Cov residues koom nrog hydrogen bonding (MD ua qauv siab nyob ≥ 10 ns) coincide nrog CLMS kev sib cuam tshuam qhov chaw (K lossis S residues) hauv qhov nyuaj, qhia tias kev sib cuam tshuam los ntawm CLMS muaj kev ntseeg siab heev.Kev ntseeg tau (Daim duab 8E ).Hauv CLMS thiab MD qauv, HLA-A residues ntawm li 190-210 thiab txog 200-220 amino acids tau pom los khi H2B thiab HMGA1, raws li (FIG. 8E).
Protein-protein kev sib cuam tshuam tsim cov kev sib txuas ua ke zoo sib xws uas kho cov kev sib txuas lus intracellular hauv cov lus teb rau qee qhov stimuli.Vim tias muaj ntau txoj hauv kev proteomics kuaj pom cov kev hloov pauv hauv tag nrho lub xeev khov kho theem ntawm cov protein, cov protein-protein kev sib cuam tshuam yuav tsum muaj cov cuab yeej ntxiv los ntes cov kev sib txuas, thiab CLMS yog ib qho cuab yeej zoo li no.Interferon signaling system yog cytokine network uas tso cai rau cov hlwb los teb rau ntau yam ntawm ib puag ncig cov kab mob thiab cov kab mob hauv nruab nrog cev, ua rau muaj kev cuam tshuam ntawm subsets ntawm interferon-inducible proteins.Peb tau thov CLMS los txiav txim seb qhov kev sib cuam tshuam ntawm cov protein-protein tshiab tuaj yeem raug txheeb xyuas ntawm cov vaj huam sib luag ntawm interferon-induced proteins.Ntiaj teb no protein cross-linking tsom xam nyob rau hauv ib tug interferon-responsive Flo-1 cell qauv yog siv los ntes cov protein complexes.Kev rho tawm ntawm tryptic peptides los ntawm cov hlwb tsis sib txuas thiab cov kab sib txuas sib txuas tso cai rau kev suav peptide, txoj hauv kev ntxiv, thiab peptide ntev faib nrog LFQ siv.Canonical interferon-inducible proteins tau txheeb pom tias yog kev tswj xyuas zoo, thaum tshiab intermolecular thiab intramolecular cross-linked adducts ntawm canonical interferon-inducible proteins xws li MX1, UP18, OAS3 thiab STAT1 tau pom.Ntau yam qauv thiab kev sib cuam tshuam hauv thaj chaw ua haujlwm tau raug tshawb xyuas.
Kev sib cuam tshuam ntawm HLA-A, MDN1 thiab H2B tau kuaj pom los ntawm kev tiv thaiv kab mob hauv Flo-1 thiab A549 hlwb kho thiab tsis kho nrog IFNα.Peb cov txiaj ntsig tau qhia tias HLA-A complexes nrog H2B nyob rau hauv IFNα-dependent yam.Peb txoj haujlwm sawv cev rau txoj kev nthuav dav rau kev tshawb nrhiav ntxiv ntawm kev sib koom ua ke ntawm ob lub complexes.Nws kuj tseem yuav nthuav kom nthuav dav CLMS txoj hauv kev rau lub vaj huam sib luag ntawm cov kab ntawm tes los txheeb xyuas cov cell-type-independent interferon-mediated protein sib cuam tshuam.Thaum kawg, peb siv MD qauv ua lwm txoj hauv kev kom nkag siab txog kev sib haum xeeb ntawm cov proteins uas koom nrog hauv H2BFS-HLA-A-HMGA1 complex, uas taug qab cov lus sib tham hauv intramolecular thiab intermolecular.Cov ntaub ntawv pov thawj los ntawm CLMS cov ntaub ntawv qhia txog qhov muaj peev xwm sib txawv ntawm H2BFS, HLA-A, thiab HMGA1 cov proteins.Qhov sib txawv ntawm qhov sib txawv ntawm cov docking protein complexes qhia ntau yam kev sib cuam tshuam zoo ib yam li cov uas tau pom hauv CLMS dataset.Ib qho ntawm lub zog tseem ceeb ntawm peb txoj kev yog tias nws tso cai rau kev txheeb xyuas yooj yim ntawm kev sib cuam tshuam cov noob polymorphic xws li HLA, yog li nws yuav nthuav kom kawm txog kev sib cuam tshuam ntawm HLA haplotype-cov protein tshwj xeeb uas nyuaj rau kev kawm.Ua ke, peb cov ntaub ntawv qhia tau hais tias CLMS tuaj yeem siv los nthuav peb txoj kev nkag siab ntawm interferon-induced signaling tes hauj lwm thiab muab lub hauv paus rau kev kawm ntau txoj hauv kev sib txuas hauv cov qog microenvironment.
