347 chitsulo chosapanga dzimbiri chophimbidwa ndi machubu a mankhwala, Kuzindikiritsidwa kwa mapuloteni amtundu wa interferon-responsive human leukocyte antigen-A (HLA-A) chaperone pogwiritsa ntchito cross-linked mass spectrometry (CLMS)

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Machubu Opanda Zitsulo 347L, Gulu la Zitsulo: SS347L

Dongosolo la ma interferon limapangitsa kuti ma cytokine ayankhe mwamphamvu kuzinthu zambiri zapathogenic ndi intrinsic pathological pathological kuchokera ku chilengedwe, zomwe zimapangitsa kuti pakhale ma subsets a interferon-inducible proteins.Tinagwiritsa ntchito DSS-mediated cross-link mass spectrometry (CLMS) kuti tizindikire kuyanjana kwatsopano kwa mapuloteni mu gawo la mapuloteni opangidwa ndi interferon.Kuphatikiza pa mapuloteni omwe amayembekezeka a interferon-inducible, tidazindikiranso ma intermolecular and intramolecular cross-linked adducts a canonical interferon-inducible proteins monga MX1, USP18, OAS3, ndi STAT1.Tidayang'ana kwambiri pakutsimikizika kwa orthogonal kwa ma network a interferon-inducible protein network omwe amapangidwa ndi mapuloteni a HLA-A (H2BFS-HLA-A-HMGA1) pogwiritsa ntchito co-immunoprecipitation ndi kafukufuku wawo wopitilira pogwiritsa ntchito mamolekyulu amphamvu.Kutengera ma conformational dynamics a protein complex adawulula malo angapo olumikizirana omwe amawonetsa kuyanjana komwe kumadziwika muzopeza za CLMS.Pamodzi, timapereka kafukufuku woyendetsa ndege wa CLMS kuti azindikire zatsopano zowonetsera zomwe zimayambitsidwa ndi interferon, ndikuyembekeza kugwiritsa ntchito kwakukulu kwa CLMS kuti tizindikire mphamvu zatsopano zamapuloteni mu chotupa microenvironment.
Chitetezo cha mthupi chisanayambike, chitetezo cha mthupi cha wolandirayo chimayambitsa kuyankha kwa antimicrobial mothandizidwa ndi gulu la alpha-helical cytokines otchedwa interferon (IFNs).Mitundu ya I IFN makalasi IFNα ndi IFNβ imayambitsa mayankho a ma cell, kuphatikizapo antiviral, proapoptotic, proinflammatory, and antiproliferative states.Kwa anthu, 13 subtypes ya IFNα imadziwika, zonse zimagwirizanitsidwa pa chromosome 91. Chodabwitsa n'chakuti IFNα2 yokha yaphunziridwa kuti igwiritsidwe ntchito kuchipatala.Posachedwapa, chidwi chapadera chaperekedwa pakufufuza pazigawo zina za IFNα.Kafukufuku waposachedwapa wasonyeza kuti IFNα14 ndi imodzi mwa isoforms yothandiza kwambiri poletsa kubwereza kwa HBV2 ndi HIV-13,4 poyerekeza ndi IFNα2 yovomerezeka.
Zatsimikiziridwa kuti makina amtundu wa I interferon receptor complexes (IFNAR1 ndi IFNAR2) amachititsa kuti phokoso la transduction cascade lokhala ndi Janus kinases TYK2 ndi JAK15,6.Izi Janus kinases phosphorylate ma transducers ndi transcriptal protein activators (STAT1 ndi STAT2) pa zotsalira za tyrosine kuti ayambitse SH2 domain-mediated heterodimerization6.Pambuyo pake, IRF9 imamanga STAT heterodimers kuti ipange trimeric complex ya IFN-stimulated factor 3 gene (ISGF3), yomwe imasunthira ku nucleus ndikupangitsa kulembedwa kwa 2000 interferon-stimulated genes (ISGs)5,6,7,8.
Ma ISG amapanga msana wa chitetezo cham'mimba, makamaka poyankha kuukira kwa ma virus.Monga njira yoyamba yodzitetezera ku matenda a mavairasi, maselo amatumiza mofulumira kuyanjana kwakukulu kwa mapuloteni a m'maselo omwe ali ndi zochitika zambiri zamoyo.Mapuloteniwa amaphatikizanso ma receptor ozindikira, mamolekyu ozindikiritsa, zinthu zolembera, ndi mapuloteni okhala ndi magwiridwe antchito a antivayirasi, komanso zowongolera zoyipa za mayankho a chitetezo chamthupi9.Zambiri pazantchito za ISG zimachokera ku zowonetsera zogwirira ntchito pogwiritsa ntchito zowonetsera mopitirira muyeso10,11 kapena njira zochepetsera jini (siRNA, RNAi ndi CRISPR)12,13 momwe ma ISG amasonyezedwa kapena kuletsedwa ndipo ntchito yawo imayesedwa pa mavairasi osiyanasiyana.Ngakhale maphunzirowa atsimikiza za antivayirasi a ma ISG pawokha, njira zoyambira zama cell za ISG iliyonse sizidziwika.Ndizovomerezeka kuti mapuloteni ambiri amalumikizana ndi ma cytokine amodzi kapena angapo kuti awonetsetse kuti akugwira ntchito mokwanira, chifukwa chake ma ISG amalumikizana mwachindunji kapena kulumikizana kwawo kumalumikizidwa ndi mapuloteni am'manja.Mwachitsanzo, kafukufuku waposachedwapa wa photocrosslinked proteomics anapeza ATPase VCP/p97 ngati bwenzi lalikulu la IFITM3 logwirizana, lomwe kuletsa kwake kumabweretsa zofooka mu lysosomal kusanja, kubweza, ndi cotransport ya IFITM3 yokhala ndi tizilombo toyambitsa matenda 14.Pogwiritsa ntchito immunoprecipitation, tinazindikira VAPA, puloteni yogwirizana ndi vesicle, monga wothandizana nawo ndi IFITM1 / 2/3 yomwe imagwirizanitsa matupi a mavairasi a cholesterol, ndipo izi zinatsimikiziridwa ndi kafukufuku wina wogwiritsa ntchito yisiti awiri-hybrid system.Thandizo la Sayansi 15, 16.
Njira yofunikira yachilengedwe yomwe imakhudzidwa ndi kupondereza kwa matenda ndi kusintha koyipa ndikuwonetsetsa kwa antigen, komwe kumayendetsedwa ndi mamolekyu akuluakulu a histocompatibility complex (MHC).Ma peptides (8-12 amino acid yaitali) kuchokera ku mapuloteni odulidwa, omwe amatha msanga kapena osokonezeka amalowetsedwa mu MHC-I heterodimer (yomwe ili ndi unyolo wa MHC-I wolemera komanso wopepuka, wotchedwa β-2-microglobulin; β2M) 17,18.Zotsatira zokhazikika za MHC-I trimers zimatengedwera ku selo pamwamba, kumene amapereka ma peptide a intracellular ku CD8 + T maselo (ma cell a cytotoxic T)17.Ma cell a T amazindikira ndikuwononga tizilombo toyambitsa matenda ndi ma cell omwe ali ndi chotupa-enieni antigen.Chifukwa chake, tizilombo toyambitsa matenda ndi ma cell chotupa nthawi zambiri amapondereza njira yowonetsera ma antigen kuti asayang'anire chitetezo chamthupi.Kuonjezera apo, MHC-I imachepetsedwa mu 40-90% ya zotupa zaumunthu ndipo nthawi zambiri zimagwirizanitsidwa ndi matenda osauka19.
Majini okhudzidwa ndi kuyankha kwa tizilombo toyambitsa matenda ayenera kusintha mofulumira pakati pa malo opumula ndi chikhalidwe cholembera.Choncho, mapuloteni angapo a m'manja amaganiziridwa kuti akugwira nawo ntchito poyankha kufunikira kwakukulu kwa IFN kwa nthawi yochepa, kuphatikizapo kukonzanso ndi kusintha kwa chromatin 20,21.Kafukufuku wambiri amayang'ana kwambiri pakuzindikiritsa anthu omwe ali ndi mapuloteni a ISG pamaso pa IFN.Maphunziro angapo a proteomic ndi transcriptomic m'ma cell cell awonetsa zotsatira za IFN pama cell cell.Komabe, ngakhale tikumvetsetsa bwino za mphamvu zomwe zimayambitsidwa ndi interferon, sitikudziwabe pang'ono za kutenga nawo mbali kwa ma ISG.Poganizira zovuta komanso nthawi zomwe zimadalira zizindikiro za interferon, pali mafunso awiri: (i) n'zotheka kukhazikika ndi kutsekera ma multiprotein complexes omwe akukhudzidwa ndi zizindikiro zofulumira, ndipo (ii) kodi kuyanjana kumeneku kungapangidwe mu danga la 3D?
