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Iro interferon signaling system inokonzera mhinduro yakasimba ye cytokine kune zvakasiyana-siyana zvepathogenic uye intrinsic pathological zviratidzo kubva kune zvakatipoteredza, zvichiita kuti kuiswa kwezvikamu zviduku zveprotein-inducible proteins.Isu takashandisa DSS-mediated cross-link mass spectrometry (CLMS) kuti tione kutsva kweprotein-protein munharaunda ye interferon-induced proteins.Pamusoro pezvinotarisirwa interferon-inducible mapuroteni, takaona zvakare novel intermolecular uye intramolecular cross-linked adducts ecanonical interferon-inducible mapuroteni akadai seMX1, USP18, OAS3, uye STAT1.Isu takatarisa pane orthogonal kusimbiswa kweiyo novel seti ye interferon-inducible protein network yakaumbwa neHLA-A mapuroteni (H2BFS-HLA-A-HMGA1) vachishandisa co-immunoprecipitation uye kwavo kuenderera mberi nekudzidza vachishandisa mamorekuru dynamics modelling.Kuenzanisira kweiyo conformational dynamics yeprotein complex yakaratidza akati wandei masaiti ekudyidzana airatidza kupindirana kwakaonekwa mune zvakawanikwa zveCLMS.Pamwe chete, tinopa chidzidzo chemutyairi weCLMS kuti tione zvitsva zvekuisa zviratidzo zvinokonzerwa neinterferon, uye tinotarisira kushandiswa kwakakura kweCLMS kuziva maitiro matsva ekubatana kweprotein mune tumor microenvironment.
Isati yatanga mhinduro yekudzivirira yekudzivirira, iyo innate defence system inosimudza antimicrobial mhinduro inoyananiswa nemhuri yeakavanda alpha-helical cytokines anonzi interferons (IFNs).Mhando I IFN makirasi IFNα uye IFNβ inogadzirisa mhinduro dzemasero, kusanganisira antiviral, proapoptotic, proinflammatory, uye antiproliferative states.Muvanhu, 13 subtypes yeIFNα inozivikanwa, yose yakabatanidzwa pane chromosome 91. Zvinoshamisa kuti IFNα2 chete yakadzidzwa kushandiswa kwekliniki.Munguva pfupi yapfuura, kutarisisa kwakabhadharwa kutsvagisa pane mamwe masubtypes eIFNα.Ongororo ichangoburwa yakaratidza kuti IFNα14 ndeimwe yeanonyanya kushanda isoforms mukudzora HBV2 uye HIV-13,4 kudzokorora zvichienzaniswa neiyo canonical IFNα2 subtype.
Zvakatarwa kuti activated type I interferon receptor complexes (IFNAR1 uye IFNAR2) inokonzera chiratidzo chekufambisa cascade inopindirana naJanus kinases TYK2 uye JAK15,6.Aya Janus kinases phosphorylate signal transducers uye transcriptal protein activators (STAT1 uye STAT2) pane tyrosine zvakasara kuti itange SH2 domain-mediated heterodimerization6.Zvadaro, IRF9 inosunga STAT heterodimers kuti iite trimeric complex yeIFN-stimulated factor 3 gene (ISGF3), iyo inotamira kune nucleus uye inoita kuti kunyorwa kwe2000 interferon-stimulated genes (ISGs)5,6,7,8.
ISGs inoumba musana weiyo innate immune system, kunyanya mukupindura kurwisa kwehutachiona.Sezvo mutsara wekutanga wekudzivirira kurwisana nehutachiona hwehutachiona, masero anokurumidza kuendesa kupindirana kwakawanda kwemapuroteni emasero ane zvakawanda zvakasiyana-siyana zvehupenyu.Aya mapuroteni anosanganisira mapatani ekucherechedzwa receptors, kusaina mamorekuru, zvinyorwa zvinyorwa, uye mapuroteni ane akananga antiviral mabasa, pamwe neasina kunaka anodzora mhinduro dzemuviri9.Ruzhinji rweruzivo rwechiitwa cheISG runobva kumasikirini anoshanda anoshandisa overexpression screens10,11 kana gene silences matekiniki (siRNA, RNAi uye CRISPR)12,13 umo maISG ega anoratidzirwa kana kuvharirwa uye basa ravo rinoedzwa pamavhairasi akasiyana.Kunyangwe izvi zvidzidzo zvakatemesa antiviral zvimiro zveISG yega yega, ari pasi pemamorekuru maitiro eISG yega yega anoramba asingazivikanwe.Zvinowanzogamuchirwa kuti mapuroteni mazhinji anodyidzana neimwe kana anopfuura cytokines kuti ave nechokwadi chekuita kwakazara, saka ingave maISG anopindirana zvakananga kana kupindirana kwavo kunoyananiswa nemapuroteni emasero.Semuenzaniso, ichangoburwa photocrosslinked proteomics study yakaratidza ATPase VCP/p97 sechinhu chikuru cheFITM3 chekubatana, iyo inhibition inotungamirira kune kukanganisa mukugadzirisa lysosomal, kudzoka, uye cotransport yeFITM3 ine viral particles 14.Tichishandisa immunoprecipitation, takaona VAPA, vesicle-associated protein, semubatsiri wekudyidzana neIFITM1/2/3 iyo inopindirana necholesterol-mediated viral maturation, uye izvi zvakasimbiswa neimwe chidzidzo uchishandisa mbiriso mbiri-hybrid system.Scientific Support 15, 16.
Iyo yakakosha biological process inobatanidzwa mukudzvinyirirwa kwehutachiona uye shanduko yakaipa ndeye antigen mharidzo, iyo inopindirana nehukuru histocompatibility complex (MHC) mamorekuru.Peptides (8-12 amino acids kureba) kubva kune yakatsemuka, nguva isati yakwana kana kuti yakashata mapuroteni anotakurwa muMHC-I heterodimer (ine MHC-I inorema uye maketani akareruka, anonzi β-2-microglobulin; β2M) 17,18.Izvo zvinoguma zvakagadzikana MHC-I trimers inotakurwa kuenda kune sero pamusoro, kwainopa intracellular peptides kuCD8 + T masero (cytotoxic T masero)17.Masero eT anoziva nekuparadza hutachiona uhu uye masero anotakura bundu-specific antigen.Nekuda kweizvozvo, hutachiona uye bundu masero anowanzo kudzvanya antigen kuratidza maitiro kuti adzivise kutariswa kwekudzivirira.Mukuwedzera, MHC-I yakaderedzwa mu40-90% yemamota evanhu uye inowanzobatanidzwa nehurombo hurombo19.
Majini anosanganisirwa mukupindura utachiona anofanira kukurumidza kushandura pakati penzvimbo yekuzorora uye mamiriro ekuita kunyorwa.Nokudaro, mapuroteni akawanda emagetsi anofungidzirwa kuti anobatanidzwa mukupindura kwepamusoro kudiwa kweIFN kwenguva pfupi, kusanganisira kugadzirisa uye kuchinjwa kwekusimudzira chromatin 20,21.Zvidzidzo zvakawanda zvakatarisana nekuzivikanwa kwega ISG mapuroteni ekudyidzana pamberi peIFN.Zvidzidzo zvakawanda zveproteomic uye transcriptomic mumuenzaniso masero akajekesa mhedzisiro yeIFN pane cellular landscape.Zvisinei, pasinei nekunzwisisa kuri kukura kwemasimba anokonzerwa ne interferon, isu tichiri kuziva zvishoma nezvekubatanidzwa kweISGs.Paunenge uchifunga nezvekuoma uye nguva-inotenderera inotenderera ye interferon signing, mibvunzo miviri inomuka: (i) zvinokwanisika here kudzikamisa uye kuteya ma-multiprotein complexes anobatanidzwa mukukurumidza kusaina, uye (ii) uku kupindirana kunogona kurongerwa munzvimbo ye3D here?
Kuti tigadzirise nyaya idzi, takashandisa disuccinimide suberate-mediated chemical cross-linking (DSS) pamwe chete ne mass spectrometry (CLMS) kuti tidzidze IFNα-induced protein interaction network uye simba rayo.DSS inowedzera covalent zvisungo pakati peproximal zvakasara zvemapuroteni uye/kana mapuroteni complexes mu vivo.Ongororo yakazotevera yeMS inoburitsa nzvimbo dzakanangana dzekuyambuka dzinoratidza kudhuze kwenzvimbo dziri mukati meprotein yakati, inonzi internal linkages, kana subunits mumaprotein complexes, anonzi interrelationships.Tichishandisa nzira iyi, takaona akati wandei maprotein-protein complexes pamwe neinterferon-induced multiprotein interaction network.Nekuenderera mberi nekuyedza chikamu chekudyidzana kutsva uku, tinoratidza kuti H2BFS (H2B histone-type FS; inozonzi H2B) uye MDN1 inoshanda sevanosunga vanobatana neHLA-A.
