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Inkqubo yokubonisa i-interferon ibangela impendulo enamandla ye-cytokine kwiimpawu ezininzi ze-pathogenic kunye ne-intrinsic pathological ezivela kwindalo, okubangelwa ukufakwa kwee-subsets ze-interferon-inducible proteins.Sasebenzisa i-DSS-mediated cross-link mass spectrometry (CLMS) ukufumanisa i-protein-protein entsha yokusebenzisana kwi-domain ye-interferon-induced proteins.Ukongeza kwiiprotheni ezilindelekileyo ze-interferon-inducible, siye safumanisa i-novel intermolecular and intramolecular cross-linked adducts of canonical interferon-inducible proteins ezifana ne-MX1, USP18, OAS3, kunye ne-STAT1.Sigxininise ekuqinisekisweni kwe-orthogonal yeseti yenoveli ye-interferon-inducible protein networks eyenziwe yi-HLA-A proteins (H2BFS-HLA-A-HMGA1) isebenzisa i-co-immunoprecipitation kunye nophando lwabo olongezelelweyo usebenzisa imodeli ye-molecular dynamics.Ukumodareyitha kotshintsho lweprotheyini eyinkimbinkimbi iveze iindawo ezininzi zokusebenzisana ezibonisa intsebenziswano echongiweyo kwiziphumo zeCLMS.Sisonke, sibonisa uphononongo olulingwayo lwe-CLMS ukuchonga ii-complexes ezintsha zokubonisa ezibangelwa yi-interferon, kwaye sijonge phambili kusetyenziso olubanzi lwe-CLMS ukuchonga amandla amatsha entsebenziswano yeprotheyini kwi-tumor microenvironment.
Ngaphambi kokuba impendulo ye-immune eguquguqukayo iqale, inkqubo yokukhusela ye-host innate iphakamisa impendulo ye-antimicrobial mediated yintsapho eyimfihlo ye-alpha-helical cytokines ebizwa ngokuba yi-interferons (IFNs).Uhlobo lwe-IFN iiklasi ze-IFNα kunye ne-IFNβ zisebenzise iimpendulo zeselula, ezibandakanya i-antiviral, i-proapoptotic, i-proinflammatory, kunye ne-antiproliferative states.Kubantu, i-13 subtypes ye-IFNα iyaziwa, yonke ihlanganiswe kwi-chromosome 91. Okumangalisayo kukuba, kuphela i-IFNα2 ifundelwe ukusetyenziswa kweklinikhi.Kungekudala, ingqalelo ekhethekileyo ihlawulwe kuphando kwezinye ii-subtypes ze-IFNα.Uphononongo olutshanje lubonise ukuba i-IFNα14 yenye ye-isoforms esebenzayo ekuthinteleni i-HBV2 kunye ne-HIV-13,4 ukuphindaphinda xa kuthelekiswa ne-canonical IFNα2 subtype.
Kuye kwasekwa ukuba uhlobo olusebenzayo lwe-interferon receptor complexes (IFNAR1 kunye ne-IFNAR2) lubangela i-cascade yokudluliselwa kwesignali edibeneyo nguJanus kinases TYK2 kunye ne-JAK15,6.Ezi ze-Janus kinases ze-phosphorylate zesignali ze-transducers kunye ne-transcriptal protein activators (STAT1 kunye ne-STAT2) kwii-residues ze-tyrosine ukuqalisa i-SH2 ye-domain-mediated heterodimerization6.Emva koko, i-IRF9 ibophelela i-STAT heterodimers ukuba yenze i-trimeric complex ye-IFN-stimulated factor 3 gene (ISGF3), etshintshela kwi-nucleus kwaye ibangele ushicilelo olungaphezulu kwe-2000 ye-interferon-stimulated genes (ISGs)5,6,7,8.
Ii-ISG zenza umqolo we-immune system, ngakumbi ekuphenduleni uhlaselo lwentsholongwane.Njengomgca wokuqala wokukhusela kwintsholongwane yentsholongwane, iiseli zithumela ngokukhawuleza ukusebenzisana okubanzi kweeprotheni zeselula kunye neentlobo ezininzi zezinto eziphilayo.Ezi proteni zibandakanya ii-receptors zokuqaphela iipatheni, iimolekyuli zokubonisa, izinto ezikhutshelweyo, kunye neeprotheni ezinemisebenzi ethe ngqo ye-antiviral, kunye nabalawuli abangalunganga beempendulo ze-immune9.Uninzi lolwazi malunga nomsebenzi we-ISG luvela kwizikrini ezisebenzayo zisebenzisa izikrini zokubonisa ngokugqithiseleyo10,11 okanye iindlela zokuthulisa imfuza (i-siRNA, i-RNAi kunye ne-CRISPR) i-12,13 apho i-ISG nganye ichazwa okanye ivinjelwe kwaye umsebenzi wabo uvavanywa kwiintsholongwane ezahlukeneyo.Nangona olu phononongo lumisele iipropathi ze-antiviral ze-ISG nganye, iindlela ezisisiseko zeemolekyuli ze-ISG nganye zihlala zingaziwa.Kuyamkelwa ngokubanzi ukuba iiproteni ezininzi zinxibelelana ne-cytokines enye okanye ngaphezulu ukuqinisekisa umsebenzi opheleleyo, ngoko ke ii-ISG zinxibelelana ngokuthe ngqo okanye ukusebenzisana kwazo kulamlwa ziiproteni zeselula.Ngokomzekelo, uphando olutshanje lwe-photocrosslinked proteomics luchonge i-ATPase VCP / p97 njengeqabane elikhulu le-IFITM3 lokusebenzisana, ukuvimbela kwakhe kukhokelela kwiziphene ekuhleleni i-lysosomal, ukuguqulwa, kunye ne-cotransport ye-IFITM3 kunye neentsholongwane ze-viral 14.Ukusebenzisa i-immunoprecipitation, sichonge i-VAPA, iprotheni ehambelana ne-vesicle, njengeqabane lokusebenzisana kunye ne-IFITM1 / 2 / 3 edibanisa i-cholesterol-mediated maturation viral maturation, kwaye oku kwaqinisekiswa ngolunye uphando usebenzisa i-yeast two-hybrid system.Inkxaso yezeNzululwazi 15, 16.
Inkqubo yebhayoloji esisiseko ebandakanyekayo ekuthinteleni usulelo kunye notshintsho olubi kukubonakaliswa kwe-antigen, elamlwa ziimolekyuli ezinkulu ze-histocompatibility complex (MHC).I-Peptides (i-8-12 i-amino acids ubude) ukusuka kwiiprotheni ezinqamlekileyo, ezipheliswe ngaphambi kwexesha okanye eziphosakeleyo zilayishwa kwi-MHC-I heterodimer (ebandakanya i-MHC-I enzima kunye ne-chain chain, ebizwa ngokuba yi-β-2-microglobulin; β2M) 17,18.Iziphumo ezizinzileyo ze-MHC-I ze-trimers zithunyelwa kwi-cell surface, apho zibonakalisa i-peptides ye-intracellular kwiiseli ze-CD8 + T (iiseli ze-cytotoxic T)17.Iiseli ze-T ziyaziqonda kwaye zitshabalalise ezi ntsholongwane kunye neeseli ezithwele i-antigen ethile.Ngenxa yoko, ii-pathogens kunye neeseli ze-tumor zihlala zicinezela inkqubo yokubonisa i-antigen ukunqanda ukujongwa komzimba.Ukongezelela, i-MHC-I iyancipha kwi-40-90% yezicubu zomntu kwaye ihlala idibaniswa ne-prognosis empofu19.
Ufuzo olubandakanyekayo ekuphenduleni kwi-pathogens kufuneka lutshintshe ngokukhawuleza phakathi kwemeko yokuphumla kunye nemeko yokubhala okusebenzayo.Ngako oko, iiprotheni ezininzi zeselula zicatshangelwa ukuba zibandakanyeke ekuphenduleni imfuno ephezulu ye-IFN kwixesha elifutshane, kubandakanywa ukulungiswa kunye nokuguqulwa komgqugquzeli we-chromatin 20,21.Uninzi lwezifundo zijolise ekuchongeni amaqabane eprotheyini ye-ISG ngamnye phambi kwe-IFN.Izifundo ezininzi zeproteomic kunye ne-transcriptomic kwiinkqubo zeeseli zemodeli ziye zacacisa umphumo we-IFN kwi-landscape yeselula.Nangona kunjalo, nangona ukuqonda okukhulayo kwe-dynamics eyenziwa yi-interferon, sisazi kancinci malunga nokubandakanyeka kwee-ISG.Xa kuqwalaselwa ubunzima kunye nexesha elixhomekeke kwi-dynamics ye-interferon signaling, kuvela imibuzo emibini: (i) ngaba kunokwenzeka ukuzinzisa kunye nokubambisa ii-multiprotein complexes ezibandakanyekayo ekuboniseni ukukhawuleza, kwaye (ii) ngaba ezi ntsebenziswano zifakwe kwimephu ye-3D?
Ukujongana nale miba, siphumeze i-disuccinimide suberate-mediated chemical cross-linking (DSS) kunye ne-mass spectrometry (CLMS) ukufunda i-IFNα-induced protein interaction network kunye ne-dynamics yayo.I-DSS yongeza amabhondi adibeneyo phakathi kwee-proximal residues zeeprotheyini kunye / okanye iiprotheyini eziyinkimbinkimbi kwi-vivo.Uhlalutyo olulandelayo lwe-MS lubonisa iisayithi ezithile ezinqamlezayo ezibonisa ukusondelelana kwendawo yemimandla ngaphakathi kweprotheni ethile, ebizwa ngokuba yi-internal linkages, okanye i-subunits kwiiprotheyini eziyinkimbinkimbi, ezibizwa ngokuba yi-interrelationships.Ukusebenzisa le ndlela, sichonge iiprotheyini ezininzi zeeprotheyini ezininzi kunye neenethiwekhi ze-interferon-induced multiprotein interaction.Ngokuphinda sivavanye iseti engaphantsi kolu nxibelelwano lutsha, sibonisa ukuba i-H2BFS (H2B histone-type FS; emva koku ibizwa ngokuba yi-H2B) kunye ne-MDN1 zisebenza njengamahlakani abopha i-HLA-A.