Flo-1 hlwb tau txais los ntawm ATCC thiab khaws cia hauv DMEM (Gibco) ntxiv nrog 1% penicillin / streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) thiab khaws cia ntawm 37 ° C thiab 5% CO2.Kev loj hlob.Cells tau loj hlob mus rau 70-80% confluence ua ntej raug kho nrog IFNα14 (tsim los ntawm Edinburgh Protein Production Facility).Tag nrho lwm cov tshuaj thiab cov tshuaj reagents tau yuav los ntawm Sigma Aldrich tshwj tsis yog sau tseg.
Flo-1 hlwb raug coj mus rau hauv 6-zoo daim hlau thiab hnub tom qab cov hlwb raug kho nrog 10 ng / ml IFNα14 rau 24 teev mus txog kwv yees li 80% confluence.Cells raug ntxuav peb zaug nrog PBS thiab ligated nrog freshly npaj DSS (Thermo Fisher Scientific) (kua hauv DMSO) hauv PBS rau 5 min ntawm 37 ° C. mus rau qhov kawg concentration ntawm 0.5 mM.DSS crosslinking cov tshuaj tiv thaiv tau hloov nrog PBS thiab cov seem DSS tau quenched los ntawm kev ntxiv 20 mM Tris (pH 8.0) hauv PBS rau 15 min ntawm 37 ° C.Cells tau sau los ntawm kev khawb thiab sau rau hauv cov hlua khi qis (Axygen).
Lub cell pellet tau lysed nrog 300 µl ntawm urea lysis tsis (8 M urea, 0.1 M Tris, pH 8.5) rau 30 min ntawm chav tsev kub nrog qee zaus co.Tag nrho cov kauj ruam centrifugation tau ua ntawm 14,000 xg ntawm 8 ° C.Centrifuge lysate rau 10 feeb thiab hloov lub supernatant mus rau lub raj tshiab.Cov seem ntshiab tau yaj nyob rau hauv 150 μl ntawm qhov thib ob lysis tsis (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) rau 30 feeb lossis ntau dua kom txog thaum tau txais cov kua dej sib xws.Lub lysate tau centrifuged rau 20 feeb thiab cov supernatant tau tov nrog cov lysate tau nyob rau hauv cov kauj ruam dhau los.Protein concentrations raug soj ntsuam siv Micro BCA assay (Thermo Fisher Scientific) raws li cov chaw tsim khoom cov lus qhia rau cov txheej txheem microplate.Cov qauv no tau khov sai sai hauv cov kua nitrogen thiab khaws cia ntawm -80 ° C.