Pofuna kuthana ndi mavutowa, tinagwiritsa ntchito disuccinimide suberate-mediated chemical cross-linking (DSS) pamodzi ndi mass spectrometry (CLMS) kuti tiphunzire za IFNα-induced protein interaction network ndi mphamvu zake.DSS imawonjezera mgwirizano pakati pa zotsalira zotsalira za mapuloteni ndi/kapena mapuloteni mu vivo.Kusanthula kotsatira kwa MS kumawulula masamba ena olumikizirana omwe amawonetsa kuyandikira kwa madera mkati mwa puloteni inayake, yotchedwa internal linkages, kapena subunits in protein complexes, yotchedwa interrelationships.Pogwiritsa ntchito njirayi, tazindikira mitundu ingapo yama protein-protein complexes komanso ma interferon-induced multiprotein interaction network.Poyesanso kagawo kakang'ono ka kuyanjana kwatsopano kumeneku, tikuwonetsa kuti H2BFS (H2B histone-type FS; yomwe pambuyo pake imatchedwa H2B) ndi MDN1 zimakhala ngati zibwenzi zomangirira za HLA-A.
Maselo a Flo-1 ndi amodzi mwa mitundu yodziwika bwino ya m'mimba ya esophageal adenocarcinoma pamene amatsanzira mbali zazikulu za zotupa zam'mero22,23.Komabe, si zotupa zonse zomwe zili ndi immunogenic, ndipo kuti tidziwe ngati maselo a Flo-1 amayankha chithandizo cha interferon, tinachitira maselo a Flo-1 ndi 10 ng / ml IFNα kwa maola 72.Maselo a Flo-1 adawonetsa kuyambika koyambirira kwa pSTAT1 ndi IRF1, kuyambira maola 2 mutalandira chithandizo ndikupitiriza kwa maola 72, ndi kuchepa kwanthawi yayitali kwa IRF1 (Chithunzi 1A).Ma ISG (MX1, IFITM1, OAS1/2, ndi ISG15) adapezeka kuti adakopeka kwambiri pambuyo pa maola 6, kutsanzira mayankho apakatikati ndi mochedwa ku IFNα (Chithunzi 1A).Pamodzi, izi zikusonyeza kuti chitsanzo cha ma cell angagwiritsidwe ntchito pophunzira mayankho a interferon.
Mayankho osiyanasiyana ofotokozera mapuloteni m'maselo a Flo-1 pambuyo pa chithandizo cha IFNα.(A) Kufotokozera kwa mapuloteni m'maselo a Flo-1 omwe amachitidwa ndi 10 ng / ml IFNα kwa 2, 6, 24, 48 ndi maola a 72 adafufuzidwa ndi immunoblot pogwiritsa ntchito ma antibodies a ISG.(B) Ma gels a SDS-PAGE a buluu a Coomassie amtundu wathunthu wa cell atalumikizana ndi DSS nthawi zosonyezedwa komanso kukhazikika.(C) Representative immunoblot anayesedwa ndi p53 (DO-1) antibody kuchokera ku zitsanzo zomwezo kuti awone kuchuluka kwa mapuloteni ogwirizanitsa.
Kuti tijambule mawonekedwe a protein mu situ, tidagwiritsa ntchito DSS, cholumikizira chomwe chimagwiritsidwa ntchito kwambiri chifukwa chakukwanira kwake kwa membrane komanso nthawi yayifupi.Nthawi yayifupi yochitapo kanthu imathandizira kupewa kupanga magulu akuluakulu a mapuloteni ophatikizika, potero kusunga bata la crosslinker.Kuti tidziwe momwe DSS ikuyendera ndikupewa kuphatikizika, tidayamba kuwonetsa ma cell ku 5, 2.5, ndi 1 mM DSS kwa mphindi 5, 10, 5, ndi 30, motsatana, ndikusanthula ma lysates ndi Coomassie-stained SDS-PAGE. (zosawonetsedwa).Ma cell lysates amawoneka kuti amalumikizana kwambiri pamlingo wotsika kwambiri komanso panthawi yaifupi kwambiri.Chifukwa chake, DSS idasinthidwa kukhala 1, 0.5, ndi 0.1 mm m'mphindi zisanu (Chithunzi 1B).Kuphatikizika koyenera kunawonedwa ndi 0.5 mM DSS kwa mphindi 5, ndipo izi zidasankhidwa m'maselo omwe amathandizidwa ndi IFNα.Kuphatikiza apo, Chithunzi 1C chikuwonetsa kutsekedwa kwa Kumadzulo komwe kumachitika pogwiritsa ntchito antibody ya p53 (DO-1) kuti awone kuchuluka kwa mapuloteni olumikizana.
Maselo a Flo-1 amathandizidwa ndi 10 ng / ml IFNα kwa maola 24 asanawonjezere crosslinker.Maselo okhudzana ndi mtanda adasinthidwa ndi masitepe awiri a proteolysis ndipo mapuloteni adakonzedwa ndi FASP (Mkuyu 2) 24,25.Ma peptides olumikizana ndi tryptic adawunikidwa ndi misa spectrometry (mkuyu 2).Mawonekedwe a MS/MS ndiye amafananizidwa ndi dongosolo la mapuloteni ndikuwerengedwa ndi MaxQuant26,27.Ma peptides olumikizidwa ndi mtanda adadziwika kuchokera kuzinthu zomwe zidapezeka pogwiritsa ntchito pulogalamu ya SIM-XL, ndipo zophatikiza zapayekha zidaphatikizidwa kukhala maukonde ovuta kugwiritsa ntchito xQuest28 ndi SIM-XL29 mapaipi otsegulira mapulogalamu apakompyuta (mkuyu 2).SIM-XL imazindikiritsa kuyanjana kwa mapuloteni-mapuloteni, maunyolo amkati ndi unyolo wapayekha muzosakaniza zosavuta kapena zovuta zamapuloteni ndipo imapereka zolemba zowonera kuyanjana kwamagulu a mapuloteni.Kuphatikiza apo, imayika kalozera aliyense ngati chiphaso cha ID malinga ndi mtundu wa MS/MS29.Mapuloteni angapo odalirika kwambiri okhudzana ndi mapuloteni ndi zovuta zadziwika, ndipo njira yatsopano yogwirizanirana yafufuzidwanso pogwiritsa ntchito co-immunoprecipitation ndi conformational kusintha kwa zovuta pogwiritsa ntchito ma molecular dynamics (MD) modelling (Fig. 2) 30, 31.
Kuwunika mwachidule kwa njira ya CLMS.Maselo a Flo-1 amathandizidwa ndi 10 ng / ml IFNα kwa maola 24 akutsatiridwa ndi in situ protein cross-linking pogwiritsa ntchito DSS yotsatiridwa ndi cell lysis ndi trypsinization.Zitsanzo zolumikizidwa ndi mtanda zidawunikidwa pogwiritsa ntchito ma spectrometer a Orbitrap mass spectrometer ndikuwonjezeranso zitsanzo za kugawikana kwa ma peptide precursors pa LC-MS/MS.Ma peptide awiri olumikizidwa adadziwika kuchokera ku mawonekedwe omwe adapezeka pogwiritsa ntchito pulogalamu ya Spectrum Recognition Machine ya Crosslinked Peptides (SIM-XL), ndipo zida zonse zidaphatikizidwa kukhala network yovuta kugwiritsa ntchito mapaipi owerengera.Zosefera kuyanjana kodzidalira kocheperako kutengera zigoli zabodza (FDR).Kuyanjana kwatsopano kwapamwamba kwa mapuloteni-mapuloteni kunatsimikiziridwanso pogwiritsa ntchito co-immunoprecipitation, ndipo kusintha kwa conformational m'mabwalowa kunayesedwa pogwiritsa ntchito ma modules a molecular dynamics (MD).
Chiwerengero cha ~ 30,500 ndi ~ 28,500 peptides chinapezedwa pogwiritsa ntchito MaxQuant mu zitsanzo zosasunthika komanso zolimbikitsa za IFNα, motero (Supplementary Table S1, Fig. 3A).Kugawika kwa peptide muzochitika zonsezi kunasonyeza kuchuluka kwa peptides akuluakulu, kusonyeza kukhalapo kwa peptide yolumikizidwa (mkuyu 3B, C).Kuonjezera apo, gawo lalikulu la ma peptide akuluakulu analipo mumtundu wa 40-55 mu zitsanzo za IFNα (mkuyu 3C).Mapu apuloteni motsutsana ndi kukula kwa log2 adawonetsa kuti mapuloteni apamwamba kwambiri opangidwa ndi interferon anali ochuluka kwambiri poyerekeza ndi zitsanzo zomwe sizinatengedwe, kuphatikiza MX1, IFIT1/3, OAS2/3, DDX58, ndi HLA-F (Chithunzi 3D).Kuwunika kwa njira zamapuloteni opitilira katatu olemetsedwa poyankha chithandizo cha IFNα pogwiritsa ntchito database ya Reactome yawonetsa kuti kuwonetsa ndi kukonza kwa antigen kwa MHC-I-mediated ndiyo njira yayikulu kwambiri (Chithunzi 3E).Mogwirizana ndi malipoti oyambirira, mayankho a antiviral omwe ali pakati pa OAS ndi ISG15 komanso IFNα / β ndi chizindikiro cha cytokine anali ena mwa njira zomwe zinayambitsidwa.Kuphatikiza apo, ma lysine- ndi serine-specific protein cross-links adadziwika kuchokera ku mawonekedwe omwe adapezedwa koyamba a MS/MS pogwiritsa ntchito SIM-XL.Kafukufuku waposachedwa wanena kuti ma ISG 104 amatenga ma virus 20 kuchokera m'makalasi 9 a virus powunika meta-mafukufuku amtundu wa ISG pamitundu 59.Komabe, kuti tithane ndi malire owerengera ma dataseti akulu, tidayamba ndi dataset yaying'ono kuti tifufuze zomwe zingachitike pakati pa mndandanda wamitundu ya IRDS yomwe idanenedwa ndi Padaria et al., ambiri omwe ndi ma ISG.
