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Uhlelo lokubonisa i-interferon ludala impendulo eqinile ye-cytokine ezinhlobonhlobo zezimpawu ze-pathogenic kanye ne-intrinsic pathological ezivela emvelweni, okuholela ekufakweni kwama-subsets amaprotheni e-interferon-inducible.Sisebenzise i-DSS-mediated cross-link mass spectrometry (CLMS) ukuze sithole ukusebenzisana okusha kwamaprotheni-protein esizindeni samaprotheni enziwe yi-interferon.Ngokungeziwe kumaprotheni alindelekile e-interferon-inducible, siphinde sahlonza izithasiselo zenoveli ze-intermolecular kanye ne-intramolecular cross-linked of canonical interferon-inducible proteins njenge-MX1, USP18, OAS3, ne-STAT1.Sigxile ekuqinisekiseni i-orthogonal yesethi yenoveli yamanethiwekhi amaprotheni e-interferon-inducible akhiwe amaprotheni e-HLA-A (H2BFS-HLA-A-HMGA1) esebenzisa i-co-immunoprecipitation kanye nocwaningo lwabo oluqhubekayo kusetshenziswa imodeli ye-molecular dynamics.Ukumodela kwe-conformational dynamics ye-protein complex kwembule amasayithi amaningana okusebenzelana abonisa ukusebenzisana okukhonjwe kokutholwe yi-CLMS.Ndawonye, ​​sethula ucwaningo lokuhlola lwe-CLMS ukuze sihlonze izinkimbinkimbi ezintsha zokusayina ezibangelwa i-interferon, futhi sibheke phambili ekusetshenzisweni okubanzi kwe-CLMS ukukhomba amandla amasha okusebenzelana kwamaprotheni ku-tumor microenvironment.
Ngaphambi kokuthi kuqale ukusabela okuguquguqukayo kokuzivikela komzimba, uhlelo lokuvikela lwangaphakathi lomsingathi lufaka impendulo elwa namagciwane elamula umndeni wama-cytokines e-alpha-helical ayimfihlo abizwa ngokuthi ama-interferon (IFNs).Amakilasi ohlobo lwe-IFN i-IFNα ne-IFNβ enza kusebenze izimpendulo zeselula, okuhlanganisa i-antiviral, proapoptotic, proinflammatory, kanye nezifunda zokuvimbela ukwanda.Kubantu, ama-subtypes angu-13 e-IFNα ayaziwa, wonke ahlanganiswe ku-chromosome 91. Ngokumangalisayo, i-IFNα2 kuphela eye yafundelwa ukusetshenziswa emtholampilo.Muva nje, ukunaka okukhethekile kukhokhwe ocwaningweni ngezinye izinhlobo ezingaphansi ze-IFNα.Ucwaningo lwakamuva lubonise ukuthi i-IFNα14 ingenye yama-isoform asebenza kahle kakhulu ekukhawuleleni i-HBV2 nokuphindaphinda kwe-HIV-13,4 uma kuqhathaniswa ne-canonical IFNα2 subtype.
Kuye kwatholakala ukuthi i-interferon receptor complexes evuliwe yohlobo lwe-I (IFNAR1 ne-IFNAR2) icupha i-cascade yokudlulisa isignali elamula u-Janus kinases TYK2 kanye ne-JAK15,6.Lezi Janus kinases phosphorylate isignali transducers kanye transcriptal amaprotheni activators (STAT1 kanye STAT2) ezinsalela tyrosine ukuqalisa SH2 domain-mediated heterodimerization6.Ngokulandelayo, i-IRF9 ibophezela ama-heterodimers e-STAT ukuthi akhe inkimbinkimbi ye-trimeric yofuzo lwe-IFN-stimulated factor 3 (ISGF3), ethuthela ku-nucleus futhi ibangele ukuloba kwezakhi zofuzo ezingaphezu kuka-2000 ezivuselelwe i-interferon (ama-ISG)5,6,7,8.
Ama-ISG akha umgogodla wamasosha omzimba azalwa nawo, ikakhulukazi ekuphenduleni ukuhlasela kwegciwane.Njengomugqa wokuqala wokuzivikela ekuthelelekeni ngegciwane, amangqamuzana athumela ngokushesha ukusebenzisana okubanzi kwamaprotheni eselula anezinhlobonhlobo zemisebenzi yebhayoloji.Lawa maphrotheni ahlanganisa ama-receptors okuqaphela iphethini, ama-molecule asayinayo, izici zokuloba, namaprotheni anemisebenzi eqondile yokulwa namagciwane, kanye nabalawuli abangemuhle bempendulo yokuzivikela komzimba9.Ulwazi oluningi ngomsebenzi we-ISG luvela kuzikrini ezisebenzayo ezisebenzisa izikrini zokuveza ngokweqile10,11 noma izindlela zokuthulisa izakhi zofuzo (i-siRNA, i-RNAi ne-CRISPR)12,13 lapho ama-ISG angawodwana evezwa khona noma avinjwe khona futhi umsebenzi wawo uhlolwa kumagciwane ahlukahlukene.Nakuba lezi zifundo zinqume izakhiwo ze-antiviral zama-ISG ngamanye, izindlela eziyisisekelo zamangqamuzana e-ISG ngayinye zihlala zingaziwa kakhulu.Kuyamukelwa ngokuvamile ukuthi amaprotheni amaningi asebenzisana ne-cytokine eyodwa noma ngaphezulu ukuze kuqinisekiswe umsebenzi ogcwele, ngakho-ke ama-ISG asebenzisana ngokuqondile noma ukusebenzisana kwawo kulamula amaphrotheni eselula.Isibonelo, ucwaningo lwakamuva lwe-photocrosslinked proteomics luhlonze i-ATPase VCP/p97 njengozakwethu omkhulu wokusebenzisana we-IFITM3, okuvimbela kwawo kuholela ekushiyekeni ekuhlungeni kwe-lysosomal, inzuzo, kanye ne-cotransport ye-IFITM3 enezinhlayiya zegciwane i-14.Ngokusebenzisa i-immunoprecipitation, sihlonze i-VAPA, iphrotheni ehambisana ne-vesicle, njengozakwethu wokusebenzelana ne-IFITM1/2/3 elamulela ukuvuthwa kwegciwane le-cholesterol, futhi lokhu kwaqinisekiswa olunye ucwaningo olusebenzisa i-yeast two-hybrid system.Ukusekelwa Kwesayensi 15, 16.
Inqubo eyisisekelo yezinto eziphilayo ehilelekile ekucindezelweni kokutheleleka kanye nokuguqulwa okulimazayo isethulo se-antigen, eqondiswa ama-molecule amakhulu e-histocompatibility complex (MHC).Ama-Peptides (ama-amino acid angu-8-12 ubude) avela kumaprotheni aqhekekile, anqanyuliwe ngaphambi kwesikhathi noma angalungile alayishwa ku-MHC-I heterodimer (ehlanganisa i-MHC-I amaketanga asindayo futhi alula, okuthiwa i-β-2-microglobulin; β2M) 17,18.I-trimers ye-MHC-I eqinile eyenza umphumela ithuthelwe endaweni yeseli, lapho iveza khona ama-peptide e-intracellular kumaseli e-CD8+ T (ama-cytotoxic T cells)17.Amaseli e-T abona futhi abhubhise lawa magciwane namaseli aphethe i-antigen ethize yesimila.Ngakho-ke, ama-pathogens namaseli e-tumor avame ukucindezela inqubo yokwethulwa kwe-antigen ukugwema ukubhekwa kwamasosha omzimba.Ngaphezu kwalokho, i-MHC-I yehliselwe phansi ku-40-90% wezimila zabantu futhi ivame ukuhlotshaniswa ne-prognosis empofu19.
Izakhi zofuzo ezihilelekile ekuphenduleni amagciwane kufanele zishintshe ngokushesha phakathi kwesimo sokuphumula kanye nesimo sokulotshwa okusebenzayo.Ngakho-ke, amaprotheni amaselula amaningana acatshangelwa ukuthi ahileleke ekuphenduleni isidingo esikhulu se-IFN ngesikhathi esifushane, kuhlanganise nokulungiswa kabusha nokuguqulwa komgqugquzeli we-chromatin 20,21.Ucwaningo oluningi lugxile ekuhlonzweni kozakwethu abangabodwana beprotheni ye-ISG lapho kukhona i-IFN.Ucwaningo oluningana lwe-proteomic kanye ne-transcriptomic kumamodeli wamaseli wamaseli luye lwacacisa umphumela we-IFN endaweni yeselula.Kodwa-ke, naphezu kokuqonda okukhulayo kokuguquguqukayo okubangelwa ama-interferon, sisazi okuncane mayelana nokubandakanyeka kwama-ISG.Uma kucatshangelwa ubunkimbinkimbi kanye nokuguquguquka okuncike esikhathini sokusayina kwe-interferon, kuphakama imibuzo emibili: (i) kungenzeka yini ukuzinzisa nokubamba ama-multiprotein complexes ahilelekile ekuboniseni amasignali okusheshayo, futhi (ii) ingabe lokhu kusebenzisana kungenziwa kumephu endaweni ye-3D?
Ukuze kubhekwane nalezi zinkinga, sisebenzise i-disuccinimide suberate-mediated chemical cross-linking (DSS) ehambisana ne-mass spectrometry (CLMS) ukuze sihlole inethiwekhi yokusebenzisana yamaprotheni e-IFNα kanye namandla ayo.I-DSS yengeza izibopho eziqinile phakathi kwezinsalela eziseduze zamaprotheni kanye/noma ama-protein complexes ku-vivo.Ukuhlaziywa kwe-MS okwalandela kuveza amasayithi athile axhumanisayo abonisa ukuba seduze kwendawo kwezifunda ngaphakathi kwephrotheni ethile, okubizwa ngokuthi ukuxhumana kwangaphakathi, noma ama-subunits ku-protein complexes, okubizwa ngokuthi i-interrelationships.Ngokusebenzisa le ndlela, sihlonze amanoveli amaningana amaprotheni-amaprotheni amanethiwekhi kanye namanethiwekhi wokusebenzisana we-interferon-induced multiprotein.Ngokuqhubeka nokuhlola isethi engaphansi yalokhu kusebenzisana okusha, sibonisa ukuthi i-H2BFS (H2B histone-type FS; kamuva ebizwa ngokuthi i-H2B) kanye ne-MDN1 zisebenza njengozakwethu ababophayo be-HLA-A.