Kwv yees li 100 μg ntawm soluble cross-linked protein tau ua tiav los ntawm kev hloov kho cov qauv kev npaj raws tu qauv (FASP) raws li tau piav qhia los ntawm Wisniewski li al.69 luv luv, cov protein yog crosslinked nrog 200 µl ntawm urea tsis (8 M urea hauv 0.1 M Tris, pH 8.5), vortexed thiab halved.Tag nrho cov kauj ruam centrifugation tau ua tiav ntawm 14,000 xg ntawm 25 ° C.Thawj ib nrab ntawm kev sib txuas ntawm cov protein lysate tau pauv mus rau 10 kDa Microcon centrifugal lim ntaus ntawv nruab nrog Ultracel-10 membrane (Merck), ua raws li centrifugation ntawm lub lim rau 25 feeb.Tom qab ntawd ntxiv ib nrab ntawm cov protein mus rau lub lim thiab rov ua cov kauj ruam tib yam.Protein rov qab tau ua los ntawm kev ntxiv 100 μl ntawm 17 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) hauv urea tsis.Rov qab tau nplawm rau ntawm lub thermomixer ntawm 600 rpm rau 30 min ntawm 37 ° C.Tsis tas li ntawd, kem tau centrifuged thiab txo cov kab sib txuas ntawm cov protein yog alkylated siv 100 μl ntawm 50 mM iodoacetamide hauv urea tsis.Cov tshuaj tiv thaiv alkylation tau nqa tawm ntawm chav tsev kub rau 20 feeb hauv qhov tsaus ntuj.Tig lub kem, ntxuav cov phab ntsa 3 zaug nrog 100 µl urea buffer, thiab ces centrifuge.Tib txoj haujlwm tau ua 3 zaug siv 100 μl ntawm 100 mM ammonium bicarbonate.Ua ntej trypsinization, hloov lub raj sau nrog ib qho tshiab.Ntxiv kev zom zaub mov tsis muaj 50 mM ammonium bicarbonate thiab 1 µl trypsin diluted hauv trypsin tsis (Promega).Qhov piv ntawm trypsin rau cov protein tau khaws cia ntawm li 1:33, thiab kev zom zaub mov tau ua rau hmo ntuj ntawm 37 ° C.Lub crosslinked peptide tau eluted los ntawm lub lim los ntawm centrifugation rau 25 feeb.Peptide rov qab zoo dua los ntawm kev ntxiv 50 μl ntawm 0.5 M NaCl rau lub lim, ua raws li centrifugation rau 25 feeb.
C18 Micro Spin kab (Harvard Apparatus) tau siv los tshem tawm cov kab txuas txuas txuas nrog tryptic peptides raws li txoj cai tau piav qhia los ntawm Bouchal li al.70 nrog kev hloov kho me me.Luv luv, C18 spin kab tau qhib nrog peb qhov ntxuav ntawm 0.1% formic acid (FA) hauv acetonitrile (AcN) (Merck) thiab ob lub ntxhua ntawm 0.1% FA.Kem tau hydrated nrog 0.1% FA rau 15 feeb.Thawb cov qauv rau hauv cov kab sib tw thiab ntxuav 3 zaug nrog 0.1% FA.Cov desalted peptides tau eluted raws li ib tug stepwise gradient siv 50%, 80% thiab 100% AcN hauv 0.1% FA.Cov qauv no tau qhuav hauv SpeedVac Plus concentrator (Eppendorf) kom txog thaum cov kua seem uas ploj lawm.Cov eluted peptides tau yaj hauv 100 μl ntawm 0.08% trifluoroacetic acid hauv 2.5% AcN thiab cov concentrations tau ntsuas ntawm NanoDrop 2000 (Thermo Scientific).Kwv yees li 1 μg ntawm crosslinked peptide ib tus qauv raug txhaj rau hauv LC-MS / MS system.
Hla-txuas peptides tau sib cais ntawm UltiMate 3000 RSLCnano LC system (Thermo Scientific) txuas nrog Orbitrap Exploris 480 huab hwm coj spectrometer (Thermo Scientific).Hla-linked peptides tau sau rau ntawm 300 µm ID, 5 hli ntev µ-pre-kem C18 ntes kab ntim nrog C18 PepMap100 sorbent thiab 5 µm PepMap sorbent (Thermo Scientific).Load lub twj tso kua mis ntws ntawm 5 µl / min 0.08% trifluoroacetic acid yaj hauv 2.5% AcN.Hla-linked peptides tau muab cais rau ntawm analytical fused silica kem nrog ib txoj kab uas hla ntawm 75 μm thiab ntev ntawm 150 mm, ntim nrog 2 μm PepMap sorbent (Thermo Scientific).Txawb theem A thiab B muaj 0.1% FA hauv dej thiab 0.1% FA hauv acetonitrile, feem.Qhov gradient pib ntawm 2.5% B thiab nce linearly mus rau 40% B dhau 90 feeb, ces mus rau 90% B nyob rau 2 feeb tom ntej.Cov theem ntawm lub xov tooj ntawm tes tau khaws cia ntawm 90% B rau 10 feeb thiab tom qab ntawd txo qis mus rau 2.5% B dhau 2 feeb.Kem tau sib npaug ntawm 2.5% B rau 8 feeb ua ntej lub voj voog tom ntej.Hla-linked peptides eluted los ntawm kab analytical kem tau ionized hauv nanoelectrospray ionization (NSI) qhov chaw thiab txhaj rau hauv Exploris 480 huab hwm coj spectrometer (Thermo Scientific).