Iiseli ze-Flo-1 yenye yezona modeli zaziwa kakhulu kwi-vitro ye-adenocarcinoma ye-esophageal njengoko zilinganisa iimpawu eziphambili ze-esophageal tumors22,23.Nangona kunjalo, akuzona zonke izicubu ze-immunogenic, kunye nokufumanisa ukuba iiseli ze-Flo-1 ziphendula unyango lwe-interferon, siphatha iiseli ze-Flo-1 kunye ne-10 ng / ml IFNα kwiiyure ze-72.Iiseli ze-Flo-1 zibonise ukuqaliswa kwangaphambili kwe-pSTAT1 kunye ne-IRF1, ukuqala iiyure ze-2 emva kokunyanga kunye nokuqhubeka kweeyure ze-72, kunye nokunciphisa ixesha elixhomekeke kumanqanaba amileyo e-IRF1 (Umfanekiso 1A).Ii-ISGs (MX1, IFITM1, OAS1/2, kunye ne-ISG15) zifunyenwe zinyanzeliswa ngamandla emva kweeyure ze-6, zilinganisa i-classic mid and late phase response to IFNα (Figure 1A).Ngokudibeneyo, ezi nkcukacha zibonisa ukuba le modeli yeselula ingasetyenziselwa ukufunda iimpendulo ze-interferon.
Iimpendulo zeeprotheyini ezihlukeneyo zokubonisa kwiiseli ze-Flo-1 emva konyango lwe-IFNα.(A) Ukubonakaliswa kweprotheyini kwiiseli ze-Flo-1 eziphathwe nge-10 ng / ml IFNα ye-2, 6, 24, 48 kunye neeyure ze-72 zahlaziywa yi-immunoblot usebenzisa i-antibodies ye-ISG ebonisiweyo.(B) I-Coomassie blue stained SDS-PAGE iigels zecell extracts ezipheleleyo emva kokudibanisa kunye ne-DSS kumaxesha abonakalisiweyo kunye nokugxininiswa.(C) Ummeli we-immunoblot uhlolwe kunye ne-p53 (DO-1) i-antibody evela kwiisampuli ezifanayo ukuvavanya iqondo leprotheni yokuwela ukudibanisa.
Ukubamba i-in situ protein interaction landscape, siye sasebenzisa i-DSS, i-agent edibanisa i-cross-link esetyenziswa ngokubanzi ngenxa yokungena kwayo kwi-membrane ephezulu kunye nexesha elifutshane lokuphendula.Ixesha elifutshane lokuphendula linceda ukukhusela ukubunjwa kwee-aggregates ezinkulu zeeprotheni ezinqamlekileyo, ngaloo ndlela kugcinwe ukuzinza kwe-crosslinker.Ukumisela i-concentration ye-DSS efanelekileyo kwaye ugweme ukuwela ukugqithisa, saqala satyhila iiseli kwi-5, 2.5, kunye ne-1 mM DSS ye-5, 10, 5, kunye nemizuzu ye-30, ngokulandelanayo, kwaye sihlalutye i-lysates ngu-Coomassie-stained SDS-PAGE (idatha ayiboniswanga) .I-cell lysates ibonakala idityaniswe kakhulu kwi-concentration ephantsi kunye nakwixesha elifutshane.Ngoko ke, i-DSS yathatyathelwa ku-1, 0.5, kunye ne-0.1 mM ngaphezu kwemizuzu emi-5 (Figure 1B).I-crosslinking efanelekileyo yabonwa nge-0.5 mM DSS kwimizuzu ye-5, kwaye le miqathango yakhethwa kwiiseli eziphathwa nge-IFNα.Ukongeza, i-Figure 1C ibonisa i-Western blot eyenziwa kusetyenziswa i-antibody ye-p53 (DO-1) ukuvavanya iqondo leprotheyini yokuwela ukudibanisa.
Iiseli ze-Flo-1 zaphathwa nge-10 ng / ml IFNα kwiiyure ze-24 ngaphambi kokuba zongeze i-crosslinker.Iiseli ezidibeneyo ezinqamlekileyo zaye zahlanjululwa ngamanyathelo amabini eproteolysis kunye neeprotheni zacutshungulwa yi-FASP (Umfanekiso 2) 24,25.I-peptide ye-tryptic edibeneyo edibeneyo ihlalutywe nge-mass spectrometry (umzobo 2).I-spectra ye-MS / MS ihambelana nokulandelelana kweprotheyini kwaye ibalwa kunye ne-MaxQuant26,27.I-peptide edibeneyo edibeneyo ichongiwe kwi-spectra efunyenweyo kusetyenziswa inkqubo ye-SIM-XL, kwaye iikhompawundi zomntu ngamnye zadityaniswa kwinethiwekhi eyinkimbinkimbi usebenzisa i-xQuest28 kunye ne-SIM-XL29 evulekileyo yemibhobho yesoftware yekhompyutha (Umfanekiso 2).I-SIM-XL ichaza i-protein-protein interactions, iintambo zangaphakathi kunye neentambo zomntu kwiimixube ezilula okanye eziyinkimbinkimbi zeprotheyini kwaye zibonelela ngezikripthi zokujonga ukusebenzisana kwiprotheni.Ukongeza, ibeka ireferensi nganye enqamlezileyo njengenqaku le-ID ngokomgangatho wespectrum weMS/MS29.Iiprotheyini ezininzi ezinokwethenjelwa kakhulu kwiiprotheyini kunye neengqungquthela ziye zachongwa, kwaye isethi entsha yokubambisana iye yaphandwa ngakumbi ngokusebenzisa i-co-immunoprecipitation kunye nokuguqulwa kokuguqulwa kwee-complexes usebenzisa i-model dynamics (MD) imodeli (Umfanekiso 2) 30, 31.
Isishwankathelo seSkimu sendlela ye-CLMS.Iiseli ze-Flo-1 zaphathwa nge-10 ng / ml IFNα kwiiyure ze-24 ezilandelwa yi-protein ye-situ cross-linking usebenzisa i-DSS elandelwa yi-cell lysis kunye ne-trypsinization.Iisampulu eziqhagamshelwe emnqamlezweni zahlalutywa kusetyenziswa i-Orbitrap mass spectrometer kunye nesampulu eyongezelelekileyo yokwahlulwa kwezandulela ze-peptide ngexesha le-LC-MS/MS.Iipeptide ezimbini ezidityanisiweyo zichongiwe kwi-spectra efunyenweyo ngokusebenzisa i-Spectrum Recognition Machine yeprogram ye-Crosslinked Peptides (SIM-XL), kwaye zonke iikhompawundi zadityaniswa kwinethiwekhi eyinkimbinkimbi usebenzisa iipayipi zokubala.Hluza unxibelelwano oluphantsi lokuzithemba olusekwe kumanqaku angeyonyani (FDR) amanqaku.Iindibaniselwano ezininzi zeprotheyini ezithembekileyo-eziphezulu zaqinisekiswa ngakumbi kusetyenziswa i-co-immunoprecipitation, kwaye utshintsho oluhambelanayo kwii-complexes luhlolwe kusetyenziswa imodeli ye-molecular dynamics (MD).
Itotali ye- ~ 30,500 kunye ne- ~ 28,500 peptides zaye zafunyanwa kusetyenziswa i-MaxQuant kwiisampuli ze-IFNα ezingakhuthazwayo kwaye zivuselelwe, ngokulandelanayo (i-Supplementary Table S1, Fig. 3A).Ukusabalalisa ubude be-peptide kwiimeko zombini kubonise umlinganiselo ophezulu weepeptide ezinkulu, ezibonisa ubukho be-peptide edibeneyo (umzobo 3B, C).Ukongezelela, inxalenye enkulu yeepeptide ezinkulu zazikhona kwi-40-55 uluhlu kwiisampuli ze-IFNα-treated (Fig. 3C).Imephu yeprotheyini ngokuchasene ne-log2 intensity ibonise ukuba iiprotheyini ezivuselelweyo ze-interferon zezona zininzi xa kuthelekiswa neesampuli ezingaphendulwanga, kubandakanywa i-MX1, IFIT1/3, OAS2/3, DDX58, kunye ne-HLA-F (Figure 3D).Uhlalutyo lweendlela zeeprotheni eziphindwe kathathu eziphuculweyo ekuphenduleni unyango lwe-IFNα usebenzisa i-database ye-Reactome yendlela ibonise ukuba i-MHC-I-mediated antigen presentation and processing yayiyeyona ndlela ihamba phambili (Figure 3E).Ngokuhambelana neengxelo zangaphambili, iimpendulo ze-antiviral ezixutywe yi-OAS kunye ne-ISG15 kunye ne-IFNα / β kunye ne-cytokine yokubonisa yayiphakathi kweendlela ezisebenzayo.Ukongezelela, i-lysine- kunye ne-serine-specific protein cross-links ichongiwe kwi-spectra ye-MS / MS efunyenwe ekuqaleni usebenzisa i-SIM-XL.Uphononongo lwakutsha nje luchaze ii-ISG ezili-104 ezithatha iintsholongwane ezingama-20 ukusuka kwiiklasi ezili-9 zentsholongwane ngohlalutyo lwemeta lwezifundo zoxinzelelo olugqithisileyo lwe-ISG kwiintlobo ezi-5 zeeseli9.Nangona kunjalo, ukoyisa imida yokubala yokuhlola iiseti zedatha enkulu, siqale ngedatha encinci yokuphonononga unxibelelwano olunokwenzeka phakathi koluhlu lwezakhi zofuzo ze-IRDS ezixelwe nguPadaria et al., uninzi lwazo zii-ISG.