Amaseli e-Flo-1 angenye yamamodeli e-in vitro aziwa kakhulu e-esophageal adenocarcinoma njengoba elingisa izici ezibalulekile zamathumba ommizo22,23.Kodwa-ke, akuwona wonke ama-tumor ane-immunogenic, futhi ukuze anqume ukuthi amaseli e-Flo-1 asabela yini ekwelapheni kwe-interferon, siphatha amaseli e-Flo-1 nge-10 ng / ml IFNα amahora angu-72.Amaseli e-Flo-1 abonise ukungeniswa kwangaphambi kwesikhathi kwe-pSTAT1 ne-IRF1, eqala amahora angu-2 ngemva kokwelashwa futhi aqhubeke amahora angu-72, ngokuncipha okuncike esikhathini kumazinga amile we-IRF1 (Umfanekiso 1A).Ama-ISG (i-MX1, IFITM1, OAS1/2, ne-ISG15) atholwe esungulwe ngokuqinile ngemva kwamahora angu-6, alingisa izimpendulo zesigaba sakudala samaphakathi nesakamuva ku-IFNα (Umfanekiso 1A).Ngokubambisana, le datha iphakamisa ukuthi le modeli yeselula ingasetshenziswa ukutadisha izimpendulo ze-interferon.
Izimpendulo zokubonisa amaprotheni ezihlukile kumaseli e-Flo-1 ngemva kokwelashwa kwe-IFNα.(A) Ukubonakaliswa kwamaprotheni kumaseli e-Flo-1 aphathwe nge-10 ng / ml IFNα ye-2, 6, 24, 48 kanye namahora we-72 kwahlaziywa nge-immunoblot kusetshenziswa ama-antibodies e-ISG abonisiwe.(B) Amajeli e-SDS-PAGE ane-Coomassie eluhlaza okwesibhakabhaka okukhishwe kweseli lonke ngemva kokuxhumanisa ne-DSS ngezikhathi ezibonisiwe nokugxilisa ingqondo.(C) I-immunoblot emele ihlolwe nge-p53(DO-1) antibody kusukela kumasampuli afanayo ukuze kuhlolwe izinga lokuxhumanisa amaprotheni.
Ukuze sithwebule ukwakheka kwezwe lokusebenzisana kwe-in situ protein, sisebenzise i-DSS, i-ejenti exhumanisa kakhulu esetshenziswa kabanzi ngenxa yokuqina kwayo kolwelwesi oluphezulu kanye nesikhathi sokusabela esifushane uma kuqhathaniswa.Isikhathi esifushane sokuphendula sisiza ukuvimbela ukwakheka kwama-aggregates amakhulu amaprotheni axhumene, ngaleyo ndlela kugcinwe ukuzinza kwe-crosslinker.Ukuze sinqume ukugxiliswa kwe-DSS okuphelele futhi sigweme ukuxhumanisa ngokweqile, siqale sadalula amaseli ku-5, 2.5, kanye ne-1 mM DSS imizuzu engu-5, 10, 5, kanye nangama-30, ngokulandelana, futhi sahlaziya ama-lysates nge-SDS-PAGE ene-Coomassie-stained. (idatha ayibonisiwe) .Ama-lysate eseli abonakala exhumene kakhulu ekugxiliseni okuphansi kakhulu nangesikhathi esifushane kakhulu.Ngakho-ke, i-DSS yalinganiswa ku-1, 0.5, kanye no-0.1 mM ngaphezu kwamaminithi angu-5 (Umfanekiso 1B).Ukuxhumanisa okufanelekile kwabonwa nge-0.5 mM DSS imizuzu engu-5, futhi lezi zimo zikhethelwe amaseli aphathwe nge-IFNα.Ngaphezu kwalokho, Umfanekiso 1C ubonisa i-blot yaseNtshonalanga eyenziwa kusetshenziswa i-p53 (DO-1) antibody ukuze kuhlolwe izinga le-protein cross-linking.
Amaseli e-Flo-1 aphathwe nge-10 ng/ml IFNα amahora angu-24 ngaphambi kokwengeza i-crosslinker.Amaseli axhumene nesiphambano abese ehlanjululwa yi-proteolysis enezinyathelo ezimbili futhi amaprotheni acutshungulwa yi-FASP (Fig. 2) 24,25.Ama-peptide e-tryptic axhumene nesiphambano ahlaziywa nge-mass spectrometry (Fig. 2).I-spectra ye-MS/MS ibe isifaniswa nokulandelana kwamaprotheni futhi ibalwa nge-MaxQuant26,27.Ama-peptide axhumene ngokuphambanayo ahlonzwe ku-spectra etholiwe kusetshenziswa uhlelo lwe-SIM-XL, futhi izinhlanganisela ngazinye zahlanganiswa zaba inethiwekhi eyinkimbinkimbi kusetshenziswa i-xQuest28 kanye ne-SIM-XL29 yomthombo ovulekile wamaphayiphi wesofthiwe yekhompuyutha (Fig. 2).I-SIM-XL ihlonza ukusebenzisana kwamaprotheni-amaprotheni, amaketanga angaphakathi namaketanga angawodwana kuzingxube zamaprotheni ezilula noma eziyinkimbinkimbi futhi inikeza imibhalo yokubuka ngeso lengqondo ukusebenzisana ezakhiweni zamaprotheni.Ngaphezu kwalokho, ilinganisa ireferensi ngayinye njengesikolo se-ID ngokuya ngekhwalithi ye-spectrum ye-MS/MS29.Ukusebenzisana okuningana okuthembeke kakhulu kwamaprotheni-amaprotheni kanye nezinkimbinkimbi kuye kwaphawulwa, futhi isethi entsha yokusebenzisana iye yaphenywa ngokuqhubekayo kusetshenziswa i-co-immunoprecipitation kanye nezinguquko ezihambisanayo ze-complex usebenzisa imodeli ye-molecular dynamics (MD) (Fig. 2) 30, 31.
Ukubuka konke okuhleliwe kwendlela ye-CLMS.Amaseli e-Flo-1 aphathwe nge-10 ng / ml IFNα amahora angu-24 alandelwa yi-in situ protein cross-linking esebenzisa i-DSS elandelwa yi-cell lysis kanye ne-trypsinization.Amasampula axhumene nesiphambano ahlaziywa kusetshenziswa i-Orbitrap mass spectrometer futhi kwaqhutshekwa nesampula ukuze kuhlukaniswe izandulela ze-peptide phakathi ne-LC-MS/MS.Ama-peptide amabili axhunyiwe ahlonzwe ku-spectra etholiwe kusetshenziswa Umshini Wokuqaphela I-Spectrum we-Crosslinked Peptides (SIM-XL) uhlelo, futhi wonke ama-compounds ahlanganiswa abe yinethiwekhi eyinkimbinkimbi kusetshenziswa amaphayiphi wokubala.Hlunga ukusebenzelana kokuzethemba okuphansi okusekelwe kumaphuzu wesilinganiso esihle esingamanga (FDR).Ukusebenzisana okusha okuningana okuthembekile okuphezulu kwamaprotheni-amaprotheni kwaqinisekiswa futhi kusetshenziswa i-co-immunoprecipitation, futhi izinguquko ezihambisanayo ezinkingeni zahlolwa kusetshenziswa imodeli ye-molecular dynamics (MD).
Isamba esingu-~30,500 kanye ne-~28,500 peptide sitholwe kusetshenziswa i-MaxQuant kumasampula e-IFNα angavuselelwe futhi avuselelwe, ngokulandelanayo (Ithebula Le-Supplementary S1, Fig. 3A).Ukusatshalaliswa kobude be-peptide kuzo zombili izimo kubonise ingxenye ephezulu yama-peptide amakhulu, okubonisa ukuba khona kwama-peptide axhumene (Fig. 3B, C).Ukwengeza, ingxenye enkulu yama-peptide amakhulu abekhona ebangeni le-40-55 kumasampuli aphethwe yi-IFNα (Fig. 3C).Ukumepha amaprotheni ngokumelene nokuqina kwe-log2 kubonise ukuthi amaprotheni avamile avuselelwe i-interferon ayemaningi kakhulu uma kuqhathaniswa namasampuli angakapheki, afaka i-MX1, IFIT1/3, OAS2/3, DDX58, ne-HLA-F (Figure 3D).Ukuhlaziywa kwezindlela zamaprotheni ezingaphezu kokuphindwe kathathu okucetshisiwe ekuphenduleni ukwelashwa kwe-IFNα usebenzisa i-Reactome pathway database kubonise ukuthi isethulo se-antigen ye-MHC-I-mediated kanye nokucubungula kwakuyindlela evelele kakhulu (Umfanekiso 3E).Ngokuvumelana nemibiko yangaphambili, izimpendulo ze-antiviral ezixhunywe yi-OAS ne-ISG15 kanye ne-IFNα / β kanye nokusayina kwe-cytokine kwakuphakathi kwezindlela ezicushiwe.Ukwengeza, i-lysine- kanye ne-serine-specific protein cross-links zikhonjwe ku-spectra etholwe ekuqaleni ye-MS/MS kusetshenziswa i-SIM-XL.Ucwaningo lwakamuva lubike ukuthi ama-ISG ayi-104 athatha amagciwane angama-20 asuka kumakilasi ayi-9 egciwane ngokuhlaziywa kwe-meta yezifundo zomuntu ngamunye ze-ISG ezinhlotsheni ezi-5 zamaseli9.Nokho, ukuze sinqobe imikhawulo yokubala yokuhlola amasethi edatha amakhulu, siqale ngedathasethi encane ukuze sihlole ukusebenzelana okungenzeka phakathi kohlu lwezakhi zofuzo ze-IRDS ezibikwe u-Padaria et al., iningi lazo okungama-ISG.