Lub Orbitrap Exploris 480 huab hwm coj spectrometer ua haujlwm hauv cov ntaub ntawv zoo sib xws.Kev luam theej duab tag nrho tau ua tiav hauv ntu hom ntawm qhov kev daws teeb meem ntawm 120,000 nrog ntau qhov chaw los ntawm m / z 350 Th rau m / z 2000 Th.Lub hom phiaj AGC normalized tau teeb tsa ntawm 300% nrog lub sijhawm nkag siab tshaj plaws ntawm 50ms.Monoisotopic ncov nrhiav tau raug tsim los rau peptides.Qhov kev txwv tsis pub so yog teem rau muaj tseeb yog tias pom muaj tsawg dhau los.Qhov tsawg kawg nkaus ionic lub zog ntawm tus precursor tau teem rau 5.0e3 thiab precursor nqi hais txog +8 tau suav nrog hauv cov kev sim.
Lub voj voog lub sij hawm ntawm qhov loj scans nyob rau hauv cov ntaub ntawv correlation hom yog teem rau 2.5 vib nas this.Dynamic mass exclusion yog teem rau 20 s tom qab thawj fragmentation ntawm lub precursor ion.Lub qhov rais precursor cais tawm tau teem rau 2 Th.Hom kev sib tsoo lub zog zoo nrog lub zog sib tsoo ruaj khov tau xaiv hauv cov ntaub ntawv nyob ntawm MS / MS scan.Kev sib tsoo zog teem rau 30%.Qhov kev daws teeb meem Orbitrap tau teem rau 15,000 thiab AGC lub hom phiaj rau 100%.Lub sijhawm txhaj tshuaj siab tshaj plaws yog teem rau 60 milliseconds.
Ua ntej taug qab cov protein-protein network hauv cov qauv sib txuas, peb tau ua tiav cov ntaub ntawv nyoos siv MaxQuant pob (version 1.6.12.0) 26,27 txhawm rau txheeb xyuas peptides / proteins hauv cov qauv.Tsis tas li ntawd, kev soj ntsuam proteomic zoo sib xws tau ua rau ntawm cov qauv tsis sib xws ntawm Flo-1 uas tau kho thiab tsis kho nrog IFNα.MS/MS cov ntaub ntawv tau tshawb fawb hauv UniProt tib neeg database (www.uniprot.org) (uploaded Lub Yim Hli 12, 2020, muaj 75,093 nkag) siv lub tshuab tshawb nrhiav hauv Andromeda27.Kev tshawb nrhiav tau ua tsis tau qhia qhov tshwj xeeb ntawm cov enzyme thiab ntau yam kev hloov pauv ntawm deamidation (N, Q) thiab oxidation (M).Precursor huab hwm coj kam rau ua tau teeb tsa ntawm 20 ppm thiab cov khoom ions ntawm 0.02 Da.Qhov pib thiab qhov siab tshaj qhov sib txawv tau teem rau 10 ppm.Qhov siab tshaj plaws ntawm peptide tau teeb tsa ntawm 4600 Da thiab qhov sib npaug sib npaug tau teeb tsa ntawm 7 thiab 25 amino acids (aa).Kev txheeb xyuas ntxiv tau ua tiav siv Perseus program (version 1.6.10.45).Cov ntsiab lus ntawm cov protein raug xam los ntawm normalizing lub spectral siv ntawm cov protein (LFQ siv; unlabeled quantification) 27 thiab cov kev siv qhov tseem ceeb tau hloov mus rau Log2.Lub hierarchical pawg ntawm cov protein uas txheeb xyuas los ntawm lawv qhov kev siv peptide tau tsim los ntawm kev siv lub pob pheatmap (v1.0.12) hauv R (v 4.1.2).Kev txheeb xyuas txoj kev txhawb nqa tau ua tiav siv Reactome pathway database rau IFNα-kho cov proteins uas tau siv ntau tshaj plaub zaug piv rau cov qauv tsis kho.