Ukuchongwa kweeprotheyini ezichazwe ngokwahlukileyo ezinqamlekileyo ekuphenduleni i-IFNα (idatha efunyenwe kwi-MaxQuant).(A) Umzobo weVenn omele inani leepeptide eziqhelekileyo kunye nezikhethekileyo ezichongiweyo kwi-IFNα14 ephathwayo kunye neesampuli ze-Flo-1 ezingaphathwanga.Ukusabalalisa ubude be-Peptide obungaphathwanga (B) kunye ne-IFNα ephathwayo (C) iisampulu ezinqamlekileyo.(D) Imephu yobushushu emele i-log2 (ukuqina kwe-LFQ) phakathi kokungaphathwanga kunye ne-IFNα14 ephathwayo iiseli ze-Flo-1.Iphaneli ekhohlo ibonisa iiprotheni ezisebenzayo kakhulu phambi kwe-IFNα.(E) I-Histogram emele i-20 iindlela ezinkulu zokutyebisa emva konyango lwe-IFNα.I-database yendlela ye-Reactome ihlalutye ngaphezu kweenguqu ezine kwiiprotheni eziphendulayo ze-IFNα.
Ukukhuthazwa kwe-ISG ye-Interferon-mediated kubhalwe kakuhle, kodwa kwinqanaba le-molecular aliqondwa kakuhle ukuba ezi proteni zifikelela njani kwinqanaba elibanzi lemisebenzi yezinto eziphilayo.Siphande unxibelelwano lweprotheyini ngokuzithemba okuphezulu phakathi kwee-ISG ezaziwayo.Okuthakazelisayo, sichonge inethiwekhi equka i-MX1, USP18, ROBO1, OAS3, kunye neeprotheni ze-STAT1 ezenza i-complex enkulu ekuphenduleni unyango lwe-IFNα (Umfanekiso 4, iThebhile S2) 32,33,34.Okubaluleke kakhulu, ezi ntsebenziswano zifunyenwe kuzo zonke i-triplicates eziphathwe nge-IFNα kwaye azifumanekanga kwiisampuli ezingaphendulwanga, ezibonisa ukuba zenziwe ngokukodwa ekuphenduleni unyango lwe-IFNα.Kuyaziwa ukuba i-STAT1 ilawula ngokubhaliweyo ukubonakaliswa kwezi ISG, kodwa ukusebenzisana kwayo nee-ISG kwinqanaba leprotheyini akuzange kufundwe.Isakhiwo sekristal se-STAT1 sibonise ukuba isizinda sayo se-helical (CCD) asibandakanyekanga ekusebenzisaneni ne-DNA okanye iiprotomers ngexesha lokubunjwa kwe-dimers35.Ezi zi-α-helices zenza isakhiwo se-helical helix esinika indawo eninzi kakhulu ye-hydrophilic ukwenzela ukusebenzisana ukuba kwenzeke i-35.Kwidatha yethu ye-CLMS, siqaphele ukuba uninzi lwentsebenziswano kunye ne-STAT1 yenzeke kwi-domain ye-SH2 eyandulela i-CCD, i-domain ye-linker, okanye i-C-terminal tail (iintsalela 700-708) (Figure 4A).Uphononongo lwangaphambili luchaze ukuba i-USP18 ibophelela kwi-CCD kunye ne-DNA-binding domain (DBD) ye-STAT2 kwaye igaywe kwi-subunit yohlobo lwe-interferon receptor IFNAR2 ukudibanisa ukuvinjelwa kohlobo lwe-interferon uphawu lwe-24.Idatha yethu ibonise ukuba i-USP18 i-domain catalytic isebenzisana ne-STAT1 DBD (Umfanekiso 4A, D), ebonisa ukuba zombini i-STAT1 kunye ne-STAT2 inokudlala indima ekutsaleni i-USP18 kwi-IFNAR2.
I-protein-protein ye-ISG network ichongiwe kwiiseli ezidityanisiweyo eziphambanayo eziphathwa nge-IFNα.(A) Isicwangciso sokusebenzisana se-2D esibonisa i-protein-protein interactions (eyenziwe kwiprogram ye-SIM-XL), kunye nemigca emele i-intermolecular interactions (i-crosslink cutoff isethi kwi-3.5).Iimpawu zeempawu ezahlukeneyo ziphawulwe ngombala wazo32: isizinda seMX1, Dynamin_N (73-249), Dynamin_M (259-547), kunye neGED (569-660).Imimandla ye-OAS3: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), kunye ne-OAS1_C (903-108).I-Domain ROBO1, Ig_3 (67-151), i-I-set (170-258), i-I-set (262-347), Ig_3 (350-432), Ig_3 (454-529), fn3 (562-646), fn3 (678-758) kunye ne-fn3 (777-864).Amabala e-STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), kunye STAT1_TAZ2bind (715–739).(B) Umbukeli wesetyhula weeprotheyini ezinqamlekileyo (MX1, UBP18, OAS3, ROBO1, kunye ne-STAT1) kunye nokusebenzisana kunye nokusebenzisana okubhalwe nge-blue and red, ngokulandelanayo.Umda we-cross-link wawubekwe kwi-3.5.Izicwangciso zedot zibonisa iindawo zokusebenzisana ze-STAT1 kunye ne-MX1 (C), i-USP18 (D), i-ROBO1 (E), kunye ne-OAS3 (F), kunye neendawo zokusebenzisana ze-K okanye ze-S phakathi kweepeptide ezimbini.Kumzobo, i-cross-link score threshold isetelwe kwi-3.0.(G) Iindawo ezahlukeneyo zokusebenzisana phakathi kwe-STAT1 kunye ne-OAS3 DI domains ezibekwe phezulu kwiiprotheyini zazo ze-PyMol (inkqubo yegraphics ye-molecular ye-PyMOL, i-version 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) kunye ne-OAS3 (pdb id: 4s3n34).inkqubo.
Ii-isoforms ezimbini ze-USP18 zichazwe kubantu, iprotheni egcweleyo ehlala ininzi kwi-nucleus, kunye ne-isoform ngaphandle kwe-N-terminal domain, i-USP18-sf, ehanjiswa ngokulinganayo kwi-cytoplasm kunye ne-nucleus 36.Ukongeza, i-N-terminus yaqikelelwa ukuba ayilungiswanga kwaye ayifuni umsebenzi we-isopeptidase okanye i-ISG1537 yokubopha.Uninzi lwentsebenziswano echongiweyo kwisifundo sethu ibekwe kwi-N-terminus yeprotheni, ebonisa ukuba olu nxibelelwano lubandakanya ubude obupheleleyo be-USP18 (Umfanekiso 4A, D) kwaye ngoko kunokwenzeka ukuba kwenzeke kwi-nucleus.Ngaphezu koko, idatha yethu ikwabonisa ukuba i-N-terminus ikhethekileyo kwi-protein-to-protein interactions.Indawo yokubopha i-IFNAR2 ifumaneka phakathi kwee-residues ze-312-368, kwaye ngokuphawulekayo, akukho nanye yeeprotheyini kwi-complex edibanisa kulo mmandla (Umfanekiso 4A) 37,38.Ezi datha zithathwe kunye zibonisa ukuba i-IFNAR2 i-domain yokubopha isetyenziswa kuphela yiprotheni ye-receptor.Ukongezelela, kuphela i-OAS3 kunye ne-ROBO1 efunyenweyo idityaniswe nemimandla ephezulu ye-N-terminus kunye ne-IFNAR2 indawo yokubopha (Umfanekiso 4A).
I-ROBO1 iyingxenye ye-immunoglobulin (Ig) ye-superfamily ye-transmembrane i-molecules yokubonisa kwaye iqulethe i-domain ye-Ig emihlanu kunye ne-fibronectin (Fn) ezintathu kummandla we-extracellular.Le mimandla ye-extracellular ilandelwa ngummandla we-membrane-proximal kunye ne-helix eyodwa ye-transmembrane 39. Ummandla we-intracellular ongakhiwanga ufumaneka kwi-C-terminus kwaye uqulethe i-motifs yokulandelelana egciniweyo eyenza i-protein ye-protein binding39.Ummandla osuka kwi-amino acids ~ 1100 ukuya kwi-1600 uphazamiseka kakhulu.Sifumene ukuba i-MX1 isebenzisana ne-ROBO1 nge-Ig, i-Fn, kunye ne-intracellular domains, ngelixa ininzi intsebenziswano kunye ne-STAT1 yenzeke phakathi kweCCD yayo, i-linker domain, kunye ne-C-terminus ye-ROBO1 (Umfanekiso 4A, E).Ngakolunye uhlangothi, ukusebenzisana kunye ne-DI, DIII, kunye ne-OAS3 imimandla yokudibanisa yasasazwa kwiprotheni ye-ROBO1 (umzobo 4A).
Intsapho yeprotheyini ye-oligoadenylate synthase (OAS) yamkela kwaye ibophe i-intracellular double-stranded RNA (dsRNA), ingena kwiinguqu ze-conformational, kwaye idibanise i-oligoadenylates ye-2',5' (2-5 As) 40.Kwafunyaniswa ukuba phakathi kwe-OASs ezintathu, i-OAS3 ibonisa ubudlelwane obuphezulu be-dsRNA kwaye idibanisa inani elincinci le-2-5 As, elinokuthi lisebenze i-RNase L kwaye ngaloo ndlela inqande ukuphindaphinda kwe-viral 41.Usapho lwe-OAS luquka i-polymerase beta (pol-β) efana ne-nucleotide transferase domains.Uphando lwangaphambili lubonise ukuba umsebenzi we-catalytic we-C-terminal domain (DIII) ixhomekeke kwi-domain ye-dsRNA-binding (DI), efunekayo ukuze kusebenze i-OAS342.Siye saqaphela ukuba imimandla ye-DI kunye ne-DII ye-OAS3 isebenzisana neCCD kunye nommandla omncinci we-junction phakathi kwe-SH2 kunye ne-STAT1 TAD (Umfanekiso 4A,F).Ukugqithisa iindawo ezinqamlekileyo ezinqamlekileyo kwisakhiwo seprotheni sibonakalise ukusebenzisana phakathi kwe-β-sheet kunye ne-DBD STAT1 loop kunye ne-pocket evulekileyo okanye i-cavity eyenziwe ngama-residu 60-75 kwi-DI domain ye-OAS3 (Fig. 4G).Ukuqhelaniswa kweeprotheni kwi-complex kwakhona kubonise ukuba akukho nanye intsebenziswano kunye ne-OAS3 iphazamise i-DNA-binding ikhono le-DI domain yayo (Fig. S1A).Ukongezelela, i-N-terminal domain ye-GTPase MX1 isebenzisana ngokubanzi kunye ne-DI kunye ne-DIII imimandla ye-OAS3 (Umfanekiso 4A).Siphinde sabona ukusebenzisana phakathi kwe-OAS1 kunye ne-MX1 kuzo zonke ezintathu eziphindaphindiweyo eziphathwe nge-IFNα, apho i-domain ye-OAS1 enye (nayo i-catalytically isebenzayo) idibene nazo zonke ezintathu i-MX1 domains (Figure S2A, B).