Ukuhlonzwa kwamaprotheni axhunywe ngokuhlukile achazwe ngokuhlukile ekuphenduleni i-IFNα (idatha etholwe ku-MaxQuant).(A) Umdwebo we-Venn omelela inombolo yama-peptide avamile futhi akhethekile akhonjwe kumasampuli e-IFNα14 alashiwe nanganakiwe e-Flo-1.Ukusatshalaliswa kobude be-peptide kwamasampula angalashiwe (B) kanye ne-IFNα aphathwe (C) axhumene.(D) Imephu yokushisa emele i-log2 (ukuqina kwe-LFQ) phakathi kwamaseli e-Flo-1 angalashwanga kanye ne-IFNα14 aphathwe.Iphaneli yesokunxele ibonisa amaprotheni asebenze kakhulu phambi kwe-IFNα.(E) I-Histogram emele izindlela ezingu-20 zokucebisa ezinkulu ngemva kokwelashwa kwe-IFNα.Isizindalwazi sendlela ye-Reactome sihlaziye izinguquko eziphindwe kane kumaphrotheni asabelayo e-IFNα amisiwe.
Ukukhuthazwa kwe-ISG ye-Interferon-mediated kubhalwe kahle, kodwa ezingeni lamangqamuzana akuqondakali kahle ukuthi la maprotheni afinyelela umvuthwandaba kanjani obanzi wemisebenzi yezinto eziphilayo.Siphenye ukusebenzisana kwamaprotheni ngezinga eliphezulu lokuzethemba phakathi kwama-ISG aziwayo.Ngokuthakazelisayo, sihlonze inethiwekhi ehlanganisa i-MX1, USP18, ROBO1, OAS3, kanye namaprotheni e-STAT1 akha inkimbinkimbi enkulu ekuphenduleni ukwelashwa kwe-IFNα (Umfanekiso 4, Ithebula S2) 32,33,34.Okubaluleke kakhulu, lokhu kusebenzisana kutholakale kuwo wonke ama-triplicates aphathwe nge-IFNα futhi awatholakalanga kumasampuli angaphenduliwe, okuphakamisa ukuthi akhiwe ngokuqondile ekuphenduleni ukwelashwa kwe-IFNα.Kuyaziwa ukuthi i-STAT1 ilawula ngokubhaliwe ukuvezwa kwalawa ma-ISG, kodwa ukusebenzisana kwayo nama-ISG ezingeni lamaphrotheni akuzange kufundwe.Isakhiwo se-crystal se-STAT1 sibonise ukuthi isizinda sayo se-helical (CCD) asibandakanyi ekusebenzelaneni ne-DNA noma ama-protomers ngesikhathi sokwakhiwa kwe-dimers35.Lawa ma-α-helices akha isakhiwo se-helical helix esinikeza indawo engaphezulu kakhulu ye-hydrophilic ukuze kusetshenziswe ukusebenzisana kwe-35.Kudatha yethu ye-CLMS, siqaphele ukuthi ukusebenzisana okuningi ne-STAT1 kwenzeke esizindeni se-SH2 esandulela i-CCD, isizinda sesixhumanisi, noma umsila we-C-terminal (izinsalela 700-708) (Umfanekiso 4A).Ucwaningo lwangaphambilini lubike ukuthi i-USP18 ibophezela ku-CCD kanye nesizinda esibophezelayo se-DNA (DBD) se-STAT2 futhi sibuthelwa ku-subunit yohlobo lwe-interferon receptor IFNAR2 ukuze kunqandwe ukuvinjelwa kohlobo I-interferon signaling 24.Idatha yethu iphinde yabonisa ukuthi isizinda se-USP18 se-catalytic sisebenzisana ne-STAT1 DBD (Umfanekiso 4A,D), iphakamisa ukuthi kokubili i-STAT1 ne-STAT2 zingadlala indima ekuheheni i-USP18 ku-IFNAR2.
Inethiwekhi ye-ISG yamaprotheni-protein ekhonjwe kumaseli axhumene kakhulu aphathwe nge-IFNα.(A) Isakhiwo sokusebenzisana se-2D esibonisa ukusebenzisana kwamaprotheni-namaprotheni (okukhiqizwe kuhlelo lwe-SIM-XL), nemigqa emele ukusebenzisana kwama-intermolecular (i-crosslink cutoff isethelwe ku-3.5).Izizinda zobunikazi obuhlukahlukene zimakwe ngombala wazo32: isizinda se-MX1, i-Dynamin_N (73–249), i-Dynamin_M (259–547), ne-GED (569–660).OAS3 izizinda: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), kanye OAS1_C (903-108).Isizinda 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) kanye ne-fn3 (777–864).Izinkambu ze-STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), kanye ne-STAT1_TAZ2bind (715–739).(B) Isibukeli esiyindilinga samaprotheni axhumene kakhulu (MX1, UBP18, OAS3, ROBO1, ne-STAT1) ngokusebenzisana nokusebenzisana okubhalwe ngokuluhlaza okwesibhakabhaka nokubomvu, ngokulandelana.Umkhawulo we-cross-link ubekwe ku-3.5.Iziqephu zamachashazi zibonisa amasayithi okusebenzisana we-STAT1 ne-MX1 (C), i-USP18 (D), i-ROBO1 (E), ne-OAS3 (F), kanye nezindawo zokusebenzisana ze-K noma ze-S phakathi kwama-peptide amabili.Emfanekisweni, umkhawulo wamaphuzu we-cross-link usethelwe ku-3.0.(G) Amasayithi okusebenzisana ahlukahlukene phakathi kwezizinda ze-STAT1 ne-OAS3 DI ezibekwe phezulu ezakhiweni zazo zamaprotheni ku-PyMol (isistimu yehluzo yamangqamuzana e-PyMOL, inguqulo 2.0 Schrödinger, LLC.);I-STAT1 (pdb id: 1bf533) kanye ne-OAS3 (pdb id: 4s3n34).) uhlelo.
Ama-isoform amabili e-USP18 achazwe kubantu, iphrotheni yobude obugcwele etholakala kakhulu ku-nucleus, kanye ne-isoform ngaphandle kwesizinda se-N-terminal, i-USP18-sf, esatshalaliswa ngokulinganayo ku-cytoplasm kanye ne-nucleus 36.Ngaphezu kwalokho, i-N-terminus yabikezelwa njengengahlelekile futhi ayidingi umsebenzi we-isopeptidase noma ukubophezela kwe-ISG1537.Ukusebenzelana okuningi okuhlonzwe ocwaningweni lwethu bekutholakala ku-N-terminus yephrotheni, okuphakamisa ukuthi lokhu kuxhumana kuhilela ubude obugcwele be-USP18 (Umfanekiso 4A, D) futhi ngaleyo ndlela kungenzeka kwenzeke ku-nucleus.Ngaphezu kwalokho, idatha yethu iphinda ibonise ukuthi i-N-terminus ikhethekile ekusebenziseni amaprotheni kuya kumaprotheni.Isayithi elibophezelayo le-IFNAR2 litholakala phakathi kwezinsalela ze-312-368, futhi ngokuphawulekayo, awekho amaprotheni ku-complex ehlanganisa lesi sifunda (Fig. 4A) 37,38.Le datha ethathwe ndawonye ibonisa ukuthi isizinda esibophezelayo se-IFNAR2 sisetshenziswa kuphela iphrotheni ye-receptor.Ukwengeza, i-OAS3 ne-ROBO1 kuphela ezitholwe zihlotshaniswa nezizinda ezikhuphuka nomfula we-N-terminus kanye ne-IFNAR2 isayithi elibophezelayo (Umfanekiso 4A).
I-ROBO1 ingeyomndeni omkhulu we-immunoglobulin (Ig) wama-molecule abonisa izimpawu ze-transmembrane futhi iqukethe izizinda ze-Ig ezinhlanu kanye nezizinda ezintathu ze-fibronectin (Fn) esifundeni esingaphandle kweseli.Lezi zizinda ezingaphandle kwe-extracellular zilandelwa isifunda se-membrane-proximal kanye ne-transmembrane helix eyodwa ye-39. Isifunda esingahleliwe se-intracellular sitholakala ku-C-terminus futhi siqukethe i-motifs yokulandelana elondoloziwe ehlanganisa i-protein binding39.Isifunda esisuka ku-amino acid ~ 1100 kuya ku-1600 siphazamisekile kakhulu.Sithole ukuthi i-MX1 isebenzisana ne-ROBO1 ngokusebenzisa i-Ig, Fn, kanye nezizinda ze-intracellular, kuyilapho ukusebenzisana okuningi ne-STAT1 kwenzeka phakathi kwe-CCD yayo, isizinda sesixhumanisi, kanye ne-C-terminus ye-ROBO1 (Fig. 4A,E).Ngakolunye uhlangothi, ukusebenzisana nezifunda ze-DI, DIII, kanye ne-OAS3 zokuxhumanisa kwasakazwa kulo lonke iphrotheni ye-ROBO1 (Fig. 4A).