Flo-1 masero ndeimwe yeanonyanya kuzivikanwa in vitro modhi yesophageal adenocarcinoma sezvaanotevedzera akakosha eesophageal tumors22,23.Zvisinei, haasi mamota ose ari immunogenic, uye kuti aone kana Flo-1 masero anopindura kune interferon kurapwa, takabata Flo-1 masero ane 10 ng / ml IFNα kwemaawa 72.Flo-1 masero akaratidza kutanga kwekutanga kwepSTAT1 uye IRF1, kutanga maawa maviri mushure mekurapa uye kuenderera mberi kwemaawa 72, nekuderera kunoenderana nenguva mumatanho akamira eRF1 (Mufananidzo 1A).ISGs (MX1, IFITM1, OAS1 / 2, uye ISG15) yakawanikwa yakanyatsokurudzirwa mushure meawa 6, kutevedzera yekare yepakati uye yekupedzisira chikamu mhinduro kuIFNα (Mufananidzo 1A).Pamwe chete, idzi data dzinoratidza kuti iyi cellular modhi inogona kushandiswa kudzidza mhinduro dze interferon.
Yakasiyana mapuroteni ekutaura mhinduro muFlo-1 masero mushure meIFNα kurapwa.(A) Protein kutaura muFlo-1 masero anobatwa ne 10 ng / ml IFNα ye2, 6, 24, 48 uye maawa 72 akaongororwa ne immunoblot achishandisa zviratidzo zve ISG zvakaratidzwa.(B) Coomassie yebhuruu yakasvibiswa SDS-PEJI gels yesero yesero yakatorwa mushure mekuyambuka-kubatanidza neDSS kwenguva dzakatarwa uye kutarisisa.(C) Representative immunoblot akaongororwa nep53 (DO-1) antibody kubva kumasampuli akafanana kuti aongorore chiyero cheprotein cross-linking.
Kubata iyo in situ protein yekudyidzana mamiriro, takashandisa DSS, inoshandiswa zvakanyanya muchinjiko-yekubatanidza mumiririri nekuda kweiyo yakakwirira membrane permeability uye ipfupi kuita nguva.Iyo ipfupi yekuita nguva inobatsira kudzivirira kuumbwa kweakakura aggregates e crosslinked mapuroteni, nekudaro kuchengetedza kugadzikana kweiyo crosslinker.Kuti titarise iyo yakakwana DSS yekumisikidza uye kudzivirira kudarika-kuyambuka, isu takatanga tafumura maseru ku5, 2.5, uye 1 mM DSS ye5, 10, 5, uye 30 maminetsi, zvichiteerana, uye kuongorora lysates neCoomassie-yakasvibiswa SDS-PAGE. (data haina kuratidzwa).Masero lysates anoita seakanyanya kuchinjika-akabatana pakudzika kwakadzika uye panguva pfupi pfupi.Naizvozvo, DSS yakanzi 1, 0.5, uye 0.1 mM pamusoro 5 maminetsi (Figure 1B).Optimal crosslinking yakaonekwa ne 0.5 mM DSS kwemaminetsi e5, uye aya mamiriro akasarudzwa kumasero anobatwa neIFNα.Pamusoro pezvo, Mufananidzo 1C unoratidza blot yekuMadokero yakaitwa uchishandisa p53 (DO-1) antibody kuongorora dhigirii reprotein cross-linking.
Flo-1 masero akabatwa ne10 ng / ml IFNα kwemaawa 24 asati awedzera crosslinker.Masero e-cross-linked akazoshandiswa ne-two-step proteolysis uye mapuroteni akashandiswa neFASP (Fig. 2) 24,25.Cross-linked tryptic peptides yakaongororwa ne mass spectrometry (Fig. 2).Iyo MS/MS spectra inozofananidzwa neprotein sequence uye inoverengerwa neMaxQuant26,27.Cross-linked peptides yakaonekwa kubva kune yakawanikwa spectra uchishandisa chirongwa cheSIM-XL, uye makemikari ega akabatanidzwa muhutano hwakaoma achishandisa xQuest28 uye SIM-XL29 open source computing software pipelines (Fig. 2).SIM-XL inozivisa kupindirana kweprotein-protein, maketani emukati uye maketani ega ega mumisanganiswa yeprotein iri nyore kana yakaoma uye inopa zvinyorwa zvekuona kupindirana mumapuroteni maitiro.Uye zvakare, inoisa imwe neimwe muchinjika-referensi seye ID mamakisi zvinoenderana neMS/MS29 spectrum mhando.Zvizhinji zvakavimbika zveprotein-protein interactions uye zvigadziriswa zvakave zvichionekwa, uye imwe itsva yekubatana yakave ichiongororwa zvakare uchishandisa co-immunoprecipitation uye conformational shanduko yezvakaoma kushandisa molecular dynamics (MD) modeling (Fig. 2) 30, 31.
Schematic overview yenzira yeCLMS.Flo-1 masero akabatwa ne 10 ng / ml IFNα kwemaawa 24 achiteverwa ne in situ protein cross-linking achishandisa DSS inoteverwa ne cell lysis uye trypsinization.Masampuli akabatanidzwa-akabatanidzwa akaongororwa achishandisa Orbitrap mass spectrometer uyezve sampled yekupatsanurwa kwepeptide precursors panguva yeLC-MS/MS.Mapeptide maviri akabatanidzwa akaonekwa kubva kune yakawanikwa spectra uchishandisa Spectrum Recognition Machine yeCrosslinked Peptides (SIM-XL) chirongwa, uye ese makomisheni akasanganiswa kuita network yakaoma uchishandisa computational mapaipi.Sefa kudyidzana kwakaderera kunoenderana nekunyepa kwakanaka mwero (FDR) zvibodzwa.Mitsva yakawanda-yakanyanya kuvimbika protein-protein kupindirana kwakawedzera kusimbiswa uchishandisa co-immunoprecipitation, uye conformational shanduko mumacomplexes yakaongororwa uchishandisa mamorekuru dynamics (MD) modelling.
Zvose zve ~ 30,500 uye ~ 28,500 peptides zvakaonekwa zvichishandisa MaxQuant mune zvisingasviki uye zvinokurudzira IFNα samples, maererano (Supplementary Table S1, Fig. 3A).Kupararira kwepeptide kureba mumatambudziko ose maviri kwakaratidza chikamu chepamusoro chepeptide yakakura, zvichiratidza kuvapo kwepamuchinjikwa-yakabatanidzwa peptides (Fig. 3B, C).Mukuwedzera, chikamu chikuru chepeptide yakakura yakanga iripo muhuwandu hwe40-55 muIFNα-yakabatwa samples (Fig. 3C).Protein mepu inopesana nelog2 intensity yakaratidza kuti mapuroteni ekare e-interferon-akakurudzira ndiwo aive akanyanya kuwanda kana achienzaniswa nemasamples asina kurapwa, anosanganisira MX1, IFIT1/3, OAS2/3, DDX58, uye HLA-F (Mufananidzo 3D).Ongororo yenzira dzemapuroteni anopfuura katatu akafumiswa mukupindura IFNα kurapwa uchishandisa Reactome nzira dhatabhesi yakaratidza kuti MHC-I-mediated antigen presentation uye kugadzirisa ndiyo yainyanya kutonga nzira (Mufananidzo 3E).Zvinoenderana nemishumo yapfuura, mhinduro dzeantiviral dzakamiririrwa neOAS uye ISG15 pamwe neIFNα / β uye cytokine kusaina yaive pakati penzira dzakaitwa.Pamusoro pezvo, lysine- uye serine-specific protein cross-links yakaonekwa kubva kune yakatanga kuwanikwa MS/MS spectra vachishandisa SIM-XL.Ongororo ichangoburwa yakashuma 104 ISGs inotora makumi maviri mavhairasi kubva ku9 hutachiona makirasi nemeta-kuongorora yemunhu ISG overexpression zvidzidzo mu5 cell marudzi9.Nekudaro, kuti tikunde iyo computational mipimo yekuongorora yakakura dataset, takatanga nediki dhataset kuti tiongorore kudyidzana kunobvira pakati peiyo IRDS genes yakashumwa naPadaria et al., mazhinji acho ari maISG.