Iiprotheyini ze-MX ziyingxenye yentsapho enkulu ye-dynein-efana ne-GTPases equlethe i-N-terminal GTPase domain edibanisa kunye ne-hydrolyzes GTP, i-domain ephakathi edibanisa ukuzihlanganisa, kunye ne-C-terminal leucine zipper esebenza njenge-GTPase (LZ). ).domain effector domain25,43.I-MX1 ibophelela kwii-subunits zeepolymerasi zentsholongwane ukuthintela ushicilelo lwejene43 yentsholongwane.I-yeast exelwe ngaphambili isikrini se-hybrid-hybrid screen sibonise ukuba i-PIAS1 ehambelana ne-MX1 inqanda i-STAT1-mediated gene activation ngokuthintela umsebenzi wokubopha i-DNA kwaye inomsebenzi we-SUMO E344,45 ligase.Apha, sibonisa ukuba i-MX1 ibophelela kwi-STAT1 (Umfanekiso we-4C, D), nangona kunjalo ukuba le ntsebenziswano ichaphazela njani i-STAT1-mediated gene activation ekuphenduleni i-IFNα idinga ukufundiswa okuqhubekayo.Ukongezelela, siye safumanisa ukuba i-MX1 isebenzisana ne-IFIT3 kunye ne-DDX60 kuzo zonke ezintathu eziphindaphindiweyo ze-IFNα-treated (Fig. S2C).
I-DDX60 yi-IFN-induced cytoplasmic helicase eye yaxelwa ngaphambili ukuba idlale indima kwi-RIG-I-i-degradation ezimeleyo ye-viral RNA46.Isebenzisana ne-RIG-I kwaye isebenze ukubonakaliswa kwayo ngendlela ye-ligand-specific 46. I-DDX60 iqukethe i-DEXD / H-Box ye-helicase domain kunye ne-C-terminal helicase domain edibanisa i-viral RNA kunye ne-DNA47.Uninzi lwentsebenziswano yayo kunye ne-MX1 kunye ne-IFIT3 yenzeke phakathi kwemimandla ende ye-N- kunye ne-C-terminal ngaphandle kwe-canonical domains okanye i-motifs (Fig. S2E, F).Nangona kunjalo, i-MX1 iphinda idibaniswe ne-DEXD / H-Box ye-helicase domain (Fig. S2E).Iiprotheyini zentsapho ye-IFIT zineekopi ze-tandem ze-helix-turn-helix motif ebizwa ngokuba yi-tetrapeptide repeat (TPR).I-IFIT3 ifunyenwe ibe yimodyuli efanelekileyo ye-RIG-I yokubonisa kwaye ngoko ke icandelo le-MAVS complex.Kuthatyathwe kunye, idatha yethu ibonisa ukuba i-IFIT3 kunye ne-DDX60 isebenzisana ngokuyinhloko kummandla phakathi kwe-TPR 3-6 ye-IFIT3 kwaye inokudlala indima kwi-RIG-I / MAVS yokubonisa (Fig. S2F).
Ngenxa yokuba ukuhlolwa kwe-proteome yonke kuyinkimbinkimbi, siye sahlola yonke i-database ye-UniProt yabantu malunga nobukho bokuphindwa kwe-IFNα-treatment.Kule replica, sifumene uthungelwano oluninzi oluthembeke kakhulu lwe-HLA-A.Uhlalutyo lweendlela zeprotheni ezichongiweyo yi-MS / MS spectra ibonise ukuba i-MHC-I-based based-antigen processing kunye nomboniso yindlela ephambili eyenziwa yi-interferon (Fig. 3D).Ngoko ke, sigxininise ekufundeni i-protein ye-protein ye-MHC-I iamolekyu kunye neqondo eliphezulu lokuzithemba kuzo zonke iisampuli ezidibeneyo.I-HLA iqukethe i-α1, i-α2 kunye ne-α3 imimandla kunye namaketanga okukhanya, kunye ne-microglobulin β2 (β2m) i-protein ye-chaperone eqhubekayo.Emva kokuba ihlanganiswe kwi-endoplasmic reticulum, i-HLA ayizinzile ngokungabikho kwe-peptide ligands50.I-groove ebopha i-peptide yenziwa yi-polymorphic ephezulu kunye ne-alphare ye-α1 kunye ne-α2 engacwangciswanga kwifom ye-non-peptide kunye ne-polymorphic α351 domain encinci.Ebukhoneni be-IFNα, sifumene ii-complexes ezimbini ze-HLA-A: enye isebenzisana ne-HMGA1 kunye ne-H2B (Umfanekiso 5, i-Table S3) kwaye enye isebenzisana ne-MDN1, LRCH4 kunye ne-H2B (Umfanekiso 6).
I-IFNα yenza inethiwekhi ye-HLA-A yokusebenzisana kunye ne-H2B (H2BFS) kunye ne-HMGA1.(A) Isicwangciso se-2D (esenziwa kwi-software ye-SIM-XL) ebonisa iintlobo ezahlukeneyo zokusebenzisana kwi-H2B-HLA-A-HMGA1 eyinkimbinkimbi: i-interlink (blue), i-interlink (ebomvu) kunye nekhonkco enye (emnyama)..Imimandla yezazisi ezahlukeneyo ikhowudi yombala32: H2B (histone; 2–102) kunye ne-MHC-I (MHC_1; 25–203, iqela C1; 210–290 kunye ne-MHC_I_C; 337–364).Umda we-cross-link wawubekwe kwi-3.5.Iimpawu ze-dot zibonisa iindawo zokusebenzisana ze-HLA-A kunye ne-H2B (B) kunye ne-HMGA1 (C), kunye neendawo zokusebenzisana ze-K okanye ze-S phakathi kweepeptide ezimbini.Kumzobo, i-cross-link score threshold isetelwe kwi-3.0.(D) Ubudlelwane phakathi kweeprotheni eziboniswe kwizakhiwo ze-H2B, i-HLA-A, kunye neeprotheni ze-HMGA1 kwiprogram ye-PyMOL.Ezi zakhiwo zenziwe imodeli kusetyenziswa i-Phyre2 iseva (http://www.sbg.bio.ic.ac.uk/phyre2) kunye nezakhiwo zetemplate ze-H2B, i-HLA-A kunye ne-HMGA1 iiprotheni zaziyi-1kx552, i-1kj349 kunye ne-2eze55, ngokulandelanayo.
I-IFNα yenza inethiwekhi ye-HLA-A yokusebenzisana kunye ne-H2B (H2BFS), i-MDN1 kunye ne-LRCH4.(A) I-Intramolecular (obomvu) kunye ne-intermolecular (blue) i-crosslinks eboniswe kwimephu edibeneyo ye-2D (eyenziwe kwi-software ye-SIM-XL) kunye ne-MDN1 emelwe njengesangqa.Umda we-cross-link wawubekwe kwi-3.5.Iimpawu zezazisi ezahlukeneyo zinekhowudi yombala32: H2B (histone; 2-102), MHC-I (MHC_1; 25-203, iqela C1; 210-290 kunye ne-MHC_I_C; 337-364) kunye ne-LRCH4 (LRR_8 (68-126), LRR_8 (137-194) kunye ne-CH (535-641)).(B) Ubudlelwane phakathi kweeprotheni eziboniswe kwizakhiwo ze-H2B, i-HLA-A, i-LRCH4, kunye neeprotheni ze-MDN1 kwiprogram ye-PyMOL.Ezi zakhiwo zenziwa imodeli kusetyenziswa i-Phyre2 iseva (http://www.sbg.bio.ic.ac.uk/phyre2) kunye nezakhiwo zetemplate 1kx552, 1kj349, 6hlu62 kunye ne-6i2665 ye-H2B, i-HLA-A, i-LRCH4 kunye neeprotheni ze-MDN1, ngokulandelelanayo.Uyilo lwamachaphaza olubonisa iisayithi ze-K okanye ze-S zokunxibelelana ze-HLA-A ene-H2B (C), i-LRCH4 (D), kunye ne-MDN1 (E).Kwizicwangciso, i-cross-link score threshold yamiselwa kwi-3.0.
Ukongeza ekugcineni ingqibelelo ye-genome, i-histone H2B nayo ibandakanyeka kulawulo loshicilelo.Iprotheni ye-H2B iqukethe i-histone domain ephakathi (HFD) eyenziwe ngama-α-helices amathathu ahlukaniswe ngama-loops kunye nomsila we-C-terminal 41,52.Uninzi lwentsebenziswano kunye ne-H2B lwenzeka kwi-α1 helix, ebonelela nge-trimerization kunye ne-HFD heterodimer (Umfanekiso 5A, B).Nangona ii-lysins zibandakanyeka kwi-DNA ebophayo, ezinye iilysin zikwazezinye iindawo ze-acetylation okanye ze-methylation.Ngokomzekelo, ii-residues ze-K43, i-K46, kunye ne-K57 ezivela kwi-H2B azibandakanyekanga kwi-DNA ebophelelayo ngokuthe ngqo, kodwa ziithagethi ze-post-transcriptional modifications ezahlukeneyo53.Ngokufanayo, ii-residues ze-K44, i-K47, kunye ne-K57 kwi-H2B zinokudlala enye indima phambi kwe-IFNα, kubandakanywa ukusebenzisana nezinye iiprotheni (umzobo 5A, B).Ukongezelela, i-extrachromosomal histone H2B ivuselela ukuphendula komzimba kwiindidi ezahlukeneyo zeeseli, zisebenza njengenzwa ye-cytosolic ukuze ibone amaqhekeza e-DNA (dsDNA) aphindwe kabini athathwe kwii-agent ezosulelayo okanye iiseli ezonakalisiweyo54.Xa kukho iintsholongwane ze-DNA, ukuchithwa kwe-H2B kuvimbele ukuveliswa kwe-IFN-β kunye ne-STAT154 phosphorylation.I-H2B yaziwa nangokungena nokuphuma kwi-nucleus ngokukhawuleza kunezinye i-core histones54.Intsebenziswano ye-H2B kunye ne-MDN1 kunye ne-LRCH4 nayo yabonwa kwiisampuli ezikhethiweyo ezingaphathwanga.Sifumene ukuba i-HLA-A isebenzisana ne-H2B kuzo zontathu iisampuli eziphathwe nge-IFNα kunye nakwisampulu enye engaphendulwanga.Ezi datha zibonisa indima ye-H2B komnye umsebenzi we-physiological ozimeleyo kummiselo wokubhaliweyo.