Umndeni wamaprotheni we-oligoadenylate synthase (OAS) wamukela futhi ubophe i-RNA (dsRNA) enemicu ephindwe kabili ye-intracellular), uba nezinguquko ezihambisanayo, futhi uhlanganise ama-oligoadenylates axhunywe ku-2′,5′ (2-5 As) 40.Kutholwe ukuthi phakathi kwama-OAS amathathu, i-OAS3 ibonisa ukuhlobana okuphezulu kakhulu kwe-dsRNA futhi ihlanganise inani elincane lika-2-5 As, elingenza kusebenze i-RNase L futhi ngaleyo ndlela inciphise ukuphindaphinda kwegciwane okungu-41.Umndeni we-OAS uqukethe izizinda ze-polymerase beta (pol-β) ezifana ne-nucleotide transferase.Ucwaningo lwangaphambilini lubonise ukuthi umsebenzi we-catalytic wesizinda se-C-terminal (DIII) uncike kusizinda esibophezelayo se-dsRNA (DI), esidingekayo ukuze kwenziwe kusebenze i-OAS342.Siqaphele ukuthi izizinda ze-DI ne-DII ze-OAS3 zisebenzisana ne-CCD kanye nendawo encane yokuhlangana phakathi kwe-SH2 ne-STAT1 TAD (Umfanekiso 4A,F).Ukwemboza amasayithi axhumanisa ahlukene esakhiweni seprotheni kwembule ukusebenzisana phakathi kwe-β-sheet ne-DBD STAT1 loop kanye nephakethe elivulekile noma umgodi owenziwe izinsalela 60-75 kusizinda se-DI se-OAS3 (Fig. 4G).Ukuma kwamaphrotheni ku-complex kuphinde kwabonisa ukuthi akukho ukusebenzisana ne-OAS3 okuphazamise ikhono lokubopha i-DNA lesizinda sayo se-DI (Fig. S1A).Ngaphezu kwalokho, isizinda se-N-terminal se-GTPase MX1 sisebenzisana kakhulu nezizinda ze-DI ne-DIII ze-OAS3 (Fig. 4A).Siphinde sabona ukusebenzisana phakathi kwe-OAS1 ne-MX1 kuzo zonke izimpinda ezintathu ezilashwe yi-IFNα, lapho isizinda esisodwa se-OAS1 (naso sisebenza ngendlela eshukumisayo) sihlangane nazo zonke izizinda ezintathu ze-MX1 (Umfanekiso S2A,B).
Amaprotheni e-MX ayingxenye yomndeni omkhulu wama-GTPases afana ne-dynein aqukethe isizinda se-N-terminal GTPase esibopha futhi sikhiphe i-GTP nge-hydrolyze, isizinda esimaphakathi esixhumanisa ukuzihlanganisa, kanye neziphu ye-C-terminal leucine esebenza njenge-GTPase (LZ). ).domain effector domain25,43.I-MX1 ibophezela kumayunithi amancane ama-polymerase egciwane ukuvimba ukulotshwa kofuzo lwegciwane43.Isikrini esibikwe ngaphambilini se-yeast two-hybrid sabonisa ukuthi i-PIAS1 ehlotshaniswa ne-MX1 ivimbela ukusebenza kofuzo lwe-STAT1-mediated ngokuvimbela umsebenzi wokubopha i-DNA futhi futhi inomsebenzi we-SUMO E344,45 ligase.Lapha, sibonisa ukuthi i-MX1 ibophezela ku-STAT1 (Umfanekiso 4C,D), nokho ukuthi lokhu kusebenzisana kuthinta kanjani ukusebenza kofuzo lwe-STAT1-mediated ekuphenduleni ku-IFNα kudinga ukufundwa okwengeziwe.Ngaphezu kwalokho, siphinde sathola ukuthi i-MX1 ihlanganyele ne-IFIT3 kanye ne-DDX60 kuzo zonke izimpinda ezintathu eziphathwe nge-IFNα (Fig. S2C).
I-DDX60 iyi-cytoplasmic helicase eyenziwe nge-IFN okuke kwabikwa ngayo ngaphambilini ukuthi ibambe iqhaza ekonakalisweni kwe-RIG-I-i-independent ye-viral RNA46.Isebenzisana ne-RIG-I futhi ivule ukusayinda kwayo ngendlela eqondene ne-ligand 46. I-DDX60 iqukethe isizinda se-helicase ye-DEXD/H-Box kanye nesizinda se-C-terminal helicase esihlanganisa i-RNA yegciwane kanye ne-DNA47.Iningi lokusebenzelana kwayo ne-MX1 kanye ne-IFIT3 kwenzeka phakathi kwezifunda ezinde ze-N- ne-C-terminal ngaphandle kwezizinda ze-canonical noma ama-motifs (Fig. S2E,F).Nokho, i-MX1 iphinde ihlotshaniswe nesizinda se-helicase ye-DEXD/H-Box (Fig. S2E).Amaprotheni omndeni we-IFIT anamakhophi e-tandem e-helix-turn-helix motif ebizwa ngokuthi i-tetrapeptide repeat (TPR).I-IFIT3 itholwe iyimoduli enhle yokusayina i-RIG-I futhi yingakho iyingxenye ye-MAVS complex.Sekuhlangene, idatha yethu iphakamisa ukuthi i-IFIT3 ne-DDX60 zisebenzisana ngokuyinhloko esifundeni esiphakathi kwe-TPR 3–6 ye-IFIT3 futhi zingadlala indima ekusayineni kwe-RIG-I/MAVS (Fig. S2F).
Uma kubhekwa ukuthi ukuhlola yonke i-proteome kugxile kakhulu ngokwekhompiyutha, sibe sesihlola yonke imininingwane yabantu ye-UniProt ukuze kube khona enye yezimpinda ezilashwe yi-IFNα.Kulesi sifaniso, sithole amanethiwekhi ambalwa okusebenzisana athembeke kakhulu e-HLA-A.Ukuhlaziywa kwezindlela zamaprotheni ezikhonjwe yi-MS/MS spectra kubonise ukuthi ukucubungula kwe-antigen okusekelwe ku-MHC-I-iyindlela eyinhloko eyenziwa yi-interferon (Fig. 3D).Ngakho-ke, sigxile ekutadisheni ukusebenzisana kwamaprotheni ama-molecule e-MHC-I ngezinga eliphezulu lokuzethemba kuwo wonke amasampula axhumene.I-HLA iqukethe izizinda ezingu-α1, α2 kanye ne-α3 namaketanga okukhanya, futhi i-microglobulin β2 (β2m) iyiprotheni ehlala njalo ye-chaperone49.Uma isihlanganiswe ku-endoplasmic reticulum, i-HLA ayizinzile lapho kungekho peptide ligands50.I-groove ebopha i-peptide yakhiwe isizinda se-α1 ne-α2 esine-polymorphic kakhulu futhi esingahlelekile ngendlela engeyona ye-peptide kanye nesizinda esingaphansi kwe-polymorphic α351.Ebukhoneni be-IFNα, sithole izingxube ezimbili ze-HLA-A: eyodwa isebenzisana ne-HMGA1 ne-H2B (Umfanekiso 5, Ithebula le-S3) kanti enye isebenzisana ne-MDN1, LRCH4 ne-H2B (Umfanekiso 6).
I-IFNα ingenisa inethiwekhi yokusebenzisana ye-HLA-A ne-H2B (H2BFS) kanye ne-HMGA1.(A) Isakhiwo se-2D (ekhiqizwe ngesofthiwe ye-SIM-XL) esibonisa izinhlobo ezihlukene zokusebenzisana kunxanxathela ye-H2B-HLA-A-HMGA1: i-interlink (blue), i-interlink (bomvu) nesixhumanisi esisodwa (esimnyama)..Izizinda zobunikazi obuhlukahlukene zinekhodi yombala32: H2B (histone; 2–102) kanye ne-MHC-I (MHC_1; 25–203, iqembu C1; 210–290 kanye ne-MHC_I_C; 337–364).Umkhawulo we-cross-link ubekwe ku-3.5.Iziqephu zamachashazi zibonisa amasayithi okusebenzelana e-HLA-A ne-H2B (B) ne-HMGA1 (C), kanye nezindawo zokusebenzisana ze-K noma ze-S phakathi kwama-peptide amabili.Emfanekisweni, umkhawulo wamaphuzu we-cross-link usethelwe ku-3.0.(D) Ubudlelwano phakathi kwamaprotheni aboniswe ezakhiweni zamaprotheni e-H2B, HLA-A, kanye ne-HMGA1 kuhlelo lwe-PyMOL.Lezi zakhiwo zenziwe imodeli kusetshenziswa iseva ye-Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) futhi izakhiwo zesifanekiso samaprotheni e-H2B, HLA-A kanye ne-HMGA1 bekuyi-1kx552, 1kj349 kanye ne-2eze55, ngokulandelana.
I-IFNα ingenisa inethiwekhi yokusebenzisana ye-HLA-A ne-H2B (H2BFS), MDN1 kanye ne-LRCH4.(A) Izixhumanisi ze-Intramolecular (red) kanye ne-intermolecular (blue) ezethulwa kumephu esebenzisanayo ye-2D (ekhiqizwe ngesofthiwe ye-SIM-XL) ene-MDN1 emelelwe njengombuthano.Umkhawulo we-cross-link ubekwe ku-3.5.Izizinda zobunikazi obuhlukene zinekhodi yombala32: H2B (histone; 2–102), MHC-I (MHC_1; 25–203, iqembu C1; 210–290 kanye ne-MHC_I_C; 337–364) kanye ne-LRCH4 (LRR_8 (68–126), LRR_8 (137–194) kanye ne-CH (535–641)).(B) Ubudlelwano phakathi kwamaprotheni aboniswe ezakhiweni zamaprotheni e-H2B, HLA-A, LRCH4, kanye ne-MDN1 kuhlelo lwe-PyMOL.Lezi zakhiwo zenziwe imodeli kusetshenziswa iseva ye-Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) enezakhiwo zesifanekiso 1kx552, 1kj349, 6hlu62 kanye ne-6i2665 yamaphrotheni e-H2B, HLA-A, LRCH4 kanye ne-MDN1, ngokulandelana.Iziqephu zechashazi ezibonisa amasayithi e-K noma e-S e-HLA-A ene-H2B (C), LRCH4 (D), kanye ne-MDN1 (E).Ezindaweni, umkhawulo wamaphuzu we-cross-link wawusethwe ukuze ube ngu-3.0.