Kuzivikanwa kwemapuroteni akapatsanurwa akasiyana-siyana mukupindura IFNα (data yakawanikwa kubva kuMaxQuant).(A) Venn dhayagiramu inomiririra nhamba yeakajairwa uye akasarudzika peptides akaonekwa muIFNα14 yakarapwa uye isina kubatwa Flo-1 samples.Peptide kureba kugoverwa kweasina kurapwa (B) uye IFNα yakabatwa (C) yakachinjika samples.(D) Mepu inopisa inomiririra log2 (LFQ intensity) pakati pekusarapwa uye IFNα14 yakarapwa Flo-1 masero.Iro kuruboshwe rinoratidza mapuroteni anonyanya kushandiswa muhupo hweIFNα.(E) Histogram inomiririra 20 nzira huru dzekupfumisa mushure mekurapa kweIFNα.Iyo Reactome nzira dhatabhesi yakaongorora zvinopfuura mana-kakapetwa shanduko mune upregulated IFNα-inopindura mapuroteni.
Interferon-mediated ISG stimulation yakanyorwa zvakanaka, asi pachikamu chemolecular hazvinzwisiswe kuti mapuroteni aya anoguma sei muhuwandu hwemabasa ehupenyu.Isu takaongorora kudyidzana kweprotein nehupamhi hwekuvimba pakati pemaISG anozivikanwa.Sezvineiwo, takaona network inosanganisira MX1, USP18, ROBO1, OAS3, uye STAT1 mapuroteni anoumba yakakura yakaoma mukupindura IFNα kurapwa (Mufananidzo 4, Tafura S2) 32,33,34.Chinonyanya kukosha, kushamwaridzana uku kwakawanikwa mumatatu ose akabatwa neIFNα uye haana kuwanikwa mumasampuli asina kutorwa, achiratidza kuti akaumbwa zvakananga mukupindura IFNα kurapwa.Izvo zvinozivikanwa kuti STAT1 transcriptally inoronga kutaura kweaya maISG, asi kudyidzana kwayo nemaISGs padanho reprotein hakuna kudzidzwa.Iyo crystal structure yeSTAT1 yakaratidza kuti iyo helical domain (CCD) haina kubatanidzwa mukubatana neDNA kana protomers panguva yekuumbwa kwe dimers35.Aya ma-α-helices anoumba chimiro chehelical helix chinopa nzvimbo yakawanda ye hydrophilic pamusoro pekuti kuwirirana kuitike 35.Mune data yedu yeCLMS, takaona kuti kushamwaridzana kwakawanda neSTAT1 kwakaitika munharaunda yeSH2 inotangira CCD, iyo linker domain, kana C-terminal muswe (masara 700-708) (Mufananidzo 4A).Chidzidzo chekare chakashuma kuti USP18 inosunga kuCCD uye DNA-binding domain (DBD) yeSTAT2 uye inonyorerwa kune subunit yemhando I interferon receptor IFNAR2 kupindira kuvharidzirwa kwemhando I interferon chiratidzo 24.Yedu data yakaratidzawo kuti USP18 catalytic domain inodyidzana neSTAT1 DBD (Mufananidzo 4A, D), zvichiratidza kuti zvese STAT1 uye STAT2 zvinogona kuita basa mukukwezva USP18 kuIFNAR2.
Protein-protein ISG network yakaonekwa mumaseru-akabatana maseru anobatwa neIFNα.(A) 2D yekudyidzana plot inoratidza maprotein-protein interactions (yakagadzirwa muSIM-XL chirongwa), ine mitsetse inomiririra intermolecular interactions (crosslink cutoff set to 3.5).Madomasi emhando dzakasiyana anoratidzwa neruvara rwawo32: MX1 domain, Dynamin_N (73–249), Dynamin_M (259–547), uye GED (569–660).OAS3 domains: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), uye 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) uye fn3 (777–864).STAT1 ndima: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), uye STAT1_TAZ2bind (715–739).(B) Circular viewer of cross-linked proteins (MX1, UBP18, OAS3, ROBO1, uye STAT1) nekubatana uye kupindirana kwakanyorwa mubhuruu uye tsvuku, maererano.Chikumbaridzo-chikumbaridzo chakaiswa pa3.5.Dot plots inoratidza STAT1 yekudyidzana nzvimbo neMX1 (C), USP18 (D), ROBO1 (E), uye OAS3 (F), pamwe neK kana S nzvimbo dzekudyidzana pakati pemapeptide maviri.Mumufananidzo, iyo cross-link mamakisi zvibodzwa yakaiswa ku 3.0.(G) Nzvimbo dzakasiyana-siyana dzekudyidzana pakati peSTAT1 neOAS3 DI madomasi akaiswa pamusoro pezvimiro zvavo zveprotein muPyMol (PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) uye OAS3 (pdb id: 4s3n34).) chirongwa.
Isoforms mbiri dze USP18 dzakatsanangurwa muvanhu, puroteni yakazara-yakareba iyo inonyanya kuwanikwa mukati memukati, uye isoform isina N-terminal domain, USP18-sf, iyo inoparadzirwa zvakaenzana mu cytoplasm uye nucleus 36.Pamusoro pezvo, iyo N-terminus yakafanotaurwa kuve isina kurongeka uye haidi isopeptidase chiitiko kana ISG1537 inosunga.Mazhinji ekudyidzana akaonekwa muchidzidzo chedu aive paN-terminus yeprotein, zvichiratidza kuti kusangana uku kunosanganisira hurefu hwakazara USP18 (Figure 4A, D) uye nekudaro zvingangoitika mukati memukati.Uyezve, data redu rinoratidzawo kuti N-terminus inonyanya kushandiswa kweprotein-to-protein.Iyo IFNAR2 inosunga nzvimbo iri pakati pezvisara 312-368, uye kunyanya, hapana mapuroteni ari mumatambudziko anosunga munharaunda iyi (Fig. 4A) 37,38.Iyi data yakatorwa pamwechete inoratidza kuti iyo IFNAR2 inosunga domain inongoshandiswa chete neiyo receptor protein.Mukuwedzera, chete OAS3 uye ROBO1 yakawanikwa yakabatana nemadomasi kumusoro kweN-terminus uye IFNAR2 inosunga nzvimbo (Mufananidzo 4A).
ROBO1 ndeye immunoglobulin (Ig) superfamily yetransmembrane inosaina mamorekuru uye ine mashanu Ig domains uye matatu fibronectin (Fn) domains mudunhu reextracellular.Aya mazita e-extracellular domains anoteverwa ne-membrane-proximal region uye imwe chete transmembrane helix 39. Nzvimbo isina kuumbwa intracellular inowanikwa paC-terminus uye ine kuchengetedzwa kutevedzana motifs iyo inopindirana effector protein binding39.Dunhu rinobva kumaamino acids ~ 1100 kusvika 1600 rinonyanya kuvhiringidzika.Takaona kuti MX1 inopindirana neROBO1 kuburikidza neIg, Fn, uye intracellular domains, nepo kushamwaridzana kwakawanda neSTAT1 kunoitika pakati peCCD yayo, linker domain, uye C-terminus yeROBO1 (Fig. 4A, E).Kune rumwe rutivi, kushamwaridzana neDI, DIII, uye OAS3 linker nzvimbo dzakagoverwa mukati meROBO1 protein (Fig. 4A).
Iyo oligoadenylate synthase (OAS) mapuroteni mhuri inogamuchira uye inosunga intracellular double-stranded RNA (dsRNA), inopinda conformational shanduko, uye synthesizes 2′,5′-yakabatanidzwa oligoadenylates (2-5 As) 40.Zvakaonekwa kuti pakati pemaOAS matatu, OAS3 inoratidza hukama hwepamusoro-soro hwe dsRNA uye inosanganisa huwandu hushoma hwe2-5 As, iyo inogona kushandura RNase L uye nekudaro kudzikamisa hutachiona hwekudzokorora 41.Mhuri yeOAS ine polymerase beta (pol-β)-yakafanana nucleotide transferase domains.Tsvagiridzo yapfuura yakaratidza kuti iyo catalytic chiitiko cheC-terminal domain (DIII) inotsamira pane dsRNA-binding domain (DI), inodiwa pakuita OAS342.Takaona kuti DI neDII madomasi eOAS3 anodyidzana neCCD uye nharaunda diki yekusangana pakati peSH2 neSTAT1 TAD (Mufananidzo 4A,F).Kufukidzira nzvimbo dzakasiyana-siyana dzekuyambuka pane puroteni chimiro kwakaratidza kuwirirana pakati pe β-sheet neDBD STAT1 loop uye homwe yakazaruka kana mhango yakaumbwa nemasara 60-75 muDI domain yeOAS3 (Fig. 4G).Maonero emapuroteni ari muhombe yakaratidzawo kuti hapana chekubatana neOAS3 chakavhiringidza DNA-binding mano eDI domain (Fig. S1A).Mukuwedzera, iyo N-terminal domain yeGTPase MX1 inopindirana zvakanyanya neDI uye DIII domains yeOAS3 (Fig. 4A).Isu takaonawo kudyidzana pakati peOAS1 neMX1 mune ese matatu IFNα-akarapwa anodzokorora, apo imwechete OAS1 domain (zvakare catalytically inoshanda) yakabatana nematatu ese eMX1 domains (Mufananidzo S2A, B).