Kuzindikiritsa mapuloteni ophatikizidwa mosiyanasiyana poyankha IFNα (deta yochokera ku MaxQuant).(A) Chithunzi cha Venn choyimira chiwerengero cha ma peptide odziwika komanso apadera omwe amadziwika mu IFNα14 omwe amachiritsidwa komanso osapatsidwa mankhwala a Flo-1.Kugawa kwautali wa Peptide kwa zitsanzo zosagwiritsidwa ntchito (B) ndi IFNα (C) zosakanikirana.(D) Mapu otentha omwe akuyimira log2 (LFQ intensity) pakati pa ma cell a Flo-1 osathandizidwa ndi IFNα14.Gulu lakumanzere likuwonetsa mapuloteni omwe akugwiritsidwa ntchito kwambiri pamaso pa IFNα.(E) Histogram yoyimira njira zazikulu za 20 zolemeretsa pambuyo pa chithandizo cha IFNα.Dongosolo la Reactome pathway database idasanthula zosinthika mopitilira kanayi pamapuloteni omvera a IFNα.
Kukondoweza kwa Interferon-mediated ISG kumalembedwa bwino, koma pamlingo wa maselo sikumvetsetseka bwino momwe mapuloteniwa amathera pa ntchito zosiyanasiyana zamoyo.Tidafufuza zokhudzana ndi mapuloteni ndi chidaliro chachikulu pakati pa ma ISG odziwika.Chochititsa chidwi n'chakuti, tinazindikira maukonde kuphatikizapo MX1, USP18, ROBO1, OAS3, ndi STAT1 mapuloteni omwe amapanga zovuta zazikulu poyankha mankhwala a IFNα (Chithunzi 4, Table S2) 32,33,34.Chofunika kwambiri, kuyanjana kumeneku kunapezeka m'magawo onse atatu omwe amachitidwa ndi IFNα ndipo sanapezeke mu zitsanzo zosagwiritsidwa ntchito, kutanthauza kuti anapangidwa makamaka poyankha mankhwala a IFNα.Zimadziwika kuti STAT1 imayang'anira mawu a ma ISG awa, koma kuyanjana kwake ndi ma ISG pamlingo wa mapuloteni sikunaphunzire.Mapangidwe a kristalo a STAT1 adawonetsa kuti chigawo chake cha helical (CCD) sichimakhudzidwa ndi DNA kapena ma protomers pakupanga dimers35.Ma α-helicewa amapanga mawonekedwe a helical helix omwe amapereka malo ochuluka kwambiri a hydrophilic kuti azitha kuyanjana 35.Mu data yathu ya CLMS, tawona kuti kuyanjana kwakukulu ndi STAT1 kunachitika mu dera la SH2 patsogolo pa CCD, malo ogwirizanitsa, kapena C-terminal mchira (zotsalira 700-708) (Chithunzi 4A).Kafukufuku wam'mbuyomu adanenanso kuti USP18 imamangiriza ku CCD ndi DNA-binding domain (DBD) ya STAT2 ndipo imatumizidwa ku subunit ya mtundu wa I interferon receptor IFNAR2 kuti ikhale mkhalapakati woletsa mtundu wa I interferon chizindikiro 24.Zambiri zathu zidawonetsanso kuti gawo lothandizira la USP18 limalumikizana ndi STAT1 DBD (Chithunzi 4A, D), kuwonetsa kuti STAT1 ndi STAT2 zitha kutengapo gawo pakukopa USP18 ku IFNAR2.
Netiweki ya protein-protein ISG yodziwika m'maselo olumikizana ndi IFNα.(A) Chiwembu chothandizirana cha 2D chosonyeza kuyanjana kwa mapuloteni-mapuloteni (opangidwa mu pulogalamu ya SIM-XL), ndi mizere yoyimira intermolecular interactions (crosslink cutoff set to 3.5).Madera amitundu yosiyanasiyana amadziwika ndi mtundu wawo32: MX1 domain, Dynamin_N (73-249), Dynamin_M (259-547), ndi GED (569-660).OAS3 madambwe: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), ndi OAS1_C (903-108).Domain ROBO1, Ig_3 (67–151), I-set (170–258), I-set (262–347), Ig_3 (350–432), Ig_3 (454–529), fn3 (562–646), fn3 (678–758) ndi fn3 (777–864).Magawo a STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), ndi STAT1_TAZ2bind (715–739).(B) Wowonera wozungulira wa mapuloteni ophatikizana (MX1, UBP18, OAS3, ROBO1, ndi STAT1) ndi machitidwe ndi machitidwe omwe amalembedwa mu buluu ndi wofiira, motsatira.Chiwopsezo cholumikizirana chinakhazikitsidwa pa 3.5.Ma dothi amawonetsa malo olumikizirana a STAT1 ndi MX1 (C), USP18 (D), ROBO1 (E), ndi OAS3 (F), komanso malo olumikizirana a K kapena S pakati pa ma peptide awiriwa.M'chithunzichi, chiwerengero cha ma cross-link scores chimayikidwa ku 3.0.(G) Malo osiyanasiyana olumikizirana pakati pa madera a STAT1 ndi OAS3 DI omwe ali pamwamba pa mapuloteni awo mu PyMol (PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) ndi OAS3 (pdb id: 4s3n34).Pulogalamu).
Ma isoforms awiri a USP18 afotokozedwa mwa anthu, mapuloteni aatali omwe amapezeka kwambiri mu nucleus, ndi isoform popanda N-terminal domain, USP18-sf, yomwe imagawidwa mofanana mu cytoplasm ndi nucleus 36.Kuphatikiza apo, N-terminus idanenedweratu kuti sinakhazikitsidwe ndipo safuna ntchito ya isopeptidase kapena kumanga ISG1537.Zambiri mwazochita zomwe zadziwika mu phunziro lathu zinali pa N-terminus ya puloteni, kutanthauza kuti kuyanjana kumeneku kumaphatikizapo USP18 wautali (Chithunzi 4A, D) ndipo motero mwinamwake kumachitika mu nucleus.Kuphatikiza apo, deta yathu ikuwonetsanso kuti N-terminus ndi yapadera pakuyanjana kwa mapuloteni ndi mapuloteni.Malo omangiriza a IFNAR2 ali pakati pa zotsalira za 312-368, ndipo makamaka, palibe mapuloteni omwe ali muzovuta amangirira kudera lino (Mkuyu 4A) 37,38.Zomwe zimatengedwa palimodzi zikuwonetsa kuti IFNAR2 yomanga domain imangogwiritsidwa ntchito ndi mapuloteni olandirira.Kuonjezera apo, OAS3 ndi ROBO1 okha adapezeka kuti akugwirizana ndi madera omwe ali pamwamba pa N-terminus ndi IFNAR2 malo omangirira (Chithunzi 4A).
ROBO1 ndi ya immunoglobulin (Ig) superfamily ya transmembrane siginecha mamolekyulu ndipo ili ndi madera asanu Ig ndi atatu fibronectin (Fn) madera m'dera extracellular.Madera owonjezerawa amatsatiridwa ndi dera la membrane-proximal ndi helix imodzi ya transmembrane 39. Dera losasinthika la intracellular lili pa C-terminus ndipo lili ndi zotsatizana zotetezedwa zomwe zimagwirizanitsa zotsatira za mapuloteni39.Chigawo chochokera ku amino acid ~ 1100 mpaka 1600 chimakhala chosokonezeka.Tinapeza kuti MX1 imagwirizana ndi ROBO1 kupyolera mu madera a Ig, Fn, ndi ma intracellular, pamene kuyanjana kwakukulu ndi STAT1 kumachitika pakati pa CCD, domain linker, ndi C-terminus ya ROBO1 (Fig. 4A, E).Kumbali ina, kuyanjana ndi zigawo za DI, DIII, ndi OAS3 zogwirizanitsa zinagawidwa mu mapuloteni a ROBO1 (Mkuyu 4A).