Ngokungeziwe ekugcineni ubuqotho be-genome, i-histone H2B nayo iyabandakanyeka ekulawuleni ukuloba.Iphrotheni ye-H2B iqukethe isizinda se-histone esimaphakathi (HFD) esakhiwe ama-α-helice amathathu ahlukaniswe amalophu kanye nomsila we-C-terminal 41,52.Iningi lokusebenzelana ne-H2B kwenzeka ku-α1 helix, ehlinzeka nge-trimerization nge-HFD heterodimer (Fig. 5A,B).Nakuba ama-lysins ebandakanyeka ekubopheni i-DNA, amanye ama-lysins nawo ayizindawo ze-acetylation noma ze-methylation.Isibonelo, izinsalela i-K43, i-K46, ne-K57 ezivela ku-H2B azibandakanyi ekubopheni i-DNA eqondile, kodwa ziyizinjongo zokuguqulwa okuhlukahlukene kwangemva kokubhala53.Ngokufanayo, izinsalela ze-K44, i-K47, ne-K57 ku-H2B zingadlala enye indima lapho kukhona i-IFNα, kuhlanganise nokusebenzisana namanye amaprotheni (Fig. 5A, B).Ngaphezu kwalokho, i-extrachromosomal histone H2B yenza kusebenze impendulo yokuzivikela komzimba ezinhlotsheni zamaseli ahlukahlukene, esebenza njengenzwa ye-cytosolic ukuze ithole izingcezu ze-DNA (dsDNA) ezinemicu ephindwe kabili ezithathwe kuma-ejenti athathelwanayo noma amaseli alimele54.Lapho kukhona amagciwane e-DNA, ukuncipha kwe-H2B kuvimbele ukukhiqizwa kwe-IFN-β kanye ne-STAT154 phosphorylation.I-H2B yaziwa nangokuthi ingena iphinde iphume ku-nucleus ngokushesha kunamanye ama-core histones54.Ukusebenzisana kwe-H2B ne-MDN1 kanye ne-LRCH4 kuphinde kwabonwa kumasampuli akhethiwe anganakiwe.Sithole ukuthi i-HLA-A isebenzisana ne-H2B kuwo wonke amasampula alashwe nge-IFNα kanye nakusampula eyodwa ephindayo engelashiwe.Le datha ibonisa indima ye-H2B komunye umsebenzi wokuphila ozimele ngaphandle kokulawula okulotshiweyo.
I-HMGA1 (iqembu eliphezulu lokuhamba i-AT-Hook 1), i-nucleoprotein encane ecebile kuma-amino acids akhuthaza izifo, ikhonjwe ngokuhambisana ne-HLA-A.Inomsila we-C-terminal one-acidic kanye nama-DBD amathathu ahlukene abizwa ngokuthi ama-AT hook ngoba abophezela emgodini omncane wesifunda esicebile nge-AT ku-dsDNA55,56.Lokhu kubophezela kubangela ukuthi i-DNA igobe noma iqonde, okuvumela izici zokulotshwa kwe-canonical ukuthi zifinyelele ukulandelana kwayo kokuvumelana.Umsila we-C kukholakala ukuthi uhileleke ekusebenzisaneni kwamaprotheni namaprotheni kanye nokubuthwa kwezinto ezibhalwe phansi, njengoba izinguquko eziguquguqukayo zokususa i-C-terminal zingakwazi ukuqalisa ukuloba57.Ngaphezu kwalokho, lesi sizinda siqukethe izingosi ze-phosphorylation eziningana ezilondoloziwe ezaziwa ngama-substrates we-kinases 58.Siqaphele ukusebenzisana kwe-HLA-A ne-H2B ne-HMGA1 ngaphandle kwesizinda se-C-terminal, okuphakamisa ukuthi isizinda se-C-terminal sisetshenziselwa kakhulu ukubophezela kwesici sokuloba (Fig. 5A, C).Amaprotheni e-HMGA ancintisana ne-histone H1 ukuze abophe i-adaptha DNA, ngaleyo ndlela andise ukufinyeleleka57.Ngokufanayo, kubonakala sengathi i-HMGA isebenzisana ne-histone H2B kanye ne-DNA yokuxhumanisa emqhudelwaneni ne-histone H1.I-HMGB1 idala ukuvezwa kwe-HLA-A, -B, kanye -C kumaseli e-dendritic, okuholela ekusebenzeni kwawo59, kodwa ukusebenzisana phakathi kwe-HMG ne-HLA akuzange kubikwe ngaphambilini.Sithole ukuthi i-HMGA1 isebenzisana nezizinda ze-α1 kanye ne-α3 ze-HLA-A, ngokuningi kokusebenzelana ngaphandle kwe-3 DBD yayo (Umfanekiso 5A, C).Ezandleni zethu, i-HLA-A itholwe yenziwe yasendaweni ku-nucleus (idatha engabonisiwe), futhi uma kubhekwa ukuthi i-H2B ne-HMGA1 nazo zikhona ku-nucleus, lokhu kusebenzisana kungenzeka kwenzeke ku-nucleus.Izengezo ezithile zikalwa phakathi kwe-H2B, i-HLA-A, ne-HMGA1 ziboniswa kuMfanekiso 5D.
Ukusebenzelana okuningi kwe-HLA-A namanye amaprotheni kwenzeka ngaphakathi kwesizinda sayo se-α1 ne-α2 kanye nesizinda se-C-terminal esiphazamisekile (Fig. 6).Kwesinye salezi zibonelo, sithole ukuthi i-HLA-A isebenzisana nomsila we-N-terminal ophazamisekile we-LRCH4 (Umfanekiso 6A,D).I-LRCH4 ilawula ukusebenza kwe-TLR4 kanye nokungeniswa kwe-cytokine ye-LPS, ngaleyo ndlela imodela impendulo yokuzivikela engaphakathi kwe-immune60,61.Iphrotheni ye-membrane eneziphindaphinda eziyisishiyagalolunye ze-leucine-rich (LRRs) kanye ne-callodulin (CH) i-homology motif ku-ectodomain yayo, ilandelwa isizinda se-transmembrane (TMD) 60, i-62.Izizinda ze-CH ziye zabikwa ukuthi zixhumanisa ukusebenzisana kwamaprotheni-amaprotheni 60.Ingxenye engaba ngu-300 yama-amino acid phakathi kwezizinda ze-LRR ne-CH iyafinyeleleka ngokuqhathaniswa kodwa iphazamisekile.Ngokusekelwe emsebenzini wezifunda eziphazamisekile njengabalamuli bamanethiwekhi amaprotheni-amaprotheni kanye nokuthuthwa kwe-vesicular 63, sithole ukuthi ukusebenzisana okuningi kwamaprotheni kwenzeka ezindaweni ezingahlelekile.Ukusebenzisana ne-MDN1 kwasakazwa kulo lonke ubude beprotheni, okuhlanganisa izizinda ze-LRR1, LRR6, CH, nezifunda ezingahleliwe, kuyilapho i-H2B iboshelwe ikakhulukazi kusizinda se-CH (Fig. 6A, B).Ngokuphawulekayo, akukho ukusebenzisana okubandakanya i-TMJ, okuphakamisa ukucaciswa kwendlela ye-CLMS (Umfanekiso 6A, B).
I-MDN1 nayo ikhonjwe njengengxenye yenethiwekhi ye-HLA-A protein (Umfanekiso 6A).Ingowomndeni we-AAA wamaprotheni (ama-ATPases ahlotshaniswa nemisebenzi ehlukene).Lesi yisizinda esifanayo se-N-terminal AAA esihlela sibe yiringi ye-hexameric futhi sisuse isici sokuhlanganisa ku-60S 64 ribosomal subunit.ibonakala ifana ne-dynein64,65,66.Ngaphezu kwalokho, isifunda esicebile se-Asp/Glu silandelwa yisizinda se-MIDAS (isayithi elincike ku-ion yensimbi).Ngenxa yobukhulu obukhulu be-MDN1 (cishe ama-amino acid angama-5600) kanye ne-homology yayo elinganiselwe enamaprotheni afundwe kahle, kuncane okwaziwayo ngesakhiwo nokusebenza kwayo kubantu.Sihlonze i-HLA-A, H2B, ne-LRCH4 njengozakwethu ababophayo be-MDN1 futhi saveza umumo wabo njengama-protein complexes ku-PyMol (Fig. 6A,B).Lawa maprotheni amathathu asebenzisana nesizinda se-AAA, isizinda sokuxhumanisa esifana ne-dynein, kanye nesizinda se-MIDAS MDN1.Embikweni wangaphambilini, ukuhlanzwa okuhambisanayo kwamaprotheni okudla kuhlonze i-MDN1 njengephrotheni ehlotshaniswa ne-histone H2B67.Ngaphezu kwalokho, ucwaningo lwakamuva luphinde lwabika ukusebenzisana phakathi kwe-MDN ne-HLA-B kumaseli e-HCT116 kusetshenziswa i-affinity-purified mass spectrometry, esekela lokho esikutholile68.Ukuhlonzwa kwale nkimbinkimbi kumasampuli aphathwe i-IFNα kuphakamisa indima ye-MDN1 ekuboniseni i-interferon.
Ngenxa yokuthi ufuzo lwe-HLA lune-polymorphic kakhulu, sikhiphe ukulandelana kufundeka imephu HLA-A, -B, kanye -C kusukela kudatha elandelanayo ye-RNA yamaseli e-Flo-1 (idatha ayiboniswa).Ukulandelana kwe-Peptide okuhambisana nokufunda kokulandelana kwembula umehluko omkhulu phakathi kwe-HLA-A, -B, kanye -C ezifundeni lapho ama-peptide axhumene kakhulu atholakala ku-HLA-A (Figure S3).Ngaphezu kwalokho, asizange sibone ukuxhumanisa amaprotheni-kuya-iphrotheni ama-molecule e-HLA-B/C anamaprotheni e-H2B/HMGA1/MDN1/LRCH4.Lokhu kuphakamisa ukuthi ukusebenzisana kwamaprotheni okutholakala phakathi kwe-HLA-A, MDN1, LRCH1 ne-HMGA1 kucaciswe nge-HLA-A.Ngaphezu kwalokho, ukuhlaziywa kwe-proteomic kwamasampuli angewona ama-crosslinked (Ithebula S4) kubonise ukuthi i-HLA-A inokufakwa okuphezulu okulandelanayo uma kuqhathaniswa ne-HLA-B noma i-HLA-C.Ama-peptide akhonjwe i-HLA-A ayephezulu ngokuqina kuwo womabili amasampula alashwe nge-IFNα nangelashiwe.