Mapuroteni eMX chikamu chemhuri yakakura yeDynein-seGTPase ine N-terminal GTPase domain inosunga uye hydrolyzes GTP, nzvimbo yepakati inopindirana nekuzvibatanidza, uye C-terminal leucine zipper inoshanda seGTPase (LZ). )domain effector domain25,43.MX1 inosungirira kuzvikamu zvehutachiona hwepolymerase kuvhara kunyorwa kweiyo viral gene43.Yakambotaurwa mbiriso mbiri-yakasanganiswa skrini yakaratidza kuti PIAS1-yakabatana MX1 inhibits STAT1-mediated gene activation nekuvharira DNA-inosunga chiitiko uye zvakare ine SUMO E344,45 ligase chiitiko.Pano, tinoratidza kuti MX1 inosungira kuSTAT1 (Mufananidzo 4C, D), zvisinei kuti kuwirirana uku kunobata sei STAT1-mediated gene activation mukupindura IFNα inoda kuwedzera kudzidza.Mukuwedzera, takaonawo kuti MX1 yakabatana neIFIT3 uye DDX60 mune zvose zvitatu IFNα-yakabatwa inodzokorora (Fig. S2C).
DDX60 ndeye IFN-induced cytoplasmic helicase iyo yakambotaurwa kuti inobata basa muRIG-I-yakasununguka kuderedzwa kwehutachiona RNA46.Inopindirana neRIG-I uye inoita kuti iite zviratidzo zvayo nenzira ye ligand-specific 46. DDX60 inosanganisira DEXD / H-Box helicase domain uye C-terminal helicase domain iyo inosunga viral RNA neDNA47.Yakawanda yekudyidzana kwayo neMX1 uye IFIT3 inoitika mukati menguva refu N- uye C-terminal matunhu asina canonical domains kana motifs (Fig. S2E, F).Zvisinei, MX1 inobatanidzwawo neDEXD / H-Box helicase domain (Fig. S2E).Mapuroteni emhuri yeIFIT ane tandem makopi einosiyana helix-turn-helix motif inonzi tetrapeptide repeat (TPR).IFIT3 yakawanikwa iri yakanaka modulator yeRIG-I kusaina uye nekudaro chikamu cheMAVS yakaoma.Takatorwa pamwechete, data yedu inoratidza kuti IFIT3 neDDX60 inodyidzana zvakanyanya munharaunda pakati peTPR 3-6 yeIFIT3 uye inogona kuita basa muRIG-I / MAVS kusaina (Fig. S2F).
Tichifunga kuti kuongorora iyo yese proteome yakanyanya kuwanda, isu takabva taongorora dhatabhesi revanhu reUniProt nekuda kwekuvapo kweiyo IFNα-yakarapwa inodzokorora.Mune ino replica, takawana akati wandei akavimbika ekudyidzana network yeHLA-A.Kuongororwa kweprotein nzira dzakaonekwa neMS / MS spectra dzakaratidza kuti MHC-I-based antigen processing uye mharidzo ndiyo nzira huru inokonzerwa ne interferon (Fig. 3D).Nokudaro, takatarisa pakudzidza kupindirana kweprotein yeMHC-I molecules nehupamhi hwekuvimba mumasampuli ose akabatanidzwa.HLA ine α1, α2 uye α3 domains uye chiedza cheni, uye microglobulin β2 (β2m) inogara iine chaperone protein49.Kana yangoungana mu endoplasmic reticulum, HLA haina kugadzikana mukusavapo kwepeptide ligands50.Iyo peptide-inosunga groove inoumbwa neiyo yakanyanya polymorphic uye isina kurongeka α1 uye α2 madomasi mune isiri-peptide fomu uye yakaderera yepolymorphic α351 domain.Muhupo hweIFNα, takaona maviri HLA-A complexes: imwe inopindirana neHMGA1 uye H2B (Mufananidzo 5, Tafura S3) uye imwe inopindirana ne MDN1, LRCH4 uye H2B (Mufananidzo 6).
IFNα inokonzera HLA-A yekudyidzana network neH2B (H2BFS) uye HMGA1.(A) 2D plot (yakagadzirwa muSIM-XL software) inoratidza marudzi akasiyana-siyana ekubatana muH2B-HLA-A-HMGA1 yakaoma: interlink (blue), interlink (tsvuku) uye imwe chete link (dema)..Domain of different identity are color coded32: H2B (histone; 2–102) neMHC-I (MHC_1; 25–203, group C1; 210–290 and MHC_I_C; 337–364).Chikumbaridzo-chikumbaridzo chakaiswa pa3.5.Dot zvirongwa zvinoratidza HLA-A yekudyidzana nzvimbo neH2B (B) uye HMGA1 (C), pamwe neK kana S nzvimbo dzekudyidzana pakati pemapeptide maviri.Mumufananidzo, iyo cross-link mamakisi zvibodzwa yakaiswa ku 3.0.(D) Ukama huri pakati pemapuroteni anoratidzwa muzvikamu zveH2B, HLA-A, uye HMGA1 mapuroteni muchirongwa chePyMOL.Aya maumbirwo akateedzerwa achishandisa Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2) uye zvimiro zvetemplate zveH2B, HLA-A uye HMGA1 mapuroteni aive 1kx552, 1kj349 uye 2eze55, zvichiteerana.
IFNα inokonzera HLA-A yekudyidzana network neH2B (H2BFS), MDN1 uye LRCH4.(A) Intramolecular (tsvuku) uye intermolecular (blue) crosslinks inoratidzwa pa 2D interactive mepu (inogadzirwa muSIM-XL software) ine MDN1 inomiririrwa sedenderedzwa.Chikumbaridzo-chikumbaridzo chakaiswa pa3.5.Domains of different identity are color coded32: H2B (histone; 2–102), MHC-I (MHC_1; 25–203, group C1; 210–290 and MHC_I_C; 337–364) and LRCH4 (LRR_8 (68–126), LRR_8 (137–194) uye CH (535–641)).(B) Ukama huri pakati pemapuroteni anoratidzwa muzvikamu zveH2B, HLA-A, LRCH4, uye MDN1 mapuroteni muchirongwa chePyMOL.Aya magadzirirwo akateedzerwa achishandisa Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2) ine template zvimiro 1kx552, 1kj349, 6hlu62 uye 6i2665 yeH2B, HLA-A, LRCH4 uye MDN1 mapuroteni, zvichiteerana.Dot zvirongwa zvinoratidza K kana S yekudyidzana nzvimbo dzeHLA-A ine H2B (C), LRCH4 (D), uye MDN1 (E).Pazvirongwa, muchinjika-chinongedzo chibodzwa chakaiswa ku3.0.