Banja la mapuloteni a oligoadenylate synthase (OAS) limavomereza ndikumanga RNA (dsRNA) ya intracellular iwiri, imasintha kusintha, ndipo imapanga 2',5'-yolumikizidwa oligoadenylates (2-5 As) 40.Zinapezeka kuti pakati pa ma OAS atatu, OAS3 imawonetsa kuyanjana kwakukulu kwa dsRNA ndipo imapanga kuchuluka kwa 2-5 As, komwe kumatha kuyambitsa RNase L ndikuchepetsa kubwereza kwa ma virus 41.Banja la OAS lili ndi ma polymerase beta (pol-β)-monga nucleotide transferase domains.Kafukufuku wam'mbuyomu wasonyeza kuti chothandizira cha C-terminal domain (DIII) chimadalira dsRNA-binding domain (DI), yomwe imafunika kuti OAS342 ayambe.Tidawona kuti magawo a DI ndi DII a OAS3 amalumikizana ndi CCD ndi kagawo kakang'ono kolumikizana pakati pa SH2 ndi STAT1 TAD (Chithunzi 4A,F).Kuphimba malo osiyanasiyana ophatikizira pamapangidwe a mapuloteni kunavumbula kuyanjana pakati pa β-sheet ndi DBD STAT1 loop ndi thumba lotseguka kapena patsekeke lopangidwa ndi zotsalira 60-75 mu DI domain ya OAS3 (Fig. 4G).Mayendedwe a mapuloteni muzovuta adawonetsanso kuti palibe kuyanjana ndi OAS3 komwe kunasokoneza DNA-binding mphamvu ya DI domain yake (Fig. S1A).Kuphatikiza apo, dera la N-terminal la GTPase MX1 limalumikizana kwambiri ndi madera a DI ndi DIII a OAS3 (mkuyu 4A).Tidawonanso kuyanjana pakati pa OAS1 ndi MX1 m'mabwereza atatu ochitidwa ndi IFNα, pomwe gawo limodzi la OAS1 (lomwe limagwiranso ntchito) lidalumikizana ndi madera onse atatu a MX1 (Chithunzi S2A, B).
Mapuloteni a MX ndi gawo la banja lalikulu la dynein-ngati GTPases omwe ali ndi N-terminal GTPase domain yomwe imamanga ndi hydrolyzes GTP, dera lapakati lomwe limagwirizanitsa kudzigwirizanitsa, ndi C-terminal leucine zipper yomwe imakhala ngati GTPase (LZ). ).domain effector domain25,43.MX1 imamangiriza ku ma subunits a viral polymerases kuti aletse kusindikiza kwa viral gene43.Chithunzi chojambulidwa kale cha yisiti chamitundu iwiri cha haibridi chinawonetsa kuti PIAS1 yolumikizidwa ndi MX1 imalepheretsa kuyambitsa kwa jini kwa STAT1 poletsa ntchito yomangirira DNA komanso ili ndi ntchito ya ligase ya SUMO E344,45.Pano, tikuwonetsa kuti MX1 imamangiriza ku STAT1 (Chithunzi 4C, D), komabe momwe kuyanjana uku kumakhudzira STAT1-mediated gene activation poyankha IFNα ikufunikira kuphunzira.Kuphatikiza apo, tapezanso kuti MX1 idalumikizana ndi IFIT3 ndi DDX60 muzobwereza zonse zitatu za IFNα (Fig. S2C).
DDX60 ndi IFN-induced cytoplasmic helicase yomwe idanenedwapo kale kuti imathandizira pakuwonongeka kodziyimira pawokha kwa RNA46 ya RNA46.Imalumikizana ndi RIG-I ndikuyambitsa chizindikiro chake m'njira yeniyeni ya ligand 46. DDX60 ili ndi DEXD / H-Box helicase domain ndi C-terminal helicase domain yomwe imamangiriza ma virus a RNA ndi DNA47.Zambiri mwazochita zake ndi MX1 ndi IFIT3 zimachitika mkati mwa zigawo zazitali za N- ndi C-terminal popanda madera ovomerezeka kapena ma motifs (mkuyu S2E, F).Komabe, MX1 imagwirizanitsidwanso ndi DEXD / H-Box helicase domain (Fig. S2E).Mapuloteni a banja la IFIT ali ndi makope a tandem a helix-turn-helix motif yotchedwa tetrapeptide repeat (TPR).IFIT3 idapezeka kuti ndi modulator yabwino ya RIG-I siginecha motero ndi gawo la zovuta za MAVS.Kuphatikizidwa pamodzi, deta yathu imasonyeza kuti IFIT3 ndi DDX60 zimagwirizanitsa makamaka m'dera la TPR 3-6 la IFIT3 ndipo likhoza kukhala ndi gawo la RIG-I / MAVS signing (Fig. S2F).
Poganizira kuti kuwunika kwa proteome yonse ndikokwanira kwambiri, tidayang'ana nkhokwe yonse ya UniProt yamunthu kuti ikhalepo imodzi mwazobwereza zochitidwa ndi IFNα.Pachifanizo ichi, tapeza maukonde angapo odalirika a HLA-A.Kusanthula kwa njira zamapuloteni zomwe zimadziwika ndi MS / MS spectra zinasonyeza kuti MHC-I-based antigen processing ndi kuwonetseratu ndiyo njira yaikulu yomwe imayambitsidwa ndi interferon (Fig. 3D).Chifukwa chake, tidayang'ana kwambiri pakuwerenga kuyanjana kwa mapuloteni a mamolekyu a MHC-I okhala ndi chidaliro chachikulu pamiyeso yonse yolumikizidwa.HLA imakhala ndi madera a α1, α2 ndi α3 ndi unyolo wopepuka, ndipo microglobulin β2 (β2m) ndi protein ya chaperone yokhazikika49.Akasonkhanitsidwa mu endoplasmic reticulum, HLA imakhala yosakhazikika pakalibe peptide ligands50.Mphepete yomanga ma peptide imapangidwa ndi madera a polymorphic kwambiri komanso osakhazikika a α1 ndi α2 mu mawonekedwe osakhala a peptide komanso dera lochepera la polymorphic α351.Pamaso pa IFNα, tidazindikira zovuta ziwiri za HLA-A: imodzi imagwirizana ndi HMGA1 ndi H2B (Chithunzi 5, Table S3) ndipo ina imagwirizana ndi MDN1, LRCH4 ndi H2B (Chithunzi 6).
IFNα imapangitsa kuti pakhale mgwirizano wa HLA-A ndi H2B (H2BFS) ndi HMGA1.(A) Chiwembu cha 2D (chopangidwa mu pulogalamu ya SIM-XL) chosonyeza mitundu yosiyanasiyana yolumikizirana mu H2B-HLA-A-HMGA1 complex: interlink (buluu), interlink (red) ndi single link (yakuda)..Madera amitundu yosiyanasiyana ali ndi mitundu32: H2B (histone; 2–102) ndi MHC-I (MHC_1; 25–203, gulu C1; 210–290 ndi MHC_I_C; 337–364).Chiwopsezo cholumikizirana chinakhazikitsidwa pa 3.5.Ma dot plots akuwonetsa malo olumikizirana a HLA-A ndi H2B (B) ndi HMGA1 (C), komanso malo olumikizirana a K kapena S pakati pa ma peptide awiriwa.M'chithunzichi, chiwerengero cha ma cross-link scores chimayikidwa ku 3.0.(D) Ubale pakati pa mapuloteni omwe amawonetsedwa m'mapangidwe a mapuloteni a H2B, HLA-A, ndi HMGA1 mu pulogalamu ya PyMOL.Mapangidwewa adapangidwa pogwiritsa ntchito seva ya Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) ndipo mapangidwe a ma template a mapuloteni a H2B, HLA-A ndi HMGA1 anali 1kx552, 1kj349 ndi 2eze55, motsatana.
IFNα imapangitsa kuti pakhale mgwirizano wa HLA-A ndi H2B (H2BFS), MDN1 ndi LRCH4.(A) Intramolecular (red) ndi intermolecular (blue) crosslinks zoperekedwa pamapu olumikizana a 2D (opangidwa mu pulogalamu ya SIM-XL) yokhala ndi MDN1 yoimiridwa ngati bwalo.Chiwopsezo cholumikizirana chinakhazikitsidwa pa 3.5.Madera amitundu yosiyanasiyana ndi amitundu32: H2B (histone; 2–102), MHC-I (MHC_1; 25–203, gulu C1; 210–290 ndi MHC_I_C; 337–364) ndi LRCH4 (LRR_8 (68–126), LRR_8 (137-194) ndi CH (535-641)).(B) Ubale pakati pa mapuloteni omwe amawonetsedwa m'mapangidwe a mapuloteni a H2B, HLA-A, LRCH4, ndi MDN1 mu pulogalamu ya PyMOL.Zomangamangazi zidapangidwa pogwiritsa ntchito seva ya Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) yokhala ndi ma template 1kx552, 1kj349, 6hlu62 ndi 6i2665 a H2B, HLA-A, LRCH4 ndi MDN1 mapuloteni, motsatana.Madontho osonyeza malo a K kapena S a HLA-A okhala ndi H2B (C), LRCH4 (D), ndi MDN1 (E).Kwa ziwembu, malire olumikizirana adayikidwa ku 3.0.