Ukuqinisekisa ukuthi ukusebenzisana okuphawulwe lapha akubangelwanga ukuxhuma okungaqondile okungaqondile kwamaphrotheni amabili eduze nendawo, siphinde saqinisekisa izici ezimbili ezintsha ezisebenzisanayo ze-HLA-A ngokwenza ama-co-immunoprecipitation assessment.Ukusebenzisana kwe-HLA-A ne-MDN1 engapheli kanye ne-H2B kutholwe kuwo womabili amaseli e-Flo-1 alashwe nge-IFNα nangelashwa (Umfanekiso 7, Umfanekiso S4).Siqinisekise ukuthi i-HLA-A yathwetshulwa yi-H2B kuma-immunoprecipitates nokuthi lokhu kuhlotshaniswa kwakungenxa yokwelashwa kwe-IFNα njengoba i-HLA-A yayingekho kumasampuli e-immunoprecipitate asuka kumaseli angalashiwe (Umfanekiso 7A).Nokho, idatha yethu iphakamisa ukuthi i-IFNα ilawula ngokuhlukile i-HLA-A ku-H2B ne-MDN1.I-IFNα idala ukuhlobana phakathi kwe-H2B ne-HLA-A, kodwa yehlisa ukuhlotshaniswa kwayo ne-MDN1.Sithole ukuthi i-MDN1 ibihlotshaniswa ne-HLA-A kuzilawuli, futhi ukungezwa kwe-IFNα kwehlise lokhu kusebenzisana ngaphandle kokungeniswa kwe-MDN1 nge-IFNα (Umfanekiso 7B,C).Ngaphezu kwalokho, i-HLA-A immunoprecipitation ithwebule i-H2B kumaseli angu-A549 (Fig. S4), iphakamisa ukuthi lokhu kusebenzisana kuzimele ohlotsheni lweseli.Ihlanganiswe ndawonye, ​​le miphumela isekela ukusebenzisana kwe-interferon-mediated kwe-HLA-A ne-H2B ne-MDN1.
I-HLA-A ihlanza ngokubambisana i-H2B ne-MDN1.I-H2B (A) emele i-endogenous endogenous H2B (A) kanye ne-MDN1 (B) ama-immunoblots ayengavimbeli umzimba kumaseli e-Flo-1 e-IFNα futhi aphenywa amasosha omzimba abonisiwe.I-Mouse kanye ne-rabbit IgG yasetshenziswa njengokulawula okungalungile.(C) Amanani ahlobene (okufakwayo) ama-antigen ahlukene aboniswa ama-immunoblots aphenywa ngokumelene namasosha omzimba abonisiwe, i-β-actin yasetshenziswa njengokulawula ukulayisha.
Izakhiwo zesakhiwo esisodwa samanethiwekhi axhumene ne-interferon athembeke kakhulu, i-H2B-HLA-A-HMGA1, zaphenywa.Sisebenzise imodeli ye-molecular dynamics njengendlela ehlukile yokuqonda ukuguquguquka kwe-conformational yamaprotheni ahilelekile kule nkimbinkimbi (Umfanekiso 8).Okucatshangelwayo okuvela kudatha ye-CLMS kuphakamisa ukuthi kungenzeka ukuhambisana okuhlukile kwe-H2B, HLA-A, ne-HMGA1 amaprotheni.Ngakho-ke, ama-complexes alandelayo angahle amodelwe ngendlela yokuncibilikisa: i-H2B-HLA-A, i-HMGA1-HLA-A, ne-H2B-HLA-A-HMGA1.Isikrini sokuqala se-protein-protein docking sisebenzisa i-MOE (i-Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) iphakamise ukuhlangana okungenzeka okuhluke phakathi kwalawa maprotheni (Fig. 8A).Ukubona ngeso lengqondo i-docking protein complex kwembule ukusebenzisana okuningana kanye nokuhambisana okungenzeka (Umfanekiso 5A, 8).Ngakho-ke, ukuguqulwa okukodwa okungenzeka kuboniswe kuMfanekiso 8A (onezixhumanisi eziphambene) futhi kwabuye kwahlolwa kusetshenziswa ipayipi lokumodela le-MD.Ngaphezu kwalokho, ukubophezela kwe-H2B noma i-HMGA1 ku-HLA-A kugqamisa ukuhlobana okuphezulu kwe-H2B kwe-HLA-A (Fig. 8A).
I-Conformational Dynamics yamanethiwekhi angaba khona phakathi kwe-H2B (H2BFS)-HLA-A, HMGA1-HLA-A, kanye ne-H2B-HLA-A-HMGA1 complexes.(A) Iphaneli yesokunxele imephu ye-2D (ekhiqizwe ngesofthiwe ye-SIM-XL) yeziphambano ze-intramolecular (red) kanye ne-intermolecular (blue) (i-crosslink cutoff isethwe ukuze ithi 3.5).Ngaphezu kwalokho, izinsalela ezixhumene ngokuphambanayo ezikhonjiwe zilebulwe ezakhiweni zamaphrotheni e-H2B, HLA-A, kanye ne-HMGA1.Ukuhambisana okuhlobene kwalawa maprotheni kwakhishwa kusetshenziswa ipayipi lokumisa elisetshenziswe kuphakheji ye-MOE.Iphaneli elingezansi kwesokunxele libonisa ukuhlangana okungaba khona okuhlukahlukene kwe-H2B-HLA-A kanye ne-HMGA1-HLA-A enenhlanganisela ehlukene yokubopha amaprotheni-amaprotheni (GBVI/WSA dG; kcal/mol).(B) Ukuchezuka okujwayelekile (RMSD) kokuma kwe-athomu (ngaphandle kwama-athomu e-hydrogen) kusakhiwo sephrotheni ngasinye.(C) Ukusebenzisana kwe-Intermolecular protein-protein hydrogen bond kusuka kuzakhiwo ezihlukahlukene ezilingisayo kucatshangelwa ukusebenzisana okuqondile kobude besikhathi ≥ 10 ns.Ibanga lokunqamula le-h-bond donor-acceptor limiswe ukuze libe ngu-3.5 Å, futhi i-engeli yokunqamula ye-donor-H-acceptor isethwe ukuze ithi ≥ 160°–180°.(D) Izinsalela ezinelebula ezakha ukusebenzisana kwe-HLA-A protein-protein nabalingani bazo abahlukene, okuhlanganisa ama-≥ 20 ns, akhishwe ku-dummy HLA-A-H2B kanye ne-HLA-A-HMGA1 complexes.Izakhiwo zamaphrotheni zimelela ukwakheka okumaphakathi kwe-100 ns MDS.(E) Ukusebenzisana phakathi kwenhlanganisela ye-HLA-A-H2B kanye ne-HLA-A-HMGA1 uma kuqhathaniswa nokusebenzisana okulandelelwa ngokulingisa kwe-H2B-HLA ngaphezu kuka-100 ns ngokusekelwe kusayithi lokusebenzisana le-K noma le-S phakathi kwama-peptide amabili.Izinkimbinkimbi /HMGA1-HLA-A/H2B-HLA-A-HMGA1.I-threshold value yokuhlolwa kwe-cross-link yabekwa ku-3.0, futhi ukusebenzisana okuqondile okuvela ku-MDS okuthatha ≥ 10 ns kwacatshangelwa.Izakhiwo zamaprotheni zibonwe ngeso lengqondo kusetshenziswa amaphakheji e-BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) kanye neMolecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).
Ukuzinza kwama-molecule e-HLA-A ngokuhamba kwesikhathi (ukuchezuka okujwayelekile; i-RMSD noma ukuchezuka okujwayelekile; i-RMSF) kubonise ukuthi ubukhona bamaprotheni e-H2B noma e-HMGA1 kumakhompleksi azinzile i-HLA-A (Umfanekiso 8B, Umfanekiso S5).Iphrotheni ye-HMGA1 ibophezela ngokuqinile kusayithi ye-B2M ye-HLA-A, idala ukuzinza kwama-amino acid e-HLA-A ku-HLA-A-HMGA1 noma i-H2B-HLA-A-HMGA1 complex (Umfanekiso 8B, Umfanekiso S5).ikakhulukazi, izinsalela ze-HLA ~60-90 kanye ne-~180-210 zitholwe zingaguquguquki kancane phambi kwe-H2B (FIG. 8B).I-H2B ne-HMGA1 zibonise ukubophezela okungcono ku-HLA-A ku-H2B-HLA-A-HMGA1 eyinkimbinkimbi uma kuqhathaniswa ne-HLA-A ebophezela ku-H2B noma i-HMGA1 iyodwa (Umfanekiso 8C,D; Ithebula S5).Izinsalela ezihilelekile ekubopheni i-hydrogen (i-MD eyimodeli yokuhlala ephezulu ≥ 10 ns) iqondana nezindawo zokusebenzisana ze-CLMS (izinsalela ze-K noma ze-S) endaweni eyinkimbinkimbi, okuphakamisa ukuthi ukusebenzisana okukhonjwe yi-CLMS kunokwethenjelwa kakhulu.Ukuthembeka (Fig. 8E).Kumodeli ye-CLMS ne-MD, izinsalela ze-HLA-A phakathi kwe-190-210 kanye ne-200-220 amino acids zitholwe zibopha i-H2B ne-HMGA1, ngokulandelana (FIG. 8E).