Pamusoro pekuchengetedza kutendeseka kweiyo genome, histone H2B inobatanidzwawo mukutonga kwekunyorwa.Iyo H2B protein ine yepakati histone domain (HFD) inoumbwa neatatu α-helices akaparadzaniswa nezvishwe uye C-terminal muswe 41,52.Yakawanda yekudyidzana neH2B inoitika mu α1 helix, iyo inopa trimerization neHFD heterodimer (Fig. 5A, B).Kunyangwe ma lysins achibatanidzwa mukusunga kweDNA, mamwe ma lysins zvakare mamwe acetylation kana methylation nzvimbo.Semuenzaniso, zvisaririra zveK43, K46, uye K57 kubva kuH2B hazvibatanidzwe mukusunga DNA yakananga, asi zvinonangwa zvakasiyana-siyana zvepashure-kunyorwa kweshanduko53.Saizvozvowo, masara eK44, K47, uye K57 muH2B anogona kuita rimwe basa pamberi peIFNα, kusanganisira kushamwaridzana nemamwe mapuroteni (Fig. 5A, B).Pamusoro pezvo, iyo extrachromosomal histone H2B inomutsa mhinduro yezvirwere mumasero akasiyana siyana, ichiita se cytosolic sensor kuona zvidimbu zveDNA (dsDNA) zvakapetwa kaviri zvakatorwa kubva kune zvinotapukira kana maseru akakuvadzwa54.Muhupo hwehutachiona hweDNA, kupera kweH2B kwakadzivisa IFN-β kugadzirwa uye STAT154 phosphorylation.H2B inozivikanwa zvakare kuti inofamba ichipinda nekubuda mukati memukati nekukurumidza kupfuura mamwe macore histones54.H2B kudyidzana ne MDN1 uye LRCH4 kwakaonekwawo mune akasarudzwa asina kurapwa samples.Takaona kuti HLA-A yakabatana neH2B mune ese matatu IFNα-akarapwa masampuli uye mune imwe isina kupindurwa kudzokorora sampu.Idzi data rinoratidza basa reH2B mune rimwe basa refiziological rakazvimiririra kubva kune transcriptal regulation.
HMGA1 (high mobility group AT-Hook 1), duku nucleoprotein yakapfuma muzvirwere-inokurudzira amino acids, yakaonekwa mukubatana neHLA-A.Iine acidic C-terminal muswe uye maDBD matatu akasiyana anonzi AT hook nekuti anosunga kune diki groove yeAT-rich region mu dsDNA55,56.Kusunga uku kunoita kuti DNA ikotame kana kutwasuka, zvichibvumira canonical transcription factors kuti iwane kutevedzana kwayo kwekubvumirana.Muswe weC-terminal unofungidzirwa kuti unobatanidzwa mukubatana kweprotein-protein uye kuunganidza zvinhu zvekunyora, sezvo C-terminal deletion mutants isingakwanisi kutanga zvinyorwa57.Uyezve, iyi domain ine akati wandei akachengetedzwa phosphorylation nzvimbo dzinozivikanwa substrates dze kinases 58.Takacherechedza kushamwaridzana kweHLA-A neH2B neHMGA1 kunze kweC-terminal domain, zvichiratidza kuti C-terminal domain inonyanya kushandiswa pakunyorera chinhu chinosunga (Fig. 5A, C).HMGA mapuroteni anokwikwidzana nehistone H1 yekubatanidza kune adapta DNA, nekudaro kuwedzera kusvikika57.Saizvozvo, zvinoita sekunge HMGA inodyidzana nehistone H2B pamwe neiyo linker DNA mumakwikwi nehistone H1.HMGB1 inokonzera kutaura kweHLA-A, -B, uye -C mumasero edendritic, zvichiita kuti vashande59, asi kuwirirana pakati peHMG neHLA hakuna kumbotaurwa.Takaona kuti HMGA1 inodyidzana neiyo α1 uye α3 domains yeHLA-A, ine yakawanda yekudyidzana kunze kwayo 3 DBD (Mufananidzo 5A, C).Mumaoko edu, HLA-A yakawanikwa ichiiswa munucleus (data isina kuratidzwa), uye yakapihwa kuti H2B neHMGA1 dziripowo munucleus, kusangana uku kungangoitika munucleus.Yakananga adducts akayerwa pakati H2B, HLA-A, uye HMGA1 inoratidzwa muFigure 5D.
Kubatana kwakawanda kweHLA-A nemamwe mapuroteni kunoitika mukati meiyo α1 uye α2 domains uye yakakanganiswa C-terminal domain (Fig. 6).Mune mumwe weiyi mienzaniso, takaona kuti HLA-A inodyidzana neiyo isina kurongeka N-terminal muswe weLRCH4 (Mufananidzo 6A,D).LRCH4 inoronga TLR4 activation uye LPS cytokine induction, nokudaro modulating innate immune immune response60,61.Iyo membrane protein ine mapfumbamwe e-leucine-rich inodzokorora (LRRs) uye calmodulin (CH) homology motif mu ectodomain yayo, inoteverwa ne transmembrane domain (TMD) 60, 62.CH domains yakashumwa kuti inopindirana neprotein-protein interactions 60.Yakatambanudzwa inosvika mazana matatu amino acids pakati peLRR neCH domains inosvikika asi isina kurongeka.Zvichienderana nebasa rematunhu asina kurongeka semurevereri weprotein-protein network uye vesicular transport 63, takaona kuti mapuroteni mazhinji ekudyidzana anoitika munzvimbo dzisina kugadzikana.Kudyidzana ne MDN1 kwakagoverwa mukati mehurefu hweprotein, kusanganisira LRR1, LRR6, CH domains, uye nzvimbo dzisina kurongeka, nepo H2B inonyanya kusungirirwa kuC domain (Fig. 6A, B).Zvinonzwisisika, hapana yekudyidzana kwaisanganisira iyo TMJ, ichiratidzira iyo chaiyo yeCLMS nzira (Mufananidzo 6A, B).
MDN1 yakaonekwawo sechikamu cheHLA-A protein network (Mufananidzo 6A).Iyo ndeyeAAA mhuri yemapuroteni (ATPases yakabatana nemabasa akasiyana).Iyi ndiyo imwechete N-terminal AAA domain inoronga kuita hexameric mhete uye inobvisa chinhu chegungano kubva ku60S 64 ribosomal subunit.inoita kunge yakafanana nedynein64,65,66.Uye zvakare, iyo Asp/Glu yakapfuma dunhu inoteverwa neiyo MIDAS domain (metal ion inoenderana saiti).Nekuda kwehukuru hukuru hweMDN1 (inoda kusvika 5600 amino acids) uye nehomology yayo ine mapuroteni akanyatso dzidza, zvishoma zvinozivikanwa nezve maumbirwo ayo uye kushanda kwayo muvanhu.Takaona HLA-A, H2B, uye LRCH4 seMDN1 vanosunga vadyidzani uye vakaratidza maitiro avo semaprotein complexes muPyMol (Fig. 6A,B).Aya mapuroteni matatu anopindirana neAAA domain, iyo dynein-like linker domain, uye pamwe iyo MIDAS MDN1 domain.Mumushumo wapfuura, kucheneswa kwehukama hwemapuroteni echiredzo kwakaratidza MDN1 seprotein yakabatana nehistone H2B67.Mukuwedzera, chidzidzo chechangobva kuitika chakashumawo kuwirirana pakati pe MDN neHLA-B mumasero eHCT116 achishandisa affinity-purified mass spectrometry, achitsigira zvatinowana68.Kuzivikanwa kweiyi yakaoma muIFNα-yakarapwa samples inoratidza basa re MDN1 mukusaina interferon.
Nekuti HLA majini ane polymorphic zvakanyanya, takatora kutevedzana kunoverenga mepu HLA-A, -B, uye -C kubva kuRNA kutevedza data yeFlo-1 masero (data isina kuratidzwa).Peptide sequences inoenderana nekutevedzana kwekuverenga kwakaratidza mutsauko wakakura pakati peHLA-A, -B, uye -C mumatunhu aive muchinjika-yakabatana peptides aive muHLA-A (Mufananidzo S3).Pamusoro pezvo, isu hatina kuona mapuroteni-kune-protein cross-linking yeHLA-B/C mamorekuru ane H2B/HMGA1/MDN1/LRCH4 mapuroteni.Izvi zvinoratidza kuti kudyidzana kweprotein kunowanikwa pakati peHLA-A, MDN1, LRCH1 neHMGA1 iHLA-A chaiyo.Pamusoro pezvo, kuongororwa kweproteomic yeasina-crosslinked samples (Table S4) yakaratidza kuti HLA-A ine yakakwirira sequence coverage kana ichienzaniswa neHLA-B kana HLA-C.Mapeptide akaonekwa eHLA-A aive akakwira mukusimba mune ese ari maviri IFNα-akarapwa uye asina kubatwa samples.