I-HMGA1 (iqela eliphezulu lokuhamba nge-AT-Hook 1), i-nucleoprotein encinci ecebileyo kwi-amino acids ekhuthaza izifo, ichongiwe ngokubambisana ne-HLA-A.Inomsila we-C-terminal ene-acidic kunye nee-DBD ezintathu ezihlukeneyo ezibizwa ngokuba yi-AT hooks kuba zibophelela kwi-groove encinci ye-AT-rich region kwi-dsDNA55,56.Oku kubophezela kubangela ukuba i-DNA igobe okanye iqondise, ivumela izinto ezibhaliweyo ze-canonical ukufikelela ukulandelelana kwayo kwemvumelwano.Umsila we-C-terminal ukholelwa ukuba ubandakanyeka kwi-protein-protein interactions kunye nokuqeshwa kwezinto ezibhaliweyo, ekubeni i-C-terminal deletion mutants ayikwazi ukuqalisa i-transcription57.Ngaphezu koko, lo mmandla uqulethe iindawo ezininzi ze-phosphorylation ezigciniweyo ezaziwa ngokuba ngama-kinases 58.Siqaphele ukusebenzisana kwe-HLA-A kunye ne-H2B kunye ne-HMGA1 ngaphandle kwe-C-terminal domain, ebonisa ukuba i-domain ye-C-terminal isetyenziselwa ikakhulu ukubopha into yokubhala (umzobo 5A, C).Iiprotheyini ze-HMGA zikhuphisana ne-histone H1 ngokubophelela kwi-adaptha ye-DNA, ngaloo ndlela yandisa ukufikeleleka57.Ngokufanayo, kubonakala ngathi i-HMGA inxibelelana ne-histone H2B kunye ne-DNA yekhonkco kukhuphiswano ne-histone H1.I-HMGB1 ibangela ukubonakaliswa kwe-HLA-A, -B, kunye -C kwiiseli ze-dendritic, ezikhokelela ekusebenzeni kwazo59, kodwa ukusebenzisana phakathi kwe-HMG kunye ne-HLA akuzange kuchazwe ngaphambili.Sifumene ukuba i-HMGA1 isebenzisana ne-α1 kunye ne-α3 imimandla ye-HLA-A, kunye nobuninzi bentsebenziswano ngaphandle kwe-3 DBD yayo (Umfanekiso 5A, C).Ezandleni zethu, i-HLA-A ifunyenwe ikwi-nucleus (idatha engaboniswa), kwaye inikwe ukuba i-H2B kunye ne-HMGA1 zikwakhona kwi-nucleus, le ntsebenziswano inokwenzeka kwi-nucleus.I-adducts ethile elinganiswa phakathi kwe-H2B, i-HLA-A, kunye ne-HMGA1 iboniswe kuMfanekiso we-5D.
Uninzi lwentsebenziswano ye-HLA-A kunye nezinye iiprotheni zenzeke ngaphakathi kwe-α1 kunye ne-α2 domains kunye ne-disordered C-terminal domain (Umfanekiso 6).Komnye wale mizekelo, sifumene ukuba i-HLA-A isebenzisana nomsila we-N-terminal ophazamisekileyo we-LRCH4 (Umfanekiso 6A, D).I-LRCH4 ilawula ukusebenza kwe-TLR4 kunye ne-LPS cytokine induction, ngaloo ndlela imodareyitha impendulo ye-immune ye-innate60,61.I-protein ye-membrane ene-9 leucine-rich repeats (LRRs) kunye ne-callodulin (CH) i-homology motif kwi-ectodomain yayo, ilandelwa yi-domain transmembrane (TMD) 60, i-62.I-CH domains ixelwe ukuba idibanise i-protein-protein interactive 60.Ukolula malunga ne-300 ye-amino acids phakathi kwe-LRR kunye ne-CH domains iyafikeleleka kodwa iphazamisekile.Ngokusekelwe kumsebenzi wemimandla ephazamisekileyo njengabalamli beeprotheni-protein networks kunye ne-vesicular transport 63, sifumene ukuba i-protein intsebenziswano eninzi iyenzeka kwimimandla ephazamisekileyo.Ukusebenzisana kunye ne-MDN1 kwasasazwa kulo lonke ubude beprotheni, kubandakanywa i-LRR1, i-LRR6, i-CH domains, kunye nemimandla engahleliwe, ngelixa i-H2B ibopheleleka kakhulu kwi-domain ye-CH (Umfanekiso 6A, B).Ngokucacileyo, akukho nanye intsebenziswano ebandakanya i-TMJ, iphakamisa ukucaciswa kwendlela ye-CLMS (Umfanekiso 6A, B).
I-MDN1 nayo ichongiwe njengenxalenye ye-HLA-A protein network (Figure 6A).Kungokwentsapho ye-AAA yeeprotheni (ATPases ezinxulumene nemisebenzi eyahlukeneyo).Le yi-domain ye-N-terminal AAA efanayo eququzelela kwindandatho ye-hexameric kwaye isuse i-asembly factor kwi-60S 64 i-ribosomal subunit.ibonakala ifana ne-dynein64,65,66.Ukongezelela, ummandla ocebileyo we-Asp / Glu ulandelwa yi-domain ye-MIDAS (indawo exhomekeke kwi-ion yensimbi).Ngenxa yobukhulu obukhulu be-MDN1 (malunga ne-5600 amino acids) kunye ne-homology yayo encinci kunye neeprotheni ezifundwe kakuhle, incinci iyaziwa malunga nesakhiwo kunye nomsebenzi wayo ebantwini.Sichonge i-HLA-A, i-H2B, kunye ne-LRCH4 njengamahlakani abophezelayo e-MDN1 kwaye satyhila ukuqhelaniswa kwazo njengeeprotheyini eziyinkimbinkimbi kwi-PyMol (Umfanekiso 6A, B).Ezi protheni zintathu zisebenzisana ne-AAA domain, i-dynein-like linker domain, kwaye mhlawumbi isizinda se-MIDAS MDN1.Kwingxelo yangaphambili, ukucocwa kobudlelwane beeprotheyini ze-bait zichonge i-MDN1 njengeprotheni ehambelana ne-histone H2B67.Ukongezelela, uphando olutshanje luye lwaxela ukusebenzisana phakathi kwe-MDN kunye ne-HLA-B kwiiseli ze-HCT116 zisebenzisa i-affinity-purified mass spectrometry, exhasa iziphumo zethu68.Ukuchongwa kolu luhlu kwiisampuli eziphathwe nge-IFNα zibonisa indima ye-MDN1 ekuboniseni i-interferon.
Ngenxa yokuba i-HLA yemfuza i-polymorphic kakhulu, sikhuphe ulandelelwano lufundeka imephu ye-HLA-A, -B, kunye -C ukusuka kwi-RNA yokulandelelanisa idatha yeeseli ze-Flo-1 (idatha ayiboniswanga).Ulandelelwano lwe-Peptide oluhambelana nokufunda ngokulandelelana lubonakalise ukungafani okuphawulekayo phakathi kwe-HLA-A, -B, kunye -C kwimimandla apho i-peptides edibeneyo edibeneyo ibekwe kwi-HLA-A (Figure S3).Ukongezelela, asizange sibone i-protein-to-protein cross-linking ye-HLA-B / C i-molecule ezine-H2B/HMGA1/MDN1/LRCH4 iiprotheyini.Oku kuphakamisa ukuba i-protein ye-protein efunyenwe phakathi kwe-HLA-A, MDN1, LRCH1 kunye ne-HMGA1 yi-HLA-A ethile.Ukongezelela, uhlalutyo lweproteomic lweesampuli ezingabonakaliyo (Itheyibhile S4) ibonise ukuba i-HLA-A inomgangatho ophezulu wokulandelelana xa kuthelekiswa ne-HLA-B okanye i-HLA-C.I-peptides echongiweyo ye-HLA-A yayiphezulu kwi-intensitet kwi-IFNα-ephathwayo kunye neesampuli ezingaphendulwanga.