Ukusebenzisana kwamaprotheni-amaprotheni kwenza amanethiwekhi esakhiwo ashukumisayo axhumanisa ukuxhumana kwe-intracellular ekuphenduleni izisusa ezithile.Ngenxa yokuthi izindlela eziningi ze-proteomics zithola izinguquko ezingeni eliphelele lesimo sephrotheni, amandla okusebenzisana kwamaprotheni-protein adinga amathuluzi engeziwe ukuze athwebule ukuxhumana okubophayo, futhi i-CLMS ithuluzi elilodwa elinjalo.Uhlelo lokubonisa i-interferon luyinethiwekhi ye-cytokine evumela amangqamuzana ukuba aphendule ezinhlobonhlobo zezimpawu zemvelo ze-pathogenic kanye ne-intrinsic pathological, okuholela ekufakweni kwama-subsets amaprotheni e-interferon-inducible.Sisebenzise i-CLMS ukuze sinqume ukuthi ukusebenzisana kwenoveli kwamaprotheni-amaprotheni kungabonakala yini phakathi kwephaneli lamaphrotheni enziwe yi-interferon.Ukuhlaziywa kokuxhumanisa amaprotheni omhlaba kumodeli yeseli ye-Flo-1 esabelayo e-interferon kusetshenziswe ukuthwebula ama-protein complexes.Ukukhishwa kwama-peptide e-tryptic kusuka kumaseli angaxhumekile futhi axhumene kuvumela ukubalwa kwe-peptide, ukucebisa indlela, kanye nokusatshalaliswa kobude be-peptide ngokuqina okuchaziwe kwe-LFQ.Amaprotheni e-Canonical interferon-inducible ahlonzwe njengokulawula kwangaphakathi okuhle, kuyilapho ama-adducts amasha e-intermolecular and intramolecular cross-linked of canonical interferon-inducible proteins afana ne-MX1, UP18, OAS3 kanye ne-STAT1 abonwa.Kuphenywe izici ezihlukahlukene zesakhiwo nokusebenzisana ezindaweni zokusebenza.
Ukusebenzisana phakathi kwe-HLA-A, MDN1 kanye ne-H2B kutholwe ngokuvimba amasosha omzimba kumaseli e-Flo-1 kanye ne-A549 alashwe futhi angelashwa nge-IFNα.Imiphumela yethu igqamisa ukuthi i-HLA-A ihlanganisa ne-H2B ngendlela encike ku-IFNα.Umsebenzi wethu umele indlela ethokozisayo yokuhlola okwengeziwe kokusebenza ndawonye kwalezi zakhiwo ezimbili.Kungajabulisa futhi ukunweba indlela ye-CLMS kuphaneli yemigqa yeseli ukuze kuhlonzwe ukusebenzisana kwamaprotheni e-interferon-mediated azimele ohlobo lweseli.Ekugcineni, sisebenzise imodeli ye-MD njengenye indlela yokuqonda ukuguquguquka kwe-conformational kwamaprotheni ahilelekile ku-H2BFS-HLA-A-HMGA1 complex, elandelela izingxoxo ze-intramolecular kanye ne-intermolecular cross-tals.Okucatshangelwayo okuvela kudatha ye-CLMS kuphakamisa ukuthi kungenzeka ukuhambisana okuhlukile kwe-H2BFS, HLA-A, ne-HMGA1 amaprotheni.Ukuvumelana okungenzeka okuhlukile phakathi kwalezi zakhiwo zamaphrotheni ezibambayo kwembule ukusebenzisana okuningana okufana nalokho okubonwe kudathasethi ye-CLMS.Enye yamandla amakhulu endlela yethu ukuthi ivumela ukukhonjwa okulula kokusebenzelana kofuzo lwe-polymorphic kakhulu njenge-HLA, ngakho-ke kuzojabulisa ukutadisha ukusebenzisana kwamaphrotheni aqondene ne-HLA haplotype okunzima ukuwafunda.Uma sihlanganiswa, idatha yethu ibonisa ukuthi i-CLMS ingasetshenziswa ukwandisa ukuqonda kwethu amanethiwekhi okusayinda abangelwa yi-interferon futhi inikeze isisekelo sokutadisha izinhlelo eziyinkimbinkimbi ze-intercellular ku-tumor microenvironment.
Amaseli e-Flo-1 atholwe ku-ATCC futhi agcinwa ku-DMEM (Gibco) engezwe ngo-1% we-penicillin/streptomycin (Invitrogen), u-10% we-fetus bovine serum (Gibco) futhi agcinwe ku-37°C naku-5% CO2.Ukufukamela.Amaseli akhuliswe afinyelela ku-70-80% wokuhlangana ngaphambi kokuphathwa nge-IFNα14 (ekhiqizwe i-Edinburgh Protein Production Facility).Wonke amanye amakhemikhali nama-reagents athengwe kwa-Sigma Aldrich ngaphandle uma kuphawulwe ngenye indlela.
Amaseli e-Flo-1 akhuliswe kumapuleti angu-6 futhi ngosuku olulandelayo amaseli aphathwa nge-10 ng / ml IFNα14 amahora angu-24 kuya cishe ku-80% confluence.Amaseli ahlanzwa kathathu nge-PBS futhi ahlanganiswa ne-DSS esanda kulungiswa (i-Thermo Fisher Scientific) (encibilikiswe ku-DMSO) ku-PBS imizuzu engu-5 ngo-37° C. kuya ekuhlanganiseni kokugcina kuka-0.5 mM.Ukusabela kwe-DSS crosslinking kwathathelwa indawo yi-PBS futhi i-DSS esele yacinywa ngokungeza u-20 mM Tris (pH 8.0) ku-PBS imizuzu engu-15 ku-37°C.Amaseli aqoqwe ngokukhuhla futhi aqoqwe kumashubhu okubopha aphansi (Axygen).
I-cell pellet yahluzwa ngo-300 µl we-urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) imizuzu engu-30 ekamelweni lokushisa nokunyakaziswa ngezikhathi ezithile.Zonke izinyathelo ze-centrifugation zenziwe ku-14,000 xg ku-8°C.I-Centrifuge i-lysate imizuzu engu-10 bese udlulisela i-supernatant ku-tube entsha.Izinhlayiya ezisele ezicacile zahlakazwa ku-150 μl wesibili se-lysis buffer (2 M urea, 2% (w/v) SDS (sodium dodecyl sulfate)) imizuzu engu-30 noma ngaphezulu kuze kutholakale isisombululo se-aqueous homogeneous.I-lysate yayiyi-centrifuged imizuzu engu-20 kanti i-supernatant yayixubene ne-lysate etholwe esinyathelweni sangaphambilini.Ukugxila kwamaprotheni kuye kwahlolwa kusetshenziswa i-Micro BCA assay (Thermo Fisher Scientific) ngokuya ngemiyalo yomkhiqizi yezinqubo ze-microplate.Amasampula aqandiswe ngokushesha ku-nitrogen ewuketshezi futhi agcinwe ku -80°C.
Cishe i-100 μg yeprotheyini encibilikayo exhumene ne-soluble yacutshungulwa kusetshenziswa i-modified filtration sample preparation protocol (FASP) njengoba kuchazwe u-Wisniewski et al.69 Kafushane, iphrotheni ixhunyaniswe no-200 µl we-urea buffer (8 M urea ku-0.1 M Tris, pH 8.5), i-vortex futhi ihhafu.Zonke izinyathelo ze-centrifugation zenziwe ku-14,000 xg ku-25°C.Ingxenye yokuqala ye-protein lysate exhunywe esiphambanweni idluliselwe kudivayisi yesihlungi se-10 kDa Microcon centrifugal efakwe i-Ultracel-10 membrane (Merck), elandelwa yi-centrifugation kusihlungi imizuzu engu-25.Bese wengeza ingxenye yesibili yephrotheni kusihlungi bese uphinda izinyathelo ezifanayo.Ukutholwa kwamaprotheni kwenziwe ngokungeza i-100 μl ye-17 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) ku-urea buffer.Ukubuyisela kuvuselelwe ku-thermomixer ku-600 rpm imizuzu engu-30 ku-37 ° C.Ngaphezu kwalokho, ikholomu yayiyi-centrifuged futhi iphrotheni encishisiwe exhunywe ngokuphambanayo yafakwa alkylated kusetshenziswa i-100 μl ye-50 mM iodoacetamide ku-urea buffer.Ukusabela kwe-alkylation kwenziwa ekamelweni lokushisa imizuzu engu-20 ebumnyameni.Zungezisa ikholomu, geza izindonga zekholomu izikhathi ezi-3 nge-100 µl urea buffer, bese uyi-centrifuge.Ukuhlinzwa okufanayo kwenziwa izikhathi ezi-3 kusetshenziswa i-100 μl ye-100 mM ammonium bicarbonate.Ngaphambi kwe-trypsinization, shintsha ishubhu yokuqoqa ufake entsha.Engeza isigcinalwazi sokugaya esiqukethe 50 mM ammonium bicarbonate kanye ne-trypsin engu-1 µl exutshwe ku-trypsin buffer (Promega).Isilinganiso se-trypsin neprotheni sagcinwa cishe ku-1:33, futhi ukusabela kokugaya kwafukanyelwa ngobusuku obungu-37° C. ekamelweni elinomswakama.I-peptide exhumene yakhishwa kusihlungi nge-centrifugation imizuzu engama-25.Ukutholwa kwe-Peptide kwathuthukiswa ngokungeza u-50 μl we-0.5 M NaCl kusihlungi, kulandele i-centrifugation imizuzu engu-25.
Amakholomu we-C18 Micro Spin (I-Harvard Apparatus) asetshenziselwa ukukhipha usawoti e-tryptic peptide elandela isimiso esichazwe u-Bouchal et al.70 ngokulungiswa okuncane.Kafushane, amakholomu e-C18 spin enziwe asebenza ngokugeza okuthathu kwe-0.1% ye-formic acid (FA) ku-acetonitrile (AcN) (Merck) kanye nokugeza okubili kwe-0.1% FA.Ikholomu ifakwe amanzi ngo-0.1% FA imizuzu engu-15.Layisha amasampula kuma-spin columns bese uwageza izikhathi ezi-3 nge-0.1% FA.Ama-peptide asuliwe ahlanjululwa ngokulandelana nge-gradient elandelanayo kusetshenziswa u-50%, 80% kanye no-100% AcN ku-0.1% FA.Amasampuli omisiwe kusigxivinisi se-SpeedVac Plus (Eppendorf) kuze kube yilapho uketshezi olusele lunyamalala ngokuphelele.Ama-peptide ahlutshiwe ahlakazwa ku-100 μl ka-0.08% we-trifluoroacetic acid ku-2.5% AcN futhi ukugxila kukalwa nge-NanoDrop 2000 (Thermo Scientific).Cishe i-1 μg ye-peptide exhumene ngesampula ijovwe ohlelweni lwe-LC-MS/MS.