Kuti tive nechokwadi chekuti kudyidzana kwaonekwa pano kwaisakonzerwa nekusanyanya kubatanidza mapuroteni maviri ari padyo nenzvimbo, takasimbisazve maviri matsva eHLA-A anodyidzana zvinhu nekuita co-immunoprecipitation assays.HLA-A kuwirirana ne endogenous MDN1 uye H2B yakaonekwa mune zvose IFNα-yakarapwa uye isina kubatwa Flo-1 masero (Mufananidzo 7, Mufananidzo S4).Isu takasimbisa kuti HLA-A yakabatwa neH2B mune immunoprecipitates uye kuti kushamwaridzana uku kwakakonzerwa neIFNα kurapwa sezvo HLA-A yakanga isipo mune immunoprecipitate samples kubva kumasero asina kurapwa (Mufananidzo 7A).Nekudaro, yedu data inoratidza kuti IFNα inodzora zvakasiyana-siyana HLA-A inosunga kuH2B uye MDN1.IFNα inokonzera kushamwaridzana pakati peH2B neHLA-A, asi inoderedza kushamwaridzana kwayo neMDN1.Takaona kuti MDN1 yakabatanidzwa neHLA-A mukutonga, uye kuwedzerwa kweIFNα kwakaderedza kusangana uku kwakasununguka kweMDN1 induction neIFNα (Mufananidzo 7B, C).Mukuwedzera, HLA-A immunoprecipitation yakabata H2B muA549 masero (Fig. S4), ichiratidza kuti kupindirana uku kwakasununguka kune rudzi rwesero.Zvakatorwa pamwechete, izvi mhedzisiro inotsigira interferon-mediated kudyidzana kweHLA-A neH2B uye MDN1.
HLA-A inonatsa H2B uye MDN1.Representative endogenous H2B (A) uye MDN1 (B) immunoblots vakanga vasina immunoprecipitated kubva kuIFNα-yakarapwa Flo-1 masero uye yakaongororwa nokuda kwezvirwere zvakaratidzwa.Mouse uye tsuro IgG yakashandiswa sekutonga kwakashata.(C) Huwandu hwehuwandu (inopinza) yemaantigen akasiyana-siyana inoratidzwa ne immunoblots probed against inoratidzwa antibodies, β-actin yakashandiswa sekutonga kurodha.
Izvo zvimiro zveimwe ye interferon-induced yakanyanya kuvimbika muchinjika-yakabatana network, H2B-HLA-A-HMGA1, yakaongororwa.Takashandisa molecular dynamics modelling seimwe nzira yekunzwisisa conformational dynamics yemapuroteni anobatanidzwa mune iyi yakaoma (Mufananidzo 8).Inferences kubva ku data yeCLMS inoratidza mukana wekusiyana kwakasiyana kweH2B, HLA-A, uye HMGA1 mapuroteni.Nokudaro, zvinotevera zvinogona kuoma zvakagadziriswa mumhepo inonyungudutsa: H2B-HLA-A, HMGA1-HLA-A, uye H2B-HLA-A-HMGA1.Yekutanga protein-protein docking screen uchishandisa iyo MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) pasuru yakaratidza zvingangoita conformations zvinosiyana pakati pemapuroteni aya (Fig. 8A).Kuonekwa kweiyo docking protein yakaoma kwakaratidza kupindirana kwakati wandei uye zvinogoneka conformations (Mufananidzo 5A, 8).Nekudaro, imwe inogoneka conformation inoratidzwa muFigure 8A (ine zvakanyorwa muchinjiko-manongedzo) uye yakazoongororwazve uchishandisa iyo MD yekuenzanisira pombi.Mukuwedzera, kusungirirwa kweH2B kana HMGA1 kuHLA-A kunoratidza hukama hwepamusoro hweH2B hweHLA-A (Fig. 8A).
Conformational dynamics yezvinogoneka network pakati peH2B (H2BFS)-HLA-A, HMGA1-HLA-A, uye H2B-HLA-A-HMGA1 complexes.(A) Paruboshwe imepu ye2D (yakagadzirwa muSIM-XL software) ye intramolecular (tsvuku) uye intermolecular (blue) crosslinks (crosslink cutoff set to 3.5).Pamusoro pezvo, zvisaririra zvekubatanidza zvakaonekwa zvakanyorwa pazvimiro zveH2B, HLA-A, uye HMGA1 mapuroteni.Izvo zvinobatanidzwa zvemapuroteni aya zvakatorwa pachishandiswa pombi yekumisa yakaiswa muMOE package.Panera rezasi rekuruboshwe rinoratidza zvakasiyana-siyana zvinogoneka zveH2B-HLA-A uye HMGA1-HLA-A complexes ine mapuroteni-protein anosunga affinities akasiyana (GBVI/WSA dG; kcal/mol).(B) Kutsauka kwakajairwa (RMSD) kwenzvimbo dzeatomu (kusanganisa maatomu ehydrogen) kune yega yega mapuroteni.(C) Intermolecular protein-protein hydrogen bond kupindirana kubva kumhando dzakasiyana-siyana dzakafananidzwa dzichifunga nezvekubatana kwakananga kwenguva ≥ 10 ns.Iyo h-bond donor-acceptor cutoff kureba yakaiswa ku3.5 Å, uye donor-H-acceptor cutoff angle yakaiswa ku ≥ 160 ° -180 °.(D) Yakanyorwa zvakasara zvinogadzira HLA-A protein-protein kudyidzana neavo vakasiyana, inotora ≥ 20 ns, yakatorwa kubva dummy HLA-A-H2B uye HLA-A-HMGA1 complexes.Mapuroteni maitiro anomiririra avhareji chimiro che100 ns MDS.(E) Kudyidzana pakati peHLA-A-H2B neHLA-A-HMGA1 complexes zvichienzaniswa nekudyidzana kunoteverwa neH2B-HLA simulation pamusoro pe100 ns zvichienderana neK kana S yekudyidzana nzvimbo pakati pemapeptide maviri.Complexes /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Chikumbaridzo kukosha kwekuongororwa kwe-cross-links kwakaiswa ku3.0, uye kupindirana kwakananga kubva kuMDS kutora ≥ 10 ns kwakatorwa.Mapuroteni maitiro akaonekwa achishandisa BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) uye Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada) mapakeji.
Kugadzikana kweHLA-A mamorekuru nekufamba kwenguva (standard deviation; RMSD kana standard deviation; RMSF) yakaratidza kuti kuvepo kweH2B kana HMGA1 mapuroteni mumisanganiswa yakagadzikana HLA-A (Mufananidzo 8B, Mufananidzo S5).Iyo HMGA1 protein inosunga zvakasimba kune B2M nzvimbo yeHLA-A, inokonzera kugadzikana kweHLA-A amino acids muHLA-A-HMGA1 kana H2B-HLA-A-HMGA1 yakaoma (Mufananidzo 8B, Mufananidzo S5).kunyanya, HLA zvakasara ~ 60-90 uye ~ 180-210 zvakawanikwa zvisinganyanyi kushanduka pamberi peH2B (FIG. 8B).H2B neHMGA1 yakaratidza zviri nani kusunga kuHLA-A muH2B-HLA-A-HMGA1 yakaoma kana ichienzaniswa neHLA-A inosunga kuH2B kana HMGA1 chete (Mufananidzo 8C,D; Tafura S5).Zvisaririra zvinobatanidzwa muhydrogen bonding (MD modeled high occupancy ≥ 10 ns) inopindirana neCLMS yekudyidzana nzvimbo (K kana S residues) mumatambudziko, zvichiratidza kuti kupindirana kunoonekwa neCLMS kwakavimbika zvikuru.Kuvimbika (Fig. 8E).MuCLMS uye MD modelling, HLA-A yakasara pakati pe190-210 uye inenge 200-220 amino acids yakawanikwa inosunga H2B neHMGA1, maererano (FIG. 8E).
Kudyidzana kweprotein-protein kunoumba dynamic structure networks inopindira intracellular communication mukupindura zvimwe zvinokurudzira.Nekuda kwekuti nzira dzakawanda dzeproteomics dzinoona shanduko muhuwandu hwakatsiga hwehuwandu hweprotein, mapuroteni-protein yekudyidzana dynamics inoda mamwe maturusi ekutora anosunga mainterface, uye CLMS chimwe chishandiso chakadaro.Iyo inonzi interferon signaling system inonzi cytokine network inobvumira masero kuti apindure kune zvakasiyana-siyana zvezvakatipoteredza pathogenic uye intrinsic pathological signals, zvichiguma nekuiswa kwezvikamu zveprotein-inducible proteins.Isu takaisa CLMS kuti tione kana riini mapuroteni-protein kudyidzana angaonekwa pakati pepanhi remapuroteni anokonzerwa ne-interferon.Global protein cross-linking analysis mune interferon-responsive Flo-1 cell model yakashandiswa kutora maprotein complexes.Kubviswa kwetryptic peptides kubva kune asina-muchinjika-yakabatana uye muchinjika-yakabatana maseru inobvumira kuverenga peptide, kuvandudza nzira, uye peptide kureba kugovera nekutsanangurwa kwakatsanangurwa LFQ intensity.Canonical interferon-inducible mapuroteni akaonekwa seakanaka mukati mekutonga, nepo itsva intermolecular uye intramolecular cross-linked adducts ye canonical interferon-inducible mapuroteni akadai seMX1, UP18, OAS3 uye STAT1 akaonekwa.Zvimiro zvakasiyana-siyana uye kupindirana munzvimbo dzekushanda zvakaongororwa.