Ukuqinisekisa ukuba intsebenziswano echongiweyo apha ibingabangelwa kukudityaniswa okuthe ngqo kweeproteni ezimbini kwindawo ekufutshane yendawo, siye saqinisekisa ngakumbi izinto ezimbini ezintsha ezisebenzisanayo ze-HLA-A ngokwenza uvavanyo lwe-co-immunoprecipitation.Ukusebenzisana kwe-HLA-A kunye ne-MDN1 engapheliyo kunye ne-H2B zifunyenwe kuzo zombini iiseli ze-Flo-1 eziphathwayo kunye ne-IFNα (Umfanekiso 7, uMzobo S4).Siye saqinisekisa ukuba i-HLA-A ibanjwe yi-H2B kwi-immunoprecipitates kwaye ukuba lo mbutho wawubangelwa unyango lwe-IFNα ekubeni i-HLA-A yayingekho kwiisampuli ze-immunoprecipitate kwiiseli ezingaphathwanga (Umfanekiso 7A).Nangona kunjalo, idatha yethu ibonisa ukuba i-IFNα ilawula ngokwahlukileyo i-HLA-A ebophelelayo kwi-H2B kunye ne-MDN1.I-IFNα ibangela ubudlelwane phakathi kwe-H2B kunye ne-HLA-A, kodwa iyanciphisa ukudibanisa kwayo ne-MDN1.Sifumene ukuba i-MDN1 idibaniswe ne-HLA-A kulawulo, kwaye ukongezwa kwe-IFNα kunciphisa le ntsebenziswano ezimeleyo kwi-MDN1 induction yi-IFNα (Umfanekiso 7B, C).Ukongezelela, i-HLA-A immunoprecipitation ithathe i-H2B kwiiseli ze-A549 (umfanekiso we-S4), ebonisa ukuba le ntsebenziswano izimeleyo kuhlobo lweseli.Zithathiwe kunye, ezi ziphumo zixhasa ukusebenzisana kwe-interferon-mediated ye-HLA-A kunye ne-H2B kunye ne-MDN1.
I-HLA-A icoca i-H2B kunye ne-MDN1.Ummeli omele i-H2B (A) kunye ne-MDN1 (B) i-immunoblots i-immunoprecipitated ukusuka kwi-IFNα-treatated Flo-1 cells kwaye ihlolisise i-antibodies ebonisiweyo.Impuku kunye nomvundla IgG zasetyenziswa njengolawulo olubi.(C) Iimali ezihambelanayo (igalelo) lee-antigens ezahlukeneyo zibonakaliswa ngama-immunoblots ahlolwe ngokuchasene nama-antibodies abonakalisiweyo, i-β-actin yayisetyenziswe njengolawulo lokulayisha.
Iimpawu zesakhiwo sesinye se-interferon-induced i-interferon ethembekileyo kakhulu yothungelwano lwe-cross-link, i-H2B-HLA-A-HMGA1, yaphandwa.Sisebenzise imodeli ye-molecular dynamics njengenye indlela yokuqonda i-conformational dynamics yeeprotheni ezibandakanyekayo kule ngxaki (Umfanekiso 8).Iimpembelelo ezivela kwiinkcukacha ze-CLMS zibonisa ukuba kunokwenzeka ukuhambelana okuhlukeneyo kweeprotheni ze-H2B, i-HLA-A, kunye ne-HMGA1.Ngoko ke, ezi zixhobo zilandelayo ezinokuthi zifakwe kwi-solvent medium: H2B-HLA-A, HMGA1-HLA-A, kunye ne-H2B-HLA-A-HMGA1.Iprotheyini yokuqala ye-protein-protein docking screen usebenzisa i-MOE (i-Molecular Operating Environment; i-Chemical Computing Group Inc., i-Montreal, i-Quebec, i-Canada) i-package iphakamise ukuhambelana okunokwenzeka okwahlukileyo phakathi kwezi proteni (Fig. 8A).Ukubonwa kweprotein ye-docking complex kuveze intsebenziswano emininzi kunye nokuhambelana okunokwenzeka (Umfanekiso 5A, 8).Ngaloo ndlela, ukuhambelana okunokwenzeka kuboniswe kuMfanekiso 8A (obhalwe i-cross-links) kwaye yaphinda yavavanywa ngokusebenzisa umbhobho we-MD wokulinganisa.Ukongezelela, ukubopha i-H2B okanye i-HMGA1 kwi-HLA-A igxininisa ubudlelwane obuphezulu be-H2B ye-HLA-A (Umfanekiso 8A).
I-Conformational dynamics yothungelwano olunokwenzeka phakathi kwe-H2B (H2BFS)-HLA-A, i-HMGA1-HLA-A, kunye ne-H2B-HLA-A-HMGA1.(A) Iphaneli yasekhohlo yimephu ye-2D (eyenziwe kwi-software ye-SIM-XL) ye-intramolecular (ebomvu) kunye ne-intermolecular (blue) crosslinks (i-crosslink cutoff isete kwi-3.5).Ukongezelela, ii-residus ezinqamlekileyo ezichongiweyo zibhalwe kwizakhiwo ze-H2B, i-HLA-A, kunye ne-HMGA1 iiprotheni.Ukuhambelana okuhambelanayo kwezi proteni kwacatshulwa kusetyenziswa umbhobho we-docking ophunyezwe kwiphakheji ye-MOE.Iphaneli esezantsi esekhohlo ibonisa ukuhambelana okunokwenzeka kwe-H2B-HLA-A kunye ne-HMGA1-HLA-A yekhompleksi ezineprotein-protein ezibophelelayo ezahlukeneyo (GBVI/WSA dG; kcal/mol).(B) Ukutenxa okusemgangathweni (RMSD) yeendawo ze-athomu (ngaphandle kwee-athomu ze-hydrogen) kwisakhiwo ngasinye seprotheni.(C) Iiprotheyini ze-Intermolecular-protein hydrogen bond bond ukusuka kwii-complex ezahlukeneyo ezifanisiweyo eziqwalasela ukusebenzisana okuthe ngqo kwexesha ≥ 10 ns.Umgama we-h-bond donor-acceptor cutoff ubekwe kwi-3.5 Å, kunye ne-donor-H-acceptor cutoff angle ibekwe ku-≥ 160 ° -180 °.(D) Iintsalela ezibhalwe phantsi zenza i-HLA-A protein-protein intsebenziswano kunye namaqabane abo ahlukeneyo, athatha i-≥ 20 ns, ekhutshwe kwi-dummy HLA-A-H2B kunye ne-HLA-A-HMGA1 complexes.Ulwakhiwo lweeprotheyini lumele ulwakhiwo oluyi-avareji lwe-100 ns MDS.(E) Ukusebenzisana phakathi kwe-HLA-A-H2B kunye ne-HLA-A-HMGA1 i-complexes xa kuthelekiswa nokusebenzisana okulandelwa yi-H2B-HLA yokulinganisa ngaphezu kwe-100 ns ngokusekelwe kwisayithi ye-K okanye ye-S yokunxibelelana phakathi kweepeptide ezimbini.Iicomplexes /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Ixabiso lomda wokuvavanya i-cross-links libekwe kwi-3.0, kwaye intsebenziswano ethile evela kwi-MDS ithatha ≥ i-10 ns ithathelwe ingqalelo.Izakhiwo zeprotheyini zabonwa kusetyenziswa i-BIOVIA Discovery Studio (i-Dassault Systèmes, i-BIOVIA Corp., i-San Diego, i-CA, i-USA) kunye ne-Molecular Operating Environment (MOE; i-Chemical Computing Group Inc., i-Montreal, Quebec, Canada) iiphakheji.
Ukuzinza kwee-molecule ze-HLA-A ngokuhamba kwexesha (ukuphambuka okuqhelekileyo; i-RMSD okanye ukuphambuka okuqhelekileyo; i-RMSF) ibonise ukuba ubukho be-H2B okanye i-HMGA1 iiprotheni kwii-complex ezizinzile i-HLA-A (Umfanekiso 8B, uMfanekiso S5).Iprotheyini ye-HMGA1 ibophelela ngokuqinileyo kwindawo ye-B2M ye-HLA-A, ibangela ukuzinza kwe-HLA-A amino acids kwi-HLA-A-HMGA1 okanye i-H2B-HLA-A-HMGA1 eyinkimbinkimbi (Umfanekiso 8B, uMfanekiso S5).ngokukodwa, i-HLA i-residus ~ 60-90 kunye ne- ~ 180-210 ifunyenwe i-flexible encinci phambi kwe-H2B (FIG. 8B).I-H2B kunye ne-HMGA1 ibonise ukubopha okungcono kwi-HLA-A kwi-H2B-HLA-A-HMGA1 eyinkimbinkimbi xa kuthelekiswa ne-HLA-A ebophelela kwi-H2B okanye i-HMGA1 yodwa (Umfanekiso 8C, D; Itheyibhile S5).Iintsalela ezibandakanyekayo kwi-hydrogen bonding (i-MD imodeli yokuhlala ephezulu ≥ 10 ns) ihambelana neendawo zokusebenzisana ze-CLMS (ii-K okanye i-S residues) kwi-complex, ebonisa ukuba ukusebenzisana okuchongiweyo yi-CLMS kunokwethenjelwa kakhulu.Ukuthembeka (Umfanekiso 8E).Kwi-CLMS kunye ne-MD modeling, i-HLA-A intsalela phakathi kwe-190-210 kunye ne-200-220 ye-amino acids yafunyanwa ukuba ibophe i-H2B kunye ne-HMGA1, ngokulandelanayo (FIG. 8E).
Iiprotheyini-iiprotheyini ezisebenzisanayo zenza iinethiwekhi eziguquguqukayo zesakhiwo ezidibanisa unxibelelwano lwe-intracellular ekuphenduleni kwi-stimuli ethile.Ngenxa yokuba iindlela ezininzi zeproteomics zibona utshintsho kwinqanaba elipheleleyo leprotheyini, i-protein-protein interaction dynamics idinga izixhobo ezongezelelweyo zokubamba i-interfaces ezibophelelayo, kwaye i-CLMS sesinye isixhobo esinjalo.Inkqubo yokubonisa i-interferon yinethiwekhi ye-cytokine evumela iiseli ukuba ziphendule uluhlu lweempawu ze-pathogenic zendalo kunye ne-intrinsic pathological signals, ekugqibeleni ekufakweni kwee-subsets ze-interferon-inducible proteins.Sasebenzisa i-CLMS ukufumanisa ukuba i-novel protein-protein interactions ingachongwa phakathi kwephaneli yeeprotheyini ze-interferon-induced.Uhlalutyo lwe-protein ye-Global cross-linking analysis kwi-interferon-responsive Flo-1 imodeli yeseli yayisetyenziselwa ukubamba iiprotheyini eziyinkimbinkimbi.Ukutsalwa kwee-peptide ze-tryptic kwiiseli ezingadityaniswanga kunye neziphambanayo zivumela ukubalwa kwe-peptide, ukutyetyiswa kwendlela, kunye nokusabalalisa ubude be-peptide kunye nobunzulu obuchaziweyo be-LFQ.Iiprotheni zeCanonical interferon-inducible zichongiwe njengolawulo lwangaphakathi olulungileyo, ngelixa i-intermolecular kunye ne-intramolecular cross-linked cross-linked adducts ye-canonical interferon-inducible proteins ezifana ne-MX1, UP18, OAS3 kunye ne-STAT1 zabonwa.Iinkalo ezahlukeneyo zolwakhiwo kunye nonxibelelwano kwiinkalo zokusebenza ziye zaphandwa.