Ama-peptide axhumene nesiphambano ahlukaniswa ngohlelo lwe-UltiMate 3000 RSLCnano LC (Thermo Scientific) oluxhunywe ku-Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Ama-peptide axhumene ngokuphambana aqoqwe ku-ID engu-300 µm, ikholomu yokuthwebula engu-5 mm ubude µ-ngaphambi kwekholomu C18 epakishwe nge-C18 PepMap100 sorbent kanye ne-5 µm PepMap sorbent (Thermo Scientific).Layisha ukugeleza kwepompo okusethwe ku-5 µl/min 0.08% trifluoroacetic acid encibilike ku-2.5% AcN.Ama-peptide axhumene ngokuphambana ahlukaniswe kukholamu yesilica ehlanganisiwe yokuhlaziya enobubanzi obungaphakathi obungu-75 μm nobude obungu-150 mm, egcwaliswe nge-2 μm PepMap sorbent (Thermo Scientific).Izigaba zamaselula A no-B zazihlanganisa u-0.1% FA emanzini kanye no-0.1% FA ku-acetonitrile, ngokulandelanayo.I-gradient iqala ku-2.5% B bese ikhuphuka ngokulandelana iye ku-40% B ngaphezu kwemizuzu engu-90, bese ifinyelela ku-90% B emizuzwini emi-2 elandelayo.Ukwakheka kwesigaba seselula kuye kwagcinwa ku-90% B imizuzu eyi-10 kwase kwehla ngokulandelanayo kwaba ngu-2.5% B emaminithini angu-2.Ikholomu ilinganiswe ku-2.5% B imizuzu engu-8 ngaphambi komjikelezo olandelayo.Ama-peptide axhumene ngokuphambana akhishwe kukholamu yokuhlaziya enziwe i-ionization emthonjeni we-nanoelectrospray ionization (NSI) futhi afakwa ku-Exploris 480 mass spectrometer (Thermo Scientific).
I-Orbitrap Exploris 480 mass spectrometer isebenze kumodi yokuhlanganisa idatha evumayo.Ukuskena okugcwele kwenziwa kumodi yesigaba ngokulungiswa okungu-120,000 ngezilungiselelo zobubanzi ukusuka ku-m/z 350 Th kuya ku-m/z 2000 Th.Okuhlosiwe okujwayelekile kwe-AGC kwamiswa ku-300% ngesikhathi sokufaka esiphezulu esingu-50ms.Ukutholwa kokuphakama kwe-Monoisotopic sekusungulwe kuma-peptide.Ipharamitha yokuphumuza umkhawulo isethwe ukuze ibe iqiniso uma kutholakala izandulela ezimbalwa kakhulu.Amandla amancane we-ionic wesandulela amiswe ukuze abe ngu-5.0e3 futhi ishaja eyandulelayo ifika ku-+8 ifakiwe ekuhlolweni.
Isikhathi somjikelezo phakathi kokuskena okukhulu kumodi yokuxhumanisa idatha sisethwe ukuze sibe amasekhondi angu-2.5.Ukukhishwa kwesisindo esinamandla kusethelwe kumasekhondi angu-20 ngemva kokuhlukana kokuqala kwe-ion eyandulelayo.Iwindi lokuhlukanisa lesandulela lisethelwe ku-2 Th.Uhlobo lwamandla ajwayelekile okushayisana ngemodi yamandla okushayisana okugxilile lukhethwe kusikena esincike kudatha ye-MS/MS.Amandla okushayisana asethelwe ku-30%.Isixazululo se-Orbitrap simiselwe ku-15,000 kanye nethagethi ye-AGC yaba ngu-100%.Isikhathi somjovo esiphezulu ngokwezifiso sisethwe kuma-millisecond angama-60.
Ngaphambi kokulandelela inethiwekhi yamaprotheni-amaprotheni kumasampuli axhumene, sicubungule amafayela aluhlaza sisebenzisa iphakheji ye-MaxQuant (inguqulo 1.6.12.0) 26,27 ukuze sihlonze ama-peptide/amaprotheni alandelekayo kumasampuli.Ukwengeza, ukuhlaziya okufanayo kwe-proteomic kwenziwa kumasampula e-Flo-1 angaxhunywanga aphathwe futhi angelashwa nge-IFNα.Idatha ye-MS/MS iseshwa kusizindalwazi sabantu se-UniProt (www.uniprot.org) (elayishwe ngomhla ka-12 Agasti 2020, iqukethe okufakiwe okungu-75,093) kusetshenziswa injini yokusesha eyakhelwe ngaphakathi i-Andromeda27.Ukusesha kwenziwa ngaphandle kokukhombisa ukucaciswa kwe-enzyme kanye nokuguqulwa okuhlukahlukene kwe-deamidation (N, Q) kanye ne-oxidation (M).I-Precursor mass tolerances isethwe ku-20 ppm nama-ion womkhiqizo ku-0.02 Da.Ukuchezuka kwesisindo sokuqala nokukhulu kwakusethelwe ku-10 ppm.Isisindo esiphezulu se-peptide sibekwe ku-4600 Da futhi ukufana kokulandelana kusethwe phakathi kwe-7 ne-25 amino acids (aa).Ukuhlaziywa kwezibalo okwengeziwe kwenziwa kusetshenziswa uhlelo lwe-Perseus (inguqulo 1.6.10.45).Okuqukethwe kwamaprotheni kubalwe ngokujwayelekile ukushuba kwe-spectral kwephrotheni (ukuqina kwe-LFQ; ubuningi obungalebuli)27 futhi amanani okuqina aguqulelwe ku-Log2.Iqoqo elilandelanayo lamaprotheni akhonjwe amandla awo e-peptide lakhiwe kusetshenziswa iphakheji ye-pheatmap (v1.0.12) ku-R (v 4.1.2).Ukuhlaziywa kokunothisa kwendlela kwenziwa kusetshenziswa isizindalwazi sendlela ye-Reactome yamaprotheni alashwe yi-IFNα aye enziwe asebenza izikhathi ezingaphezu kwezine uma kuqhathaniswa namasampuli angalashwanga.
Ukuhlonzwa kwe-lysine (K) noma i-serine (S) yamakhemikhali e-crosslinks akhethekile we-protein complexes eqashwe i-LC-MS/MS kwenziwa kusetshenziswa umshini wokuhlonza i-spectroscopic (SIM-XL) wama-peptide axhumene kakhulu (SIM-XL)29.Okokuqala, ukusebenzisana okungenzeka phakathi kwe-interferon-associated (IFN) i-DNA yesiginesha yokuphikiswa kokulimala komonakalo (IRDS) kuphenywe kusetshenziswa idathasethi yedatha yeprotheyini ye-IRDS echazwe ku-Padariya et al.28.Ukuhlola zonke izimo nokuphindaphinda kwayo yonke i-UniProt yomuntu kunamandla kakhulu, ngakho yonke isizindalwazi somuntu se-UniProt (www.uniprot.org) (kulandwe ngomhla ka-12 Agasti 2020, siqukethe okufakiwe okungu-75,093) ngokumelene nezimpinda ezilashwe yi-IFNα.Esinye sezihlungi zokusebenzelana okuphezulu.Lokhu kusebenzisana kokubaluleka okuphezulu okutholiwe kwanwetshwa futhi kwahlolwa kukho konke ukuphindaphinda nezimo.
Ku-SIM-XL, i-DSS yayisetshenziselwa i-crosslinker (XL) kanye nokushintshwa kwesisindo se-XL nokuguqulwa kwesisindo kusethelwe ku-138.06 no-156.07, ngokulandelanayo.Amasayithi wokusabela ahlanganayo alandelayo ayacatshangelwa: i-KK, i-KS ne-KN-TERM, ngaphandle kwama-ion wezintatheli.Kokubili i-precursor ne-fragment ppm zasethelwe ku-20 futhi umkhawulo we-Xrea wawubekwe ku-0.15.I-Trypsin yayibhekwa njengecacile ngokuphelele, futhi indlela yokuhlukanisa i-C-trap (HCD) enamandla amakhulu yasetshenziswa.Umkhawulo wokunciphisa we-XCorr DB kanye nenani eliphansi lama-peptide okunciphisa i-DB aguquguqukayo amiswe ku-2.5 no-2, ngokulandelanayo.Amanye amapharamitha yilawa: Amathuba e-monoisotope kanye nokunqamuka kokuqondana okukhulu, ubuncane bezinsalela ze-AA ezi-4 ngomucu ngamunye kanye nokushajwa komucu okuphezulu, kanye nobukhulu obu-3 bokuhlukaniswa okugejiwe.Amamephu we-2D athungiwe ahlaziywe ku-(SIM-XL) futhi ukumelwa kwesithombe kwe-xQuest28 kwasetshenziswa ukwakha amamephu e-2D.Amaphrotheni axhumanisa ezakhiweni zamaprotheni anikezwa nge-PyMol (PyMOL Molecular Graphics System, inguqulo 2.0 Schrödinger, LLC).
Izakhiwo zamamodeli wephrotheni zidalwe kusetshenziswa iseva ye-Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)11 kusetshenziswa izimiso zokumodela kwe-homology nokusebenzisa "Indlela ye-Markov Efihliwe".I-Phyre2 ikhiqiza izakhiwo zemodeli ngokusekelwe ekulandeleni ukulandelana nezakhiwo ezaziwayo zamaphrotheni.Kumaprotheni e-H2BFS, HLA-A, HMGA1, LRCH4, kanye ne-MDN1, izakhiwo zesifanekiso 1kx552, 1kj349, 2eze55, 6hlu62, kanye ne-6i2665 zisetshenzisiwe.Ngaphezu kwalokho, ukwakheka kwe-AlphaFold71 MX1, UBP18 kanye ne-ROBO1 nakho kucatshangelwe.Isakhiwo samaprotheni sabonwa kusetshenziswa iphakheji ye-BIOVIA Discovery Studio Visualizer (i-Dassault Systèmes, BIOVIA, San Diego, CA, USA) kanye nephakheji ye-Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).