Kev txheeb xyuas cov lysine (K) lossis serine (S) cov tshuaj sib txuas ntawm cov protein ntau saib xyuas los ntawm LC-MS / MS tau ua los ntawm kev siv lub tshuab spectroscopic identification (SIM-XL) rau cross-linked peptides (SIM-XL)29.Ua ntej, muaj peev xwm sib cuam tshuam ntawm interferon-associated (IFN) DNA puas tiv thaiv kos npe (IRDS) noob tau tshawb xyuas siv IRDS protein dataset tau piav qhia hauv Padariya li al.28.Kev tshuaj xyuas tag nrho cov xwm txheej thiab rov ua dua ntawm tag nrho tib neeg UniProt yog suav nrog, yog li tag nrho tib neeg UniProt database (www.uniprot.org) (downloaded 12 Lub Yim Hli 2020, muaj 75,093 nkag) tawm tsam IFNα-kho rov ua dua.Ib qho ntawm cov ntxaij lim dej rau kev ntseeg siab sib cuam tshuam.Cov kev sib cuam tshuam tseem ceeb no tau nthuav dav thiab sim hauv txhua qhov kev rov ua dua thiab cov xwm txheej.
Hauv SIM-XL, DSS tau siv rau crosslinker (XL) thiab XL qhov hnyav hloov thiab hloov pauv qhov hnyav tau teem rau 138.06 thiab 156.07, raws li.Cov chaw sib txuas sib txuas hauv qab no raug suav hais tias: KK, KS thiab KN-TERM, tsis muaj cov neeg sau xov xwm ions.Ob qho tib si precursor thiab fragment ppm tau teem rau 20 thiab Xrea qhov pib tau teem rau 0.15.Trypsin tau raug suav hais tias yog qhov tshwj xeeb, thiab muaj zog C-trap (HCD) fragmentation txoj kev tau ua tiav.XCorr dynamic DB txo qhov pib thiab qhov tsawg kawg nkaus ntawm peptides rau dynamic DB txo tau teem rau 2.5 thiab 2, feem.Lwm yam tsis muaj xws li: monoisotope probability thiab peak coincidence cutoff, yam tsawg kawg nkaus 4 AA residues ib strand thiab siab tshaj plaws strand nqi, thiab 3 maxima ntawm plam splits.Cov txiaj ntsig tau ua tiav 2D daim duab qhia tau txheeb xyuas hauv (SIM-XL) thiab xQuest28 cov duab kos duab tau siv los tsim 2D daim duab qhia.Protein crosslinks ntawm cov qauv protein muaj nyob rau hauv PyMol (PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC).
Cov qauv qauv tsim tau tsim los siv Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2)11 siv cov qauv ntawm homology qauv thiab kev siv ntawm "Hidden Markov Method".Phyre2 tsim cov qauv qauv raws li kev sib txuas ua ke nrog cov qauv protein paub.Rau H2BFS, HLA-A, HMGA1, LRCH4, thiab MDN1 cov proteins, cov qauv qauv 1kx552, 1kj349, 2eze55, 6hlu62, thiab 6i2665 tau siv.Tsis tas li ntawd, cov qauv ntawm AlphaFold71 MX1, UBP18 thiab ROBO1 kuj tau txiav txim siab.Cov qauv protein tau pom zoo siv BIOVIA Discovery Studio Visualizer pob (Dassault Systèmes, BIOVIA, San Diego, CA, USA) thiab pob Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).