Ukusebenzisana phakathi kwe-HLA-A, i-MDN1 kunye ne-H2B yafunyanwa nge-immunoblotting kwi-Flo-1 kunye neeseli ze-A549 eziphathwe kwaye zingaphathwa nge-IFNα.Iziphumo zethu zibonisa ukuba i-HLA-A idibanisa ne-H2B ngendlela exhomekeke kwi-IFNα.Umsebenzi wethu ubonisa indlela enika umdla yokuphonononga ngakumbi ukudityaniswa kwezi zakhiwo zimbini.Kwakhona kuya kuba nomdla ukwandisa indlela ye-CLMS kwiphaneli yemigca yeeseli ukuchonga i-cell-type-independent interferon-mediated protein interactions.Okokugqibela, sisebenzise imodeli ye-MD njengenye indlela yokuqonda ukuguquguquka kweeprotheyini ezibandakanyekayo kwi-H2BFS-HLA-A-HMGA1 complex, elandelela umkhondo we-intramolecular kunye ne-intermolecular cross-talk.Iimpembelelo ezivela kwiinkcukacha ze-CLMS zibonisa ukuba kunokwenzeka ukuhambelana okuhlukeneyo kweeprotheni ze-H2BFS, i-HLA-A, kunye ne-HMGA1.Ukulungelelaniswa okunokwenzeka okwahlukileyo phakathi kwezi zixhobo zeprotheyini ze-docking zibonise intsebenziswano emininzi efana naleyo ibonwa kwi-dataset ye-CLMS.Awona mandla aphambili endlela yethu kukuba ivumela ukuchongwa ngokulula kokunxibelelana kofuzo lwepolymorphic efana ne-HLA, ke kuya kuba ngumdla ukufunda ukusebenzisana kwe-HLA haplotype-specific proteins ekunzima ukuyifunda ngenye indlela.Kuthatyathwe kunye, idatha yethu ibonisa ukuba i-CLMS ingasetyenziselwa ukwandisa ukuqonda kwethu i-interferon-induced signaling networks kunye nokubonelela ngesiseko sokufunda iinkqubo eziyinkimbinkimbi ze-intercellular kwi-tumor microenvironment.
Iiseli ze-Flo-1 zifunyenwe kwi-ATCC kwaye zigcinwe kwi-DMEM (Gibco) zongezwa nge-1% penicillin / streptomycin (Invitrogen), i-10% ye-fetal bovine serum (Gibco) kwaye igcinwe kwi-37 ° C kunye ne-5% CO2.Ukufukamela.Iiseli zakhuliswa kwi-70-80% ye-confluence ngaphambi kokuba iphathwe nge-IFNα14 (eyenziwe yi-Edinburgh Protein Production Facility).Zonke ezinye iikhemikhali kunye nee-reagents zathengwa kwi-Sigma Aldrich ngaphandle kokuba kuchazwe ngenye indlela.
Iiseli ze-Flo-1 zazikhuliswe kwiiplate ze-6-well kwaye ngosuku olulandelayo iiseli zaphathwa nge-10 ng / ml IFNα14 kwiiyure ze-24 malunga ne-80% yokudibanisa.Iiseli zihlanjwe kathathu nge-PBS kwaye zixutywe nge-DSS esanda kulungiswa (i-Thermo Fisher Scientific) (ichithwe kwi-DMSO) kwi-PBS kwi-5 min kwi-37 ° C. ukuya kwi-concentration yokugqibela ye-0.5 mM.I-DSS crosslinking reaction yatshintshwa nge-PBS kunye ne-DSS eseleyo yacinywa ngokudibanisa i-20 mM Tris (pH 8.0) kwi-PBS kwi-15 min kwi-37 ° C.Iiseli zaqokelelwa ngokukhuhla kwaye ziqokelelwe kwiibhubhu zokubopha eziphantsi (i-Axygen).
I-cell pellet yahlanjululwa nge-300 µl ye-urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) imizuzu engama-30 kwiqondo lobushushu begumbi kunye nokungcangcazela ngamanye amaxesha.Onke amanyathelo e-centrifugation enziwe kwi-14,000 xg kwi-8°C.I-Centrifuge i-lysate imizuzu eyi-10 kwaye idlulisele i-supernatant kwi-tube entsha.Iincinci ezisele ezicacileyo zachithwa kwi-150 μl yesibini ye-lysis buffer (2 M urea, 2% (w / v) i-SDS (i-sodium dodecyl sulfate)) imizuzu engama-30 okanye ngaphezulu de kufunyenwe isisombululo se-aqueous homogeneous.I-lysate yayiyi-centrifuged imizuzu engama-20 kwaye i-supernatant yayixutywe kunye ne-lysate efunyenwe kwisinyathelo sangaphambili.Iiprotheyini ezigxininisiweyo zavavanywa kusetyenziswa i-Micro BCA assay (i-Thermo Fisher Scientific) ngokwemiyalelo yomenzi weenkqubo ze-microplate.Iisampulu zakhawuleza zakhenkcezwa kwinitrogen engamanzi zaze zagcinwa ku -80°C.
Ngokumalunga ne-100 μg yeprotheyini edibeneyo edibeneyo e-soluble yacutshungulwa kusetyenziswa iprotocol yokulungiswa kwesampula yokucoca (FASP) njengoko ichazwe nguWisniewski et al.69 Ngokufutshane, iprotheni idityaniswe ne-200 µl ye-urea buffer (8 M urea ku-0.1 M Tris, pH 8.5), i-vortex kwaye isiqingatha.Onke amanyathelo e-centrifugation enziwe kwi-14,000 xg kwi-25°C.Isiqingatha sokuqala seprotheni ye-lysate edibeneyo idluliselwe kwi-10 kDa Microcon isixhobo sokucoca i-centrifugal exhotywe nge-Ultracel-10 membrane (Merck), ilandelwa yi-centrifugation kwi-filter ye-25 imizuzu.Emva koko yongeza isiqingatha sesibini seprotheni kwisihlunu kwaye uphinde ulandele amanyathelo afanayo.Ukubuyiselwa kweprotheyini kwenziwa ngokudibanisa i-100 μl ye-17 mM tris (2-carboxyethyl) iphosphine hydrochloride (TCEP) kwi-urea buffer.Ukubuyiswa kwavuselelwa kwi-thermomixer kwi-600 rpm kwimizuzu engama-30 kwi-37 ° C.Ukongeza, ikholamu yayiyi-centrifuged kwaye iprotheni encitshisiweyo edibeneyo yayiyi-alkylated isebenzisa i-100 μl ye-50 mM iodoacetamide kwi-urea buffer.Ukusabela kwe-alkylation kwenziwa kwindawo yokushisa kwemizuzu engama-20 ebumnyameni.Jikelezisa ikholamu, hlamba iindonga zekholamu ka-3 nge-100 µl urea buffer, uze ube yi-centrifuge.Umsebenzi ofanayo wenziwa amaxesha ama-3 kusetyenziswa i-100 μl ye-100 mM ammonium bicarbonate.Ngaphambi kwe-trypsinization, buyisela ityhubhu yokuqokelela entsha.Yongeza isithinteli sokugaya esiqulathe i-50 mM ammonium bicarbonate kunye ne-1 µl ye-trypsin exutywe kwi-trypsin buffer (Promega).Umlinganiselo we-trypsin kunye neprotheni ugcinwe malunga ne-1:33, kwaye iintshukumo zokwetyisa zafukanywa ngobusuku obungama-37 ° C. kwigumbi elifumileyo.I-peptide edityanisiweyo yakhutshwa kwisihluzo nge-centrifugation imizuzu engama-25.I-peptide recovery iphuculwe ngokudibanisa i-50 μl ye-0.5 M NaCl kwisihluzo, ilandelwa yi-centrifugation imizuzu engama-25.
Iikholamu zeC18 Micro Spin (Izixhobo zeHarvard) zasetyenziselwa ukukhupha iipeptide zetryptic eziqhagamshelwe ngokunqamlezileyo ngokulandela inkqubo echazwe nguBouchal et al.70 ngohlengahlengiso olungephi.Ngokufutshane, iikholomu ze-C18 ze-spin zenziwe zasebenza kunye nokuhlamba okuthathu kwe-0.1% ye-asidi ye-formic (FA) kwi-acetonitrile (AcN) (i-Merck) kunye nokuhlamba ezimbini kwe-0.1% FA.Ikholomu ifakwe i-hydrated nge-0.1% FA kwimizuzu ye-15.Layisha iisampuli kwiikholamu ze-spin kwaye uhlambe amaxesha e-3 nge-0.1% FA.Iipeptide ezichithiweyo zaye zahluthwa ngokulandelelanayo ngethambeka elihamba ngamanyathelo kusetyenziswa i-50%, i-80% kunye ne-100% ye-AcN kwi-0.1% ye-FA.Iisampulu zomiswa kwi-SpeedVac Plus concentrator (Eppendorf) de ulwelo olushiyekileyo lwanyamalala ngokupheleleyo.I-peptides eluted yanyibilika kwi-100 μl ye-0.08% ye-trifluoroacetic acid kwi-2.5% ye-AcN kwaye imilinganiselo yalinganiswa kwi-NanoDrop 2000 (i-Thermo Scientific).Ngokumalunga ne-1 μg ye-peptide edibeneyo kwisampulu nganye yafakwa kwi-LC-MS/MS inkqubo.