Kudyidzana pakati peHLA-A, MDN1 neH2B kwakaonekwa ne immunoblotting muFlo-1 uye A549 masero akabatwa uye asina kubatwa neIFNα.Mhedzisiro yedu inoratidza kuti HLA-A yakaoma neH2B nenzira inotsamira paIFNα.Basa redu rinomiririra nzira inonakidza yekuenderera mberi nekuongorora kwekubatanidzwa kwenzvimbo mbiri idzi.Zvingave zvakare zvinonakidza kuwedzera nzira yeCLMS kune mapaneru emitsara yesero kuona sero-mhando-yakazvimirira interferon-mediated protein kudyidzana.Pakupedzisira, takashandisa MD modelling seimwe nzira yekunzwisisa conformational dynamics yemapuroteni akabatanidzwa muH2BFS-HLA-A-HMGA1 yakaoma, iyo yakateedzera intramolecular uye intermolecular cross-talks.Inferences kubva ku data yeCLMS inoratidza mukana wekusiyana kwakasiyana kweH2BFS, HLA-A, uye HMGA1 mapuroteni.Izvo zvinogoneka zvakasiyana siyana pakati peaya docking maprotein complexes akaburitsa akati wandei mabatiro akafanana neanoonekwa muCLMS dataset.Rimwe remasimba makuru enzira yedu nderekuti inobvumira kuzivikanwa nyore kwekudyidzana kwakanyanya polymorphic majini akadai seHLA, saka zvichave zvinonakidza kudzidza kudyidzana kweHLA haplotype-chaiwo mapuroteni ayo anonetsa kudzidza.Takatorwa pamwechete, data yedu inoratidza kuti CLMS inogona kushandiswa kuwedzera kunzwisisa kwedu kwe interferon-induced siginacha network uye inopa hwaro hwekudzidza zvakanyanya kuomarara intercellular masisitimu mune tumor microenvironment.
Flo-1 masero akawanikwa kubva kuATCC uye akachengetwa muDMEM (Gibco) akawedzerwa ne1% penicillin / streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) uye yakachengetwa pa 37 ° C uye 5% CO2.Incubation.Masero akakura kusvika ku70-80% confluence asati abatwa neIFNα14 (yakagadzirwa neEdinburgh Protein Production Facility).Ese mamwe makemikari uye reagents akatengwa kubva kuSigma Aldrich kunze kwekunge zvataurwa neimwe nzira.
Flo-1 masero akagadzirwa mu6-tsime mahwendefa uye zuva rakatevera masero akabatwa ne 10 ng / ml IFNα14 kwemaawa 24 kusvika anenge 80% confluence.Masero akashambidzwa katatu nePBS uye akabatanidzwa neDSS ichangobva kugadzirwa (Thermo Fisher Scientific) (yakanyungudutswa muDMSO) muPBS ye5 min pa 37 ° C. kusvika kuhuwandu hwekupedzisira hwe 0.5 mM.Iyo DSS crosslinking reaction yakatsiviwa nePBS uye yakasara DSS yakadzimwa nekuwedzera 20 mM Tris (pH 8.0) muPBS kwemaminetsi gumi nemashanu pa37 ° C.Masero akaunganidzwa nekukweshwa uye akaunganidzwa mumachubhu akadzika anosunga (Axygen).
Sero pellet rakaiswa lysed ne 300 µl ye urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) kwemaminetsi makumi matatu patembiricha yekamuri pamwe nekuzununguka pano neapo.Matanho ese ecentrifugation akaitwa pa14,000 xg pa8°C.Centrifuge the lysate kwemaminitsi gumi uye endesa iyo supernatant kune imwe chubhu.Izvo zvakasara zvakajeka zvimedu zvakanyungudutswa mu 150 μl yechipiri lysis buffer (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) kwemaminitsi makumi matatu kana kupfuura kusvikira homogeneous aqueous solution yawanikwa.Iyo lysate yaive centrifuged kwemaminetsi makumi maviri uye iyo supernatant yakasanganiswa neiyo lysate yakawanikwa munhanho yapfuura.Mapuroteni akaiswa akaongororwa uchishandisa Micro BCA assay (Thermo Fisher Sayenzi) zvinoenderana nemirairo yemugadziri yemamicroplate maitiro.Sampuli dzakakurumidza kuomeswa nechando mumvura ine nitrogen uye yakachengetwa pa -80 ° C.
Inenge 100 μg yeprotein yakanyungudika yakasanganiswa yakagadziriswa uchishandisa yakagadziridzwa filtration sample preparation protocol (FASP) sezvakatsanangurwa naWisniewski et al.69 Muchidimbu, puroteni yakabatanidzwa ne 200 µl ye urea buffer (8 M urea mu 0.1 M Tris, pH 8.5), yakavharwa uye nepakati.Matanho ese ecentrifugation akaitwa pa14,000 xg pa25°C.Hafu yekutanga ye-cross-linked protein lysate yakaendeswa kune 10 kDa Microcon centrifugal filter device ine Ultracel-10 membrane (Merck), inoteverwa ne centrifugation pane sefa kwemaminitsi makumi maviri nemashanu.Wobva wawedzera hafu yechipiri yeprotein kune sefa uye dzokorora matanho akafanana.Kupora kweprotein kwakaitwa nekuwedzera 100 μl ye17 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) muurea buffer.Kudzoreredza kwakamutswa pane thermomixer pa600 rpm kwemaminetsi makumi matatu pa37 ° C.Pamusoro pezvo, iyo column yaive centrifuged uye iyo yakaderedzwa muchinjika-yakabatanidzwa protein yaive alkylated ichishandisa 100 μl ye50 mM iodoacetamide muurea buffer.Iyo alkylation reaction yakaitwa pakamuri tembiricha kwemaminitsi makumi maviri murima.Tenderedza mbiru, geza madziro ekoramu katatu ne 100 µl urea buffer, wozoisa centrifuge.Kuvhiya kwakafanana kwakaitwa katatu pachishandiswa 100 μl ye100 mM ammonium bicarbonate.Usati waedza trypsinization, tsiva chubhu yekuunganidza neitsva.Wedzera digestion buffer ine 50 mM ammonium bicarbonate uye 1 µl trypsin yakadirwa mutrypsin buffer (Promega).Reshiyo yetrypsin kune mapuroteni yakachengetedzwa panenge 1:33, uye maitiro ekugaya akaiswa mukati mehusiku pa 37 ° C. mukamuri ine mwando.Iyo crosslinked peptide yakaburitswa kubva kune sefa ne centrifugation kwemaminitsi makumi maviri nemashanu.Peptide kupora kwakagadziridzwa nekuwedzera 50 μl ye0.5 M NaCl kune sefa, ichiteverwa necentrifugation kwemaminitsi makumi maviri nemashanu.
C18 Micro Spin columns (Harvard Apparatus) yakashandiswa kubvisa muchinjika-yakabatana tryptic peptides ichitevera gwara rakatsanangurwa naBouchal et al.70 nedikindurudzo diki.Muchidimbu, C18 spin columns yakashandiswa nekugezwa katatu kwe0.1% formic acid (FA) muacetonitrile (AcN) (Merck) uye kushambidza maviri kwe0.1% FA.Iyo column yakanyoroveswa ne 0.1% FA kwemaminetsi gumi nemashanu.Isa sampuli mumaspin column uye geza katatu ne0.1% FA.Mapeptide akabviswa munyu akajekeswa zvakatevedzana aine nhanho gradient achishandisa 50%, 80% uye 100% AcN mu0.1% FA.Iwo masampula akaomeswa muSpeedVac Plus concentrator (Eppendorf) kudzamara mvura yasara yanyangarika zvachose.Mapeptides akanyungudutswa akanyungudutswa mu100 μl ye0.08% trifluoroacetic acid mu2.5% AcN uye iwo maconcentration akayerwa paNanoDrop 2000 (Thermo Scientific).Inenge 1 μg ye crosslinked peptide per sampuli yakaiswa muLC-MS/MS system.
Muchinjikwa-yakabatana peptides akapatsanurwa pane UltiMate 3000 RSLCnano LC system (Thermo Sayenzi) yakabatana neOrbitrap Exploris 480 mass spectrometer (Thermo Scientific).Muchinjikwa-yakabatana peptides akaunganidzwa pa300 µm ID, 5 mm kureba µ-pre-column C18 yekutora column yakazara neC18 PepMap100 sorbent uye 5 µm PepMap sorbent (Thermo Scientific).Takura pombi inoyerera yakaiswa pa5 µl/min 0.08% trifluoroacetic acid yakanyungudika mu2.5% AcN.Muchinjikwa-akabatanidzwa peptides akapatsanurwa pane analytical fused silica column ine mukati dhayamita ye75 μm uye kureba kwe150 mm, yakazadzwa ne2 μm PepMap sorbent (Thermo Sayenzi).Nharembozha zvikamu A uye B zvaisanganisira 0.1% FA mumvura uye 0.1% FA muacetonitrile, zvichiteerana.Iyo gradient inotangira pa2.5% B uye inowedzera zvakatevedzana kusvika ku40% B pamusoro pe90 maminetsi, yozosvika pa90% B pamaminetsi maviri anotevera.Iyo mobile phase yekuumbwa yakachengetwa pa90% B kwemaminetsi gumi uye yakadzikira zvine mutsetse kusvika 2.5% B pamusoro pemaminitsi maviri.Iyo column yakaenzana pa2.5% B kwemaminetsi masere pamberi pechikamu chinotevera.Muchinjikwa-akabatanidzwa peptides akarutswa kubva pakuongorora koramu akaiswa ionized mune nanoelectrospray ionization (NSI) sosi uye akaiswa muExploris 480 mass spectrometer (Thermo Scientific).
Iyo Orbitrap Exploris 480 mass spectrometer yakashandiswa mune yakanaka data correlation mode.Kuongororwa kwakazara kwakaitwa muchikamu chechikamu pakugadziriswa kwe120,000 nemhando dzakasiyana kubva m/z 350 Th kusvika m/z 2000 Th.Iyo yakajairwa AGC tarisiro yakaiswa pa300% neyakakura yekuisa nguva ye50ms.Monoisotopic peak yekuona yakagadziriswa peptides.Iyo yekumanikidza kuzorora paramende inoiswa kune chokwadi kana yakawandisa precursors inowanikwa.Iyo shoma ionic simba reiyo precursor yakaiswa ku 5.0e3 uye precursor charge inoti kusvika ku +8 yakaverengerwa muzviedzo.
Nguva yekutenderera pakati pema scans makuru mu data correlation mode yakaiswa kumasekonzi 2.5.Dynamic mass kubviswa kwakaiswa kuma 20s mushure mekutanga kupatsanurwa kweiyo precursor ion.Iyo precursor yekuzviparadzanisa hwindo yakaiswa ku2 Th.Iyo mhando yeyakajairwa kudhumhana simba neyakagadzika kudhumhana simba modhi yakasarudzwa mune data inotsamira MS/MS scan.Simba rekudhumhana rakaiswa ku30%.Iyo Orbitrap resolution yakaiswa ku15,000 uye iyo AGC yakanangana ne100%.Iyo yakajairwa yakawanda jekiseni nguva inoiswa ku60 milliseconds.
Tisati taronda network yeprotein-protein mumasamples akabatanidzwa, takagadzirisa mafaera mambishi tichishandisa MaxQuant package (version 1.6.12.0)26,27 kuona mapeptides/maprotein anowanikwa mumasampuli.Mukuwedzera, kuongorora kwakafanana kweproteomic kwakaitwa pane uncrosslinked Flo-1 samples yakabatwa uye isina kubatwa neIFNα.Data yeMS/MS yakatsvaga muUniProt human database (www.uniprot.org) (yakaiswa Nyamavhuvhu 12, 2020, ine 75,093 zvinyorwa) uchishandisa yakavakirwa-mukati yekutsvaga injini Andromeda27.Kutsvaga kwakaitwa pasina kuratidza kujeka kweiyo enzyme uye kwakasiyana shanduko ye deamidation (N, Q) uye oxidation (M).Precursor mass tolerances yakaiswa pa20 ppm uye zvigadzirwa ions pa0.02 Da.Kutsauka kwekutanga uye kwepamusoro kwakaiswa ku10 ppm.Iyo yakanyanya kuwanda yepeptide yakaiswa pa4600 Da uye kutevedzana kwakafanana kwakaiswa pakati pe7 ne25 amino acids (aa).Kumwe kuongorora kwenhamba kwakaitwa pachishandiswa chirongwa chePerseus (version 1.6.10.45).Protein yemukati yakaverengerwa nekujairisa iyo spectral intensity yeprotein (LFQ intensity; unlabed quantification) 27 uye huwandu hwekusimba hwakashandurwa kuita Log2.A hierarchical clustering yemaprotein anoonekwa neawo peptide intensity akavakwa pachishandiswa pheatmap (v1.0.12) package muR (v 4.1.2).Nzira yekuvandudza nzira yakaitwa uchishandisa Reactome nzira dhatabhesi yeIFNα-yakarapwa mapuroteni ayo aive akapetwa kanopfuura mana akaitwa kana achienzaniswa neasina kubatwa samples.
Kuzivikanwa kwe lysine (K) kana serine (S) chaiyo yemakemikari crosslinks yeprotein complexes inotariswa neLC-MS/MS yakaitwa pachishandiswa spectroscopic identification machine (SIM-XL) ye-cross-linked peptides (SIM-XL)29.Kutanga, zvinogona kuitika pakati pe interferon-associated (IFN) DNA damage resistance signature (IRDS) majini akaongororwa achishandisa IRDS protein dataset inotsanangurwa muPadariya et al.28.Kuongorora mamiriro ese uye kudzokorora kwemunhu wese UniProt kwakakura zvakanyanya, saka iyo yese yemunhu UniProt dhatabhesi (www.uniprot.org) (yakatorwa 12 Nyamavhuvhu 2020, ine 75,093 zvinyorwa) zvichipesana neIFNα-yakarapwa inodzokorora.Imwe yemasefa ekuvimbana kwepamusoro.Uku kupindirana kwepamusoro-kukosha kwakawanikwa kwakawedzerwa uye kwakaedzwa mukudzokorora kwese uye mamiriro.
MuSIM-XL, DSS yakashandiswa kune crosslinker (XL) uye XL uremu kuchinja uye kushandura uremu kuchinja kwakaiswa ku138.06 uye 156.07, zvichiteerana.Aya anotevera crosslinking reaction saiti anotariswa: KK, KS uye KN-TERM, pasina reporter ions.Zvese zviri zviviri precursor uye fragment ppm zvakaiswa ku20 uye Xrea chikumbaridzo chakaiswa ku0.15.Trypsin yaionekwa seyakanyatsojeka, uye yakakwira-simba C-trap (HCD) kupatsanura nzira yakaitwa.Iyo XCorr dynamic DB yekudzikisa chikumbaridzo uye huwandu hushoma hwemapeptide ekuderedza DB kudzikiswa kwakaiswa ku2.5 ne2, zvichiteerana.Mamwe ma paramita ndeaya: mukana we monoisotope uye peak coincidence cutoff, mashoma 4 AA masara patambo uye yakanyanya tambo yekuchaja, uye 3 maxima eakapotsa akatsemuka.Iwo akasonwa mamepu e2D akaongororwa mu (SIM-XL) uye xQuest28 graphical inomiririra yakashandiswa kuvaka iyo 2D mepu.Maprotein crosslinks pazvimiro zveprotein anopiwa muPyMol (PyMOL Molecular Graphics System, shanduro 2.0 Schrödinger, LLC).
Protein model structures dzakagadzirwa uchishandisa Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2)11 vachishandisa nheyo dzehomology modeling uye kushandiswa kwe "Hidden Markov Method".Phyre2 inogadzira zvimiro zvemhando zvinoenderana nekutevedzana kurongeka neanozivikanwa mapuroteni zvimiro.Kune H2BFS, HLA-A, HMGA1, LRCH4, uye MDN1 mapuroteni, template zvimiro 1kx552, 1kj349, 2eze55, 6hlu62, uye 6i2665 zvakashandiswa.Pamusoro pezvo, chimiro cheAlphaFold71 MX1, UBP18 uye ROBO1 chakatariswa zvakare.Chimiro cheprotein chakaonekwa pachishandiswa BIOVIA Discovery Studio Visualizer package (Dassault Systèmes, BIOVIA, San Diego, CA, USA) uye Molecular Operating Environment package (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).