Kuphatikiza pa kusunga umphumphu wa genome, histone H2B imakhudzidwanso ndi kayendetsedwe ka kalembedwe.Puloteni ya H2B imakhala ndi malo apakati a histone (HFD) opangidwa ndi ma α-helice atatu olekanitsidwa ndi malupu ndi C-terminal mchira 41,52.Zambiri zokhudzana ndi H2B zimachitika mu α1 helix, yomwe imapereka trimerization ndi HFD heterodimer (Mkuyu 5A, B).Ngakhale ma lysins amatenga nawo gawo pakumanga kwa DNA, ma lysins ena ndi malo ena acetylation kapena methylation.Mwachitsanzo, zotsalira za K43, K46, ndi K57 zochokera ku H2B sizimangiriridwa mwachindunji ndi DNA, koma ndi zolinga za kusintha kosiyanasiyana kwa pambuyo polemba53.Mofananamo, zotsalira za K44, K47, ndi K57 mu H2B zingakhale ndi gawo lina pamaso pa IFNα, kuphatikizapo kuyanjana ndi mapuloteni ena (Mkuyu 5A, B).Kuphatikiza apo, extrachromosomal histone H2B imayambitsa kuyankha kwa chitetezo chamthupi m'maselo osiyanasiyana, kukhala ngati sensa ya cytosolic kuti izindikire zidutswa za DNA (dsDNA) zamitundu iwiri zomwe zimachokera ku mankhwala opatsirana kapena ma cell owonongeka54.Pamaso pa mavairasi a DNA, kuchepa kwa H2B kunalepheretsa kupanga IFN-β ndi STAT154 phosphorylation.H2B imadziwikanso kuti imatuluka ndi kulowa mkati mwachangu kuposa ma core histones54.Kuyanjana kwa H2B ndi MDN1 ndi LRCH4 kunawonedwanso mu zitsanzo zosasankhidwa zosasankhidwa.Tidapeza kuti HLA-A idalumikizana ndi H2B m'miyeso yonse itatu yothandizidwa ndi IFNα komanso mu chitsanzo chimodzi chobwereza chosasinthidwa.Deta iyi ikuwonetsa gawo la H2B mu ntchito ina yokhudzana ndi thupi popanda kutsata malamulo olembedwa.
HMGA1 (gulu loyendetsa kwambiri AT-Hook 1), nucleoprotein yaing'ono yolemera mu amino acid omwe amalimbikitsa matenda, yadziwika mogwirizana ndi HLA-A.Ili ndi acidic C-terminal mchira ndi ma DBD atatu osiyana otchedwa AT hooks chifukwa amamangiriza ku groove yaying'ono ya dera la AT-rich ku dsDNA55,56.Kumangiriza kumeneku kumapangitsa DNA kupindika kapena kuwongoka, kulola kuti zinthu zolembedwa m'mawu ovomerezeka zizitha kutsata ndondomeko yake.Mchira wa C-terminal umakhulupirira kuti umakhudzidwa ndi mapuloteni-mapuloteni komanso kulembera zinthu zolembera, popeza C-terminal deletion mutants sangathe kuyambitsa transcript57.Komanso, derali lili ndi malo angapo otetezedwa a phosphorylation omwe amadziwika kuti ma kinases 58.Tinawona kuyanjana kwa HLA-A ndi H2B ndi HMGA1 kunja kwa dera la C-terminal, kutanthauza kuti dera la C-terminal limagwiritsidwa ntchito makamaka polemba zinthu zolembera (Fig. 5A, C).Mapuloteni a HMGA amapikisana ndi histone H1 kuti amangirire ku adapter DNA, motero amakulitsa kupezeka57.Mofananamo, zikuwoneka kuti HMGA imalumikizana ndi histone H2B pamodzi ndi DNA yolumikizira mpikisano ndi histone H1.HMGB1 imayambitsa kufotokozera kwa HLA-A, -B, ndi -C m'maselo a dendritic, zomwe zimatsogolera ku activation59, koma kuyanjana pakati pa HMG ndi HLA sikunanenedwe kale.Tinapeza kuti HMGA1 imagwirizana ndi madera a α1 ndi α3 a HLA-A, ndi machitidwe ambiri kunja kwa 3 DBD (Chithunzi 5A, C).M'manja mwathu, HLA-A idapezeka kuti ili mu nyukiliya (deta yosawonetsedwa), ndipo chifukwa H2B ndi HMGA1 ziliponso pamphuno, kuyanjana uku kumachitika mu nucleus.Zowonjezera zenizeni zoyezedwa pakati pa H2B, HLA-A, ndi HMGA1 zikuwonetsedwa mu Chithunzi 5D.
Zochita zambiri za HLA-A ndi mapuloteni ena zimachitika mkati mwa madera ake a α1 ndi α2 ndi malo osokonezeka a C-terminal (mkuyu 6).Mu chimodzi mwa zitsanzozi, tapeza kuti HLA-A imagwirizana ndi N-terminal mchira wa LRCH4 wosokonezeka (Chithunzi 6A,D).LRCH4 imayang'anira TLR4 activation ndi LPS cytokine induction, potero kusinthira kuyankha kwa chitetezo chamthupi60,61.Ndi mapuloteni a membrane omwe ali ndi ma leucine-rich kubwereza (LRRs) ndi calmodulin (CH) homology motif mu ectodomain yake, yotsatiridwa ndi transmembrane domain (TMD) 60, 62.Madera a CH adanenedwa kuti amalumikizana ndi mapuloteni-mapuloteni 60.Kutalikirana kwa ma amino acid pafupifupi 300 pakati pa madera a LRR ndi CH ndikosavuta kufikako koma kusokonezeka.Malingana ndi ntchito ya madera osokonezeka monga oyimira pakati pa mapuloteni-mapuloteni okhudzana ndi mapuloteni ndi kayendedwe ka vesicular 63, tapeza kuti kuyanjana kwa mapuloteni ambiri kumachitika m'madera osokonezeka.Kuyanjana ndi MDN1 kunagawidwa muutali wonse wa mapuloteni, kuphatikizapo madera a LRR1, LRR6, CH, ndi madera osadziwika, pamene H2B makamaka amamangidwa ku CH domain (Mkuyu 6A, B).Mwachidziwitso, palibe kuyanjana komwe kunaphatikizapo TMJ, kuwonetsa kutsimikizika kwa njira ya CLMS (Chithunzi 6A, B).
MDN1 yadziwikanso ngati gawo la HLA-A protein network (Chithunzi 6A).Ndilo gulu la AAA la mapuloteni (ATPases okhudzana ndi ntchito zosiyanasiyana).Ili ndi gawo lomwelo la N-terminal AAA lomwe limapanga mphete ya hexameric ndikuchotsa chinthu cholumikizira ku 60S 64 ribosomal subunit.zikuwoneka ngati zofanana ndi dynein64,65,66.Kuphatikiza apo, dera lolemera la Asp/Glu limatsatiridwa ndi dera la MIDAS (malo odalira zitsulo).Chifukwa cha kukula kwakukulu kwa MDN1 (pafupifupi 5600 amino acid) ndi ma homology ake ochepa okhala ndi mapuloteni ophunziridwa bwino, zochepa zimadziwika za kapangidwe kake ndi ntchito mwa anthu.Tidazindikira HLA-A, H2B, ndi LRCH4 ngati omangirira MDN1 ndipo tidawulula momwe amayendera ngati ma protein complexes mu PyMol (Mkuyu 6A,B).Mapuloteni atatuwa amalumikizana ndi dera la AAA, dynein-like linker domain, ndipo mwina dera la MIDAS MDN1.Mu lipoti lapitalo, kuyeretsedwa kogwirizana kwa mapuloteni a nyambo kunazindikiritsa MDN1 ngati mapuloteni okhudzana ndi histone H2B67.Kuphatikiza apo, kafukufuku waposachedwa adawonetsanso kuyanjana pakati pa MDN ndi HLA-B m'maselo a HCT116 pogwiritsa ntchito ma affinity-purified mass spectrometry, kuchirikiza zomwe tapeza68.Kuzindikiritsa zovutazi mu zitsanzo zothandizidwa ndi IFNα zimasonyeza udindo wa MDN1 mu chizindikiro cha interferon.
Chifukwa majini a HLA ndi a polymorphic kwambiri, tidatenga masanjidwe owerengera HLA-A, -B, ndi -C kuchokera ku data yotsatizana ya RNA ya ma cell a Flo-1 (zambiri zomwe sizinawonetsedwe).Mayendedwe a peptide omwe amayenderana ndi kuwerengera adawonetsa kusiyana kwakukulu pakati pa HLA-A, -B, ndi -C m'magawo omwe ma peptide olumikizana adapezeka mu HLA-A (Chithunzi S3).Kuphatikiza apo, sitinawone kulumikizana kwa mapuloteni ndi mapuloteni a mamolekyu a HLA-B/C okhala ndi mapuloteni a H2B/HMGA1/MDN1/LRCH4.Izi zikusonyeza kuti kuyanjana kwa mapuloteni omwe amapezeka pakati pa HLA-A, MDN1, LRCH1 ndi HMGA1 ndi HLA-A yeniyeni.Kuphatikiza apo, kusanthula kwa proteomic kwa zitsanzo zosagwirizana (Table S4) kunawonetsa kuti HLA-A ili ndi kufalikira kopitilira muyeso poyerekeza ndi HLA-B kapena HLA-C.Ma peptides odziwika a HLA-A anali okwera kwambiri m'masampuli omwe amathandizidwa ndi IFNα komanso osasankhidwa.