Iipeptide eziqhagamshelweyo zahlulwa kwi-UltiMate 3000 RSLCnano LC system (Thermo Scientific) eqhagamshelwe kwi-Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Iipeptide ezidityaniswe ngokunqamlezileyo zaqokelelwa kwi-ID ye-300 µm, i-5 mm ubude µ-phambi kwekholamu ye-C18 yokubamba ikholamu epakishwe nge-C18 PepMap100 sorbent kunye ne-5 µm ye-PepMap sorbent (i-Thermo Scientific).Layisha ukuhamba kwempompo kwi-5 µl/min 0.08% i-trifluoroacetic acid echithwe kwi-2.5% AcN.I-peptides edibeneyo edibeneyo yahlula kwikholamu ye-silica edibeneyo yohlalutyo kunye nobubanzi obungaphakathi be-75 μm kunye nobude be-150 mm, ezaliswe nge-2 μm PepMap sorbent (i-Thermo Scientific).Izigaba zeselula A kunye ne-B zibandakanya i-0.1% FA emanzini kunye ne-0.1% FA kwi-acetonitrile, ngokulandelanayo.Ithambeka liqala ku-2.5% B lize linyuke ngokulandelelana ukuya kuma-40% B kwimizuzu engama-90, emva koko liye kuma-90% B kwimizuzu emi-2 elandelayo.Ukuqulunqwa kwesigaba esihambayo kwagcinwa kwi-90% B kwimizuzu eyi-10 kwaye emva koko yehla ngokulandelelana ukuya kwi-2.5% B kwimizuzu emi-2.Ikholamu ilinganiswe kwi-2.5% B kwimizuzu eyi-8 phambi komjikelo olandelayo.I-peptides edibeneyo edibeneyo ekhutshwe kwikholamu yokuhlalutya i-ionized kwi-nanoelectrospray ionization (NSI) umthombo kwaye ifakwe kwi-Exploris 480 mass spectrometer (i-Thermo Scientific).
I-Orbitrap Exploris 480 i-spectrometer ye-mass spectrometer isebenze kwimodi yokulungelelaniswa kwedatha efanelekileyo.Ukuskena okupheleleyo kwenziwa kwimodi yecandelo kwisisombululo se-120,000 kunye nezicwangciso zoluhlu ukusuka ku-m / z 350 Th ukuya ku-m / z 2000 Th.Ithagethi eqhelekileyo ye-AGC yamiselwa kuma-300% ngelona xesha liphezulu lokufaka i-50ms.Ukufunyaniswa kwencopho ye-Monoisotopic kusekwe kwiipeptides.Iparamitha yokuphumla isithintelo isetelwe ekubeni yinyani ukuba ii-precursor ezimbalwa zifunyenwe.Amandla amancinci e-ionic we-precursor amiselwe kwi-5.0e3 kwaye i-precursor charge ithi ukuya kuthi ga kwi +8 ibandakanyiwe kwiimvavanyo.
Ixesha lomjikelo phakathi kweskeni ezinkulu kwimodi yokulungelelaniswa kwedatha yamiselwa imizuzwana eyi-2.5.Ukukhutshwa kobunzima obuguquguqukayo kumiselwe kwimizuzu engama-20 emva kokuqhekeka kokuqala kweyoni eyandulelayo.Ifestile yokwahlula eyandulelayo yayimiselwe ku-2 Th.Uhlobo lwamandla ongquzulwano oluqhelekileyo olunemowudi yamandla ongquzulwano olusisigxina lukhethwe kwidatha exhomekeke kwi-MS/MS scan.Amandla okudibana amiselwe kuma-30%.Isisombululo se-Orbitrap simiselwe kwi-15,000 kunye ne-AGC ekujoliswe kuyo kwi-100%.Ixesha lesiko lokutofa limiselwe kwi-60 milliseconds.
Ngaphambi kokulandelela i-protein-protein network kwi-samples-linked-linked-samples, sicubungule iifayile eziluhlaza sisebenzisa iphakheji ye-MaxQuant (inguqulo ye-1.6.12.0) i-26,27 ukuchonga i-peptides / iiprotheni ezilandelwayo kwiisampuli.Ukongezelela, uhlalutyo olufanayo lweproteomic lwenziwa kwiisampuli ze-Flo-1 ezingaxhunywanga eziphathwe kwaye zingaphathwa nge-IFNα.Idatha ye-MS/MS yakhangelwa kwiziko ledatha le-UniProt (www.uniprot.org) (elayishwe nge-12 ka-Agasti 2020, iqulethe amangenelo angama-75,093) kusetyenziswa injini yokukhangela eyakhelwe-ngaphakathi iAndromeda27.Ukukhangela kwaqhutyelwa ngaphandle kokubonisa ukuchaneka kwe-enzyme kunye nokuguqulwa okuhlukeneyo kwe-deamidation (N, Q) kunye ne-oxidation (M).I-precursor mass tolerances yamiselwa kwi-20 ppm kunye ne-ion yemveliso kwi-0.02 Da.Ukutenxa kokuqala kunye nobukhulu obukhulu kumiselwe kwi-10 ppm.Ubuninzi be-peptide bubekwe kwi-4600 Da kwaye ukulandelelana ukufana kubekwe phakathi kwe-7 kunye ne-25 ye-amino acids (aa).Uhlalutyo olongezelelweyo lwamanani lwenziwa kusetyenziswa inkqubo yePerseus (uguqulelo 1.6.10.45).Umxholo weprotheyini ubalwa ngokuqhelanisa ubunzulu beprotheyini (ukuqina kwe-LFQ; ubungakanani obungabhalwanga) i-27 kwaye amaxabiso okuqina aguqulelwa kwi-Log2.Iqoqo le-hierarchical yeeprotheyini ezichongiweyo yi-peptide intensity yazo yakhiwe kusetyenziswa i-pheatmap (v1.0.12) ipakethe kwi-R (v 4.1.2).Uhlalutyo lokutyetyiswa kwendlela lwenziwa kusetyenziswa i-Reactome pathway database ye-IFNα-treated proteins eziye zasebenza ngaphezu kwamaxesha amane xa kuthelekiswa neesampuli ezingaphendulwanga.
Ukuchongwa kwe-lysine (K) okanye i-serine (S) i-crosslinks yekhemikhali ethile ye-protein complexes ebekwe esweni yi-LC-MS / MS yenziwa ngokusebenzisa umatshini wokuchonga we-spectroscopic (SIM-XL) kwiipeptide ezidibeneyo (SIM-XL)29.Okokuqala, ukusebenzisana okunokwenzeka phakathi kwe-interferon-associated (IFN) i-DNA yesignesha yokumelana nomonakalo (IRDS) i-genes yaphandwa ngokusebenzisa i-IRDS yedatha yeprotheyini echazwe kwi-Padariya et al.28.Ukuhlola zonke iimeko kunye nokuphindaphinda kwe-UniProt yonke yomntu i-computingly computationally, ngoko ke yonke i-database ye-UniProt yabantu (www.uniprot.org) (ekhutshelwe i-12 Agasti 2020, iqulethe i-75,093 yamangeno) ngokuchasene nokuphindaphinda kwe-IFNα.Esinye sezihluzi zonxibelelwano oluthembeke kakhulu.Olu nxibelelwano lubaluleke kakhulu olufunyenweyo lwandiswa kwaye lwavavanywa kulo lonke uphindaphindo kunye neemeko.
Kwi-SIM-XL, i-DSS yayisetyenziselwa i-crosslinker (i-XL) kunye ne-XL ye-weight shift shift and modification weight shift ibekwe kwi-138.06 kunye ne-156.07, ngokulandelanayo.Ezi ndawo zilandelayo zokusabela ezinqamlezileyo ziyaqwalaselwa: i-KK, i-KS kunye ne-KN-TERM, ngaphandle kweeyoni zentatheli.Zombini i-precursor kunye ne-fragment ppm zamiselwa kwi-20 kwaye i-Xrea threshold yamiselwa ku-0.15.I-Trypsin yayicatshangelwa njengento ecacileyo ngokupheleleyo, kwaye i-high-energy C-trap (HCD) indlela yokuqhekeka iphunyeziwe.I-XCorr DB dynamic reduction threshold kunye nenani eliphantsi leepeptide zokunciphisa iDB eziguquguqukayo zimiselwe kwi-2.5 kunye ne-2, ngokulandelelanayo.Ezinye iiparameters zezi: ukubakho kwe-monoisotope kunye nencopho ye-coincidence cutoff, ubuncinane be-4 AA intsalela ye-strand kunye nentlawulo ephezulu ye-strand, kunye ne-3 maxima yokwahlukana okuphosiweyo.Iziphumo ezithunyiweyo zeemephu ze-2D zahlalutywa kwi-(SIM-XL) kunye nomboniso we-xQuest28 womzobo wasetyenziswa ukwakha iimephu ze-2D.Iiprotheyini ezinqamlezayo kwizakhiwo zeprotheni zinikezelwa kwi-PyMol (i-PyMOL ye-Molecular Graphics System, i-version 2.0 Schrödinger, LLC).
Izakhiwo zeeprotheyini zeprotheyini zenziwe kusetyenziswa i-Phyre2 iseva (http://www.sbg.bio.ic.ac.uk/phyre2)11 isebenzisa imigaqo ye-homology yokulinganisa kunye nokuphunyezwa kwe-"Hidden Markov Method".I-Phyre2 ivelisa imodeli yezakhiwo ezisekelwe kulandelelwano kunye nezakhiwo zeprotheyini ezaziwayo.I-H2BFS, i-HLA-A, i-HMGA1, i-LRCH4, kunye neeprotheni ze-MDN1, izakhiwo ze-template 1kx552, 1kj349, 2eze55, 6hlu62, kunye ne-6i2665 zisetyenzisiwe.Ukongeza, ubume be-AlphaFold71 MX1, UBP18 kunye ne-ROBO1 nayo yaqwalaselwa.Isakhiwo seprotheyini sibonakaliswe kusetyenziswa iphakheji ye-BIOVIA Discovery Studio Visualizer (Dassault Systèmes, BIOVIA, San Diego, CA, USA) kunye ne-Molecular Operating Environment package (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).