ʻO 347 mau mea hoʻoheheʻe i hoʻopaʻa ʻia i ke kila kila, ʻike ʻia o ka interferon-responsive human leukocyte antigen-A (HLA-A) chaperone proteins me ka hoʻohana ʻana i ka cross-linked mass spectrometry (CLMS)

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Hōʻike huahana

ʻO ke kila kila 347L nā paipu huila, ka papa kila: SS347L

Hoʻokomo ka ʻōnaehana hōʻailona interferon i ka pane cytokine ikaika i kahi ākea o nā hōʻailona pathological pathogenic a intrinsic mai ke kaiapuni, e hopena i ka hoʻokomo ʻana i nā subsets o nā protein interferon-inducible.Ua noi mākou i ka DSS-mediated cross-link mass spectrometry (CLMS) e ʻike i nā pilina protein-protein hou i loko o ke kahua o nā protein i hoʻokomo ʻia e interferon.Ma waho aʻe o nā protein interferon-inducible i manaʻo ʻia, ua ʻike pū mākou i nā mea hou intermolecular a intramolecular cross-linked addducts o canonical interferon-inducible proteins e like me MX1, USP18, OAS3, a me STAT1.Ua kālele mākou i ka hōʻoia orthogonal o kahi pūʻulu hou o nā pūnaewele protein interferon-inducible i hoʻokumu ʻia e nā protein HLA-A (H2BFS-HLA-A-HMGA1) me ka hoʻohana ʻana i ka co-immunoprecipitation a me kā lākou aʻo hou