Kuwonetsetsa kuti kuyanjana komwe kwadziwika pano sikunali chifukwa cha kulumikizana kosagwirizana kwa mapuloteni awiri moyandikana kwambiri, tidatsimikiziranso zinthu ziwiri zatsopano za HLA-A zomwe zimagwira ntchito poyesa ma co-immunoprecipitation.Kuyanjana kwa HLA-A ndi endogenous MDN1 ndi H2B kunapezeka m'maselo a Flo-1 omwe amathandizidwa ndi IFNα (Chithunzi 7, Chithunzi S4).Tinatsimikizira kuti HLA-A inagwidwa ndi H2B mu immunoprecipitates komanso kuti mgwirizanowu unali chifukwa cha chithandizo cha IFNα popeza HLA-A inalibe mu zitsanzo za immunoprecipitate kuchokera ku maselo osatulutsidwa (Chithunzi 7A).Komabe, deta yathu imasonyeza kuti IFNα imayendetsa mosiyana HLA-A kumangiriza ku H2B ndi MDN1.IFNα imayambitsa mgwirizano pakati pa H2B ndi HLA-A, koma imachepetsa kuyanjana kwake ndi MDN1.Tidapeza kuti MDN1 idalumikizidwa ndi HLA-A pakuwongolera, ndipo kuwonjezera kwa IFNα kunachepetsa kuyanjana kumeneku popanda kulowetsedwa kwa MDN1 ndi IFNα (Chithunzi 7B, C).Kuphatikiza apo, HLA-A immunoprecipitation inagwira H2B m'maselo a A549 (mkuyu. S4), kutanthauza kuti kuyanjana kumeneku sikudalira mtundu wa selo.Kuphatikizidwa, zotsatirazi zimathandizira kuyanjana kwa interferon-mediated kwa HLA-A ndi H2B ndi MDN1.
HLA-A imayeretsa H2B ndi MDN1.Oimira amkati a H2B (A) ndi MDN1 (B) ma immunoblots anali osatetezedwa kuchokera ku maselo a Flo-1 omwe amathandizidwa ndi IFNα ndikufufuza ma antibodies owonetsedwa.IgG ya mbewa ndi kalulu idagwiritsidwa ntchito ngati njira yoletsa.(C) Kuchulukana (kulowetsa) kwa ma antigen osiyanasiyana kumawonetsedwa ndi ma immunoblots omwe amafufuzidwa motsutsana ndi ma antibodies owonetsedwa, β-actin idagwiritsidwa ntchito ngati kuwongolera.
Mapangidwe a imodzi mwa maukonde odalirika kwambiri opangidwa ndi interferon, H2B-HLA-A-HMGA1, adafufuzidwa.Tidagwiritsa ntchito mawonekedwe a mamolekyulu ngati njira ina kuti timvetsetse kusinthika kwa mapuloteni omwe amakhudzidwa ndi zovutazi (Chithunzi 8).Malingaliro ochokera ku data ya CLMS akuwonetsa kuthekera kwa ma conformations osiyanasiyana a mapuloteni a H2B, HLA-A, ndi HMGA1.Choncho, zovuta zotsatirazi zomwe zingatheke zinapangidwira muzitsulo zosungunulira: H2B-HLA-A, HMGA1-HLA-A, ndi H2B-HLA-A-HMGA1.Chophimba choyambirira cha mapuloteni-mapuloteni ogwiritsira ntchito pulogalamu ya MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) phukusi linanena kuti zingakhale zosiyana pakati pa mapuloteniwa (Mkuyu 8A).Kuwona kwa mapuloteni a docking adawulula kuyanjana kangapo ndi ma conformations zotheka (Chithunzi 5A, 8).Chifukwa chake, kusinthika kumodzi kukuwonetsedwa mu Chithunzi 8A (cholembedwa maulalo) ndipo idawunikidwanso pogwiritsa ntchito payipi yachitsanzo ya MD.Kuphatikiza apo, kumanga kwa H2B kapena HMGA1 ku HLA-A kumawonetsa kuyanjana kwapamwamba kwa H2B kwa HLA-A (Mkuyu 8A).
Kusintha kosinthika kwa maukonde zotheka pakati pa H2B (H2BFS)-HLA-A, HMGA1-HLA-A, ndi H2B-HLA-A-HMGA1 zovuta.(A) Gulu lakumanzere ndi mapu a 2D (opangidwa mu pulogalamu ya SIM-XL) ya intramolecular (red) ndi intermolecular (blue) crosslinks (crosslink cutoff set to 3.5).Kuphatikiza apo, zotsalira zolumikizirana zodziwika zimalembedwa pamapangidwe a mapuloteni a H2B, HLA-A, ndi HMGA1.Zogwirizana ndi mapuloteniwa zidachotsedwa pogwiritsa ntchito payipi yolumikizira yomwe idakhazikitsidwa mu phukusi la MOE.Gulu lakumanzere lakumanzere likuwonetsa mitundu ingapo ya ma H2B-HLA-A ndi HMGA1-HLA-A okhala ndi ma protein omwe amamangirira mapuloteni osiyanasiyana (GBVI/WSA dG; kcal/mol).(B) Kupatuka kokhazikika (RMSD) kwa malo a atomiki (kupatula ma atomu a haidrojeni) pamapuloteni aliwonse.(C) Intermolecular mapuloteni-mapuloteni a haidrojeni chomangira cholumikizira kuchokera kumitundu yosiyanasiyana yofananira poganizira kuyanjana kwanthawi yayitali ≥ 10 ns.Kutalika kwa h-bond donor-acceptor cutoff kunayikidwa ku 3.5 Å, ndipo mbali ya donor-H-acceptor cutoff inayikidwa ku ≥ 160 ° -180 °.(D) Zotsalira zolembedwa zomwe zimapanga kuyanjana kwa mapuloteni a HLA-A ndi okondedwa awo, zoyambira ≥ 20 ns, zotengedwa ku dummy HLA-A-H2B ndi HLA-A-HMGA1 zovuta.Mapangidwe a mapuloteni amayimira pafupifupi 100 ns MDS.(E) Kuyanjana kwapakati pa ma HLA-A-H2B ndi ma HLA-A-HMGA1 kuyerekeza ndi machitidwe omwe amatsatiridwa ndi kuyerekezera kwa H2B-HLA kupitilira 100 ns kutengera malo olumikizirana a K kapena S pakati pa ma peptide awiriwa.Zovuta /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Mlingo wocheperako pakuwunika kwa maulalo olumikizirana adayikidwa ku 3.0, ndipo kuyanjana kwapadera kuchokera ku MDS kutenga ≥ 10 ns kudaganiziridwa.Mapangidwe a mapuloteni adawonetsedwa pogwiritsa ntchito BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) ndi phukusi la Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).
Kukhazikika kwa mamolekyu a HLA-A pakapita nthawi (kupatuka kokhazikika; RMSD kapena kupatuka kokhazikika; RMSF) kunawonetsa kuti kupezeka kwa mapuloteni a H2B kapena HMGA1 m'macomplexes adakhazikika HLA-A (Chithunzi 8B, Chithunzi S5).Puloteni ya HMGA1 imamangiriza mwamphamvu ku malo a B2M a HLA-A, kupangitsa kukhazikika kwa ma amino acid a HLA-A mu HLA-A-HMGA1 kapena H2B-HLA-A-HMGA1 complex (Chithunzi 8B, Chithunzi S5).makamaka, zotsalira za HLA ~ 60-90 ndi ~ 180-210 zinapezeka kuti sizingasinthe kwambiri pamaso pa H2B (FIG. 8B).H2B ndi HMGA1 zinawonetsa kumangiriza bwino ku HLA-A mu zovuta za H2B-HLA-A-HMGA1 poyerekeza ndi HLA-A yomangiriza ku H2B kapena HMGA1 yokha (Chithunzi 8C,D; Table S5).Zotsalira zomwe zimakhudzidwa ndi hydrogen bonding (MD modeled high occupancy ≥ 10 ns) zimagwirizana ndi malo ochezera a CLMS (K kapena S zotsalira) muzovuta, kutanthauza kuti kuyanjana komwe kumadziwika ndi CLMS ndi kodalirika kwambiri.Kudalirika (mkuyu 8E).Mu CLMS ndi MD modelling, zotsalira za HLA-A pakati pa 190-210 ndi pafupifupi 200-220 amino acid zinapezeka kuti zimamanga H2B ndi HMGA1, motsatira (FIG. 8E).
Kuyanjana kwa mapuloteni-mapuloteni kumapanga maukonde osinthika omwe amalumikizana ndi ma intracellular kuyankhulana potengera zovuta zina.Chifukwa njira zambiri zamapuloteni zimazindikira kusintha kwa kuchuluka kwa protein yokhazikika, mphamvu zama protein-mapuloteni zimafunikira zida zowonjezera kuti zigwire zolumikizira zomangira, ndipo CLMS ndi chida chimodzi chotere.Dongosolo lozindikiritsa la interferon ndi netiweki ya cytokine yomwe imalola ma cell kuyankha kuzinthu zosiyanasiyana zachilengedwe za pathogenic ndi intrinsic pathological sign, zomwe zimafika pachimake pakulowetsedwa kwa magawo a mapuloteni a interferon-inducible.Tinagwiritsa ntchito CLMS kuti tidziwe ngati kuyanjana kwatsopano kwa mapuloteni-mapuloteni kungadziwike pakati pa gulu la mapuloteni opangidwa ndi interferon.Kusanthula kwa mapuloteni apadziko lonse lapansi mumtundu wa Flo-1 woyankha wa interferon kunagwiritsidwa ntchito kujambula mapuloteni.Kutulutsa ma peptide a tryptic kuchokera m'maselo osalumikizana ndi ophatikizika amalola kuwerengera ma peptide, kupititsa patsogolo njira, ndi kugawa kwautali wa peptide ndikutanthauzira mwamphamvu kwa LFQ.Mapuloteni a Canonical interferon-inducible adadziwika kuti ali ndi mphamvu zoyendetsera mkati, pamene ma intermolecular ndi intramolecular cross-linked adducts a canonical interferon-inducible proteins monga MX1, UP18, OAS3 ndi STAT1 adawonedwa.Mawonekedwe osiyanasiyana apangidwe ndi kuyanjana m'malo ogwirira ntchito afufuzidwa.
Kuyanjana pakati pa HLA-A, MDN1 ndi H2B kunazindikirika ndi immunoblotting mu Flo-1 ndi A549 maselo ochiritsidwa ndi osagwiritsidwa ntchito ndi IFNα.Zotsatira zathu zikuwonetsa kuti HLA-A imakhala ndi H2B m'njira yodalira IFNα.Ntchito yathu ikuyimira njira yosangalatsa yowunikiranso kugwirizanitsa kwa magawo awiriwa.Zingakhalenso zosangalatsa kukulitsa njira ya CLMS ku gulu la mizere ya maselo kuti muzindikire kuyanjana kwa mapuloteni amtundu wa interferon-mediated protein.Pomaliza, tidagwiritsa ntchito ma MD modelling ngati njira ina yomvetsetsa kusinthika kwa mapuloteni omwe amakhudzidwa ndi zovuta za H2BFS-HLA-A-HMGA1, zomwe zimatsata ma intramolecular ndi intermolecular cross-tals.Malingaliro ochokera ku data ya CLMS akuwonetsa kuthekera kwa ma conformations osiyanasiyana a mapuloteni a H2BFS, HLA-A, ndi HMGA1.Kusiyana kosiyana komwe kungatheke pakati pa mapuloteni a docking awa kunavumbulutsa zochitika zingapo zofanana ndi zomwe zimawonedwa mu dataset ya CLMS.Chimodzi mwazamphamvu kwambiri panjira yathu ndikuti imalola kuzindikirika kosavuta kwa majini omwe amalumikizana kwambiri ndi ma polymorphic monga HLA, chifukwa chake zidzakhala zosangalatsa kuphunzira kuyanjana kwa mapuloteni a HLA haplotype omwe ndi ovuta kuphunzira.Kuphatikizidwa pamodzi, deta yathu ikuwonetsa kuti CLMS ingagwiritsidwe ntchito kukulitsa kumvetsetsa kwathu kwa ma interferon-induced signing network ndikupereka maziko ophunzirira machitidwe ovuta kwambiri a intercellular mu chotupa microenvironment.
Maselo a Flo-1 anapezedwa kuchokera ku ATCC ndipo amasungidwa mu DMEM (Gibco) yowonjezeredwa ndi 1% penicillin/streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) ndi kusungidwa pa 37°C ndi 5% CO2.Incubation.Maselo anakula mpaka 70-80% confluence asanalandire chithandizo ndi IFNα14 (opangidwa ndi Edinburgh Protein Production Facility).Mankhwala ena onse ndi ma reagents adagulidwa ku Sigma Aldrich pokhapokha atadziwika.
Maselo a Flo-1 adakula mu mbale za 6-chitsime ndipo tsiku lotsatira maselowo anachitidwa ndi 10 ng / ml IFNα14 kwa maola 24 mpaka pafupifupi 80% confluence.Maselo adatsukidwa katatu ndi PBS ndikugwirizanitsa ndi DSS (Thermo Fisher Scientific) yokonzedwa mwatsopano (kusungunuka mu DMSO) mu PBS kwa 5 min pa 37 ° C. mpaka kumapeto kwa 0.5 mM.DSS crosslinking reaction idasinthidwa ndi PBS ndipo DSS yotsalira idazimitsidwa ndikuwonjezera 20 mM Tris (pH 8.0) mu PBS kwa mphindi 15 pa 37 ° C.Maselo anasonkhanitsidwa ndi kukanda ndikusonkhanitsidwa m'machubu otsika (Axygen).
Selo pellet idakulungidwa ndi 300 µl ya urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) kwa mphindi 30 kutentha kwa chipinda ndikugwedezeka kwakanthawi.Masitepe onse a centrifugation adachitidwa pa 14,000 xg pa 8 ° C.Centrifuge the lysate kwa mphindi 10 ndikusamutsa supernatant ku chubu chatsopano.Tinthu tating'onoting'ono tomwe tatsala timasungunuka mu 150 μl yachiwiri ya lysis buffer (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) kwa mphindi 30 kapena kuposerapo mpaka yankho lamadzimadzi lokhala ndi homogeneous litapezeka.The lysate anali centrifuged kwa mphindi 20 ndipo supernatant anali kusakaniza ndi lysate anapeza mu sitepe yapita.Kuchuluka kwa mapuloteni kunayesedwa pogwiritsa ntchito kuyesa kwa Micro BCA (Thermo Fisher Scientific) molingana ndi malangizo a wopanga ma microplate.Zitsanzozo zimawumitsidwa mwachangu mu nayitrogeni wamadzimadzi ndikusungidwa pa -80 ° C.
Pafupifupi 100 μg ya puloteni yosungunuka yosungunuka idakonzedwa pogwiritsa ntchito njira yosinthira sampuli yokonzekera (FASP) monga momwe Wisniewski et al.69 Mwachidule, puloteniyi imalumikizidwa ndi 200 µl ya urea buffer (8 M urea mu 0.1 M Tris, pH 8.5), yovunditsidwa ndi kugawa pakati.Masitepe onse a centrifugation adachitidwa pa 14,000 xg pa 25 ° C.Theka loyamba la mapuloteni a lysate olumikizidwa ndi mtanda adasamutsidwa ku chipangizo cha 10 kDa Microcon centrifugal chokhala ndi nembanemba ya Ultracel-10 (Merck), ndikutsatiridwa ndi centrifugation pa fyuluta kwa mphindi 25.Kenaka yikani theka lachiwiri la mapuloteni ku fyuluta ndikubwereza masitepe omwewo.Kuchira kwa mapuloteni kunachitidwa powonjezera 100 μl ya 17 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) mu urea buffer.Kubwezeretsa kunalimbikitsidwa pa thermomixer pa 600 rpm kwa mphindi 30 pa 37 ° C.Kuphatikiza apo, gawoli lidapangidwa ndi centrifuged ndipo puloteni yochepetsedwa yolumikizidwa idapangidwa alkylated pogwiritsa ntchito 100 μl ya 50 mM iodoacetamide mu urea buffer.The alkylation anachita ikuchitika firiji kwa mphindi 20 mu mdima.Tembenuzani mzatiwo, sambani makoma azagawo katatu ndi 100 µl urea buffer, ndiyeno centrifuge.Opaleshoni yomweyi idachitika katatu pogwiritsa ntchito 100 μl ya 100 mm ammonium bicarbonate.Isanafike trypsinization, sinthani chubu chosonkhanitsira ndi chatsopano.Onjezani chosungira chimbudzi chokhala ndi 50 mM ammonium bicarbonate ndi 1 µl trypsin yosungunuka mu trypsin buffer (Promega).Chiŵerengero cha trypsin ndi mapuloteni chinasungidwa pafupifupi 1:33, ndipo machitidwe a chimbudzi anakulungidwa usiku wonse pa 37 ° C. m'chipinda chonyowa.Peptide yolumikizidwa idachotsedwa mu fyuluta ndi centrifugation kwa mphindi 25.Kuchira kwa peptide kunasinthidwa powonjezera 50 μl ya 0.5 M NaCl ku fyuluta, kutsatiridwa ndi centrifugation kwa mphindi 25.
Mipingo ya C18 Micro Spin (Harvard Apparatus) idagwiritsidwa ntchito pochotsa mchere wa tryptic peptides wolumikizana motsatira ndondomeko yofotokozedwa ndi Bouchal et al.70 ndi zosintha zazing'ono.Mwachidule, mizati ya C18 spin adatsegulidwa ndi kutsuka katatu kwa 0.1% formic acid (FA) mu acetonitrile (AcN) (Merck) ndi kutsuka kawiri kwa 0.1% FA.Mzerewu udathiridwa madzi ndi 0.1% FA kwa mphindi 15.Kwezani zitsanzo muzitsulo zozungulira ndikutsuka katatu ndi 0.1% FA.Ma peptides ochotsedwa adasinthidwa motsatizana ndi kutsika pang'ono pogwiritsa ntchito 50%, 80% ndi 100% AcN mu 0.1% FA.Zitsanzozo zidawumitsidwa mu SpeedVac Plus concentrator (Eppendorf) mpaka madzi otsalawo atazimiririka.Ma peptides ochotsedwa adasungunuka mu 100 μl ya 0.08% trifluoroacetic acid mu 2.5% AcN ndipo zokhazikika zidayezedwa pa NanoDrop 2000 (Thermo Scientific).Pafupifupi 1 μg ya peptide yophatikizika pachitsanzo chilichonse idayikidwa mu LC-MS/MS system.
Ma peptides olumikizana ndi mtanda adalekanitsidwa pa UltiMate 3000 RSLCnano LC system (Thermo Scientific) yolumikizidwa ndi Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Ma peptide olumikizana ndi mtanda adasonkhanitsidwa pa ID ya 300 µm, 5 mm kutalika µ-pre-column C18 column yodzaza ndi C18 PepMap100 sorbent ndi 5 µm PepMap sorbent (Thermo Scientific).Kwezani mpope wotuluka pa 5 µl/mphindi 0.08% trifluoroacetic acid wosungunuka mu 2.5% AcN.Ma peptides olumikizana ndi mtanda adalekanitsidwa pagawo lowunikira la silika lokhala ndi mainchesi amkati a 75 μm ndi kutalika kwa 150 mm, lodzaza ndi 2 μm PepMap sorbent (Thermo Scientific).Magawo am'manja A ndi B anali ndi 0.1% FA m'madzi ndi 0.1% FA mu acetonitrile, motsatana.Kutsika kumayambira pa 2.5% B ndikuwonjezeka motsatira 40% B pa mphindi 90, kenako mpaka 90% B pa mphindi ziwiri zotsatira.Kuphatikizika kwa gawo la mafoni kunasungidwa pa 90% B kwa mphindi 10 kenako kutsika motsatira mpaka 2.5% B pa mphindi ziwiri.Mzerewu unayanjanitsidwa pa 2.5% B kwa mphindi 8 musanayambe kuzungulira.Ma peptides olumikizidwa ndi mtanda omwe adachotsedwa pamndandanda wowunikira adayikidwa mu gwero la nanoelectrospray ionization (NSI) ndikubayidwa mu Exploris 480 mass spectrometer (Thermo Scientific).
Orbitrap Exploris 480 mass spectrometer imagwira ntchito molumikizana bwino ndi data.Kujambula kwathunthu kunachitika mumayendedwe agawo pa kusamvana kwa 120,000 ndi zoikamo zosiyanasiyana kuchokera m/z 350 Th mpaka m/z 2000 Th.Cholinga cha AGC chokhazikika chinakhazikitsidwa pa 300% ndi nthawi yowonjezereka ya 50ms.Kuzindikira kwapamwamba kwa monoisotopic kwakhazikitsidwa kwa peptides.Parameter yopumula imayikidwa kuti ikhale yowona ngati ma precursor ochepa apezeka.Mphamvu yocheperako ya ionic ya kalambulabwalo idakhazikitsidwa ku 5.0e3 ndipo malipoti oyambira mpaka +8 adaphatikizidwa pazoyeserera.
Nthawi yozungulira pakati pa masikelo akulu mumachitidwe olumikizana ndi data idakhazikitsidwa kukhala masekondi 2.5.Kupatulapo kwamphamvu kunakhazikitsidwa ku 20s pambuyo pa kugawikana koyamba kwa ayoni woyambira.Zenera lodzipatula lotsogola linakhazikitsidwa ku 2 Th.Mtundu wa mphamvu zowombana zokhazikika zokhala ndi mphamvu zogundana zokhazikika zidasankhidwa potengera deta ya MS/MS.Mphamvu zogundana zakhazikitsidwa ku 30%.Chigamulo cha Orbitrap chinakhazikitsidwa ku 15,000 ndi cholinga cha AGC ku 100%.Nthawi yokwanira yojambulira imayikidwa ku 60 milliseconds.
Tisanatsatire ma netiweki a protein-protein mu zitsanzo zolumikizidwa, tidakonza mafayilo aiwisi pogwiritsa ntchito phukusi la MaxQuant (mtundu 1.6.12.0) 26,27 kuti tidziwe ma peptides/mapuloteni omwe amatha kutsatiridwa m'zitsanzozo.Kuphatikiza apo, kusanthula kofananira kwa proteinomic kunkachitika pazitsanzo za Flo-1 zosagwirizana komanso zosagwiritsidwa ntchito ndi IFNα.Zambiri za MS/MS zidafufuzidwa munkhokwe ya anthu ya UniProt (www.uniprot.org) (yomwe idakwezedwa pa Ogasiti 12, 2020, ili ndi zolembedwa 75,093) pogwiritsa ntchito injini yosakira yomangidwamo Andromeda27.Kusakaku kunachitika popanda kuwonetsa kutsimikizika kwa enzyme ndikusintha kosiyanasiyana kwa deamidation (N, Q) ndi oxidation (M).Kulekerera kwakukulu kwa precursor kunayikidwa pa 20 ppm ndi ma ions azinthu ku 0.02 Da.Kupatuka koyambirira komanso kopitilira muyeso kunayikidwa ku 10 ppm.Kulemera kwakukulu kwa peptide kunayikidwa pa 4600 Da ndipo kufanana kwake kunayikidwa pakati pa 7 ndi 25 amino acid (aa).Kusanthula kwina kowerengera kunachitika pogwiritsa ntchito pulogalamu ya Perseus (mtundu 1.6.10.45).Mapuloteni adawerengedwa posintha kuchuluka kwa puloteni (LFQ intensity; unlabeled quantification)27 ndipo mphamvu zake zidasinthidwa kukhala Log2.Kuphatikizika kwamapuloteni odziwika ndi kuchuluka kwawo kwa peptide kudapangidwa pogwiritsa ntchito phukusi la pheatmap (v1.0.12) mu R (v 4.1.2).Kusanthula kopindulitsa kwa njira kunachitika pogwiritsa ntchito nkhokwe ya Reactome pathway ya mapuloteni opangidwa ndi IFNα omwe anali atatsegulidwa kupitilira kanayi poyerekeza ndi zitsanzo zosasinthidwa.
Kuzindikiritsa lysine (K) kapena serine (S) mankhwala ophatikizika a protein complexes omwe amawunikidwa ndi LC-MS/MS kunachitika pogwiritsa ntchito makina ozindikiritsa a spectroscopic (SIM-XL) a peptides olumikizana ndi mtanda (SIM-XL)29.Choyamba, zotheka kuyanjana pakati pa interferon-associated (IFN) DNA damage resistance resistance signature (IRDS) majini adafufuzidwa pogwiritsa ntchito IRDS protein dataset yofotokozedwa Padariya et al.28.Kuwunika zochitika zonse ndi kubwereza kwa UniProt yonse yaumunthu ndizovuta kwambiri, kotero kuti database yonse ya UniProt yaumunthu (www.uniprot.org) (yotsitsidwa pa 12 August 2020, ili ndi zolemba 75,093) motsutsana ndi kubwereza kochitidwa ndi IFNα.Chimodzi mwazosefera za kudalirana kwakukulu.Kuyanjana kwakukulu kumeneku komwe kunapezedwa kunakulitsidwa ndikuyesedwa muzobwereza ndi zochitika zonse.
Mu SIM-XL, DSS idagwiritsidwa ntchito pa crosslinker (XL) ndi kusintha kwa kulemera kwa XL ndi kusintha kosintha kulemera kunayikidwa ku 138.06 ndi 156.07, motsatira.Masamba otsatirawa amaganiziridwa: KK, KS ndi KN-TERM, opanda ma ion atolankhani.Ma precursor ndi fragment ppm adayikidwa ku 20 ndipo malire a Xrea adayikidwa ku 0.15.Trypsin ankaonedwa kuti ndi yeniyeni, ndipo njira yogawanitsa C-trap (HCD) yamphamvu kwambiri inakhazikitsidwa.The XCorr dynamic DB kuchepetsa malire ndi chiwerengero chochepa cha ma peptide ochepetsera DB osinthika adayikidwa ku 2.5 ndi 2, motsatana.Magawo ena ndi: kuthekera kwa monoisotope ndi kudulidwa kwakukulu kwangochitika mwangozi, zosachepera 4 AA zotsalira pa chingwe chilichonse komanso kuchuluka kwa zingwe, ndi maxima atatu odukaduka ophonya.Mamapu osokedwa a 2D adawunikidwa mu (SIM-XL) ndipo mawonekedwe a xQuest28 adagwiritsidwa ntchito kupanga mamapu a 2D.Maprotein crosslinks pamagulu a mapuloteni amaperekedwa mu PyMol (PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC).
Mapangidwe amtundu wa mapuloteni adapangidwa pogwiritsa ntchito seva ya Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)11 pogwiritsa ntchito mfundo za homology modeling ndi kukhazikitsa "Hidden Markov Method".Phyre2 imapanga zomangira zachitsanzo potengera kutsatana kotsatana ndi mapuloteni odziwika.Pamapuloteni a H2BFS, HLA-A, HMGA1, LRCH4, ndi MDN1, mapangidwe a template 1kx552, 1kj349, 2eze55, 6hlu62, ndi 6i2665 adagwiritsidwa ntchito.Kuphatikiza apo, mapangidwe a AlphaFold71 MX1, UBP18 ndi ROBO1 adaganiziridwanso.Mapangidwe a mapuloteni adawonetsedwa pogwiritsa ntchito phukusi la BIOVIA Discovery Studio Visualizer (Dassault Systèmes, BIOVIA, San Diego, CA, USA) ndi phukusi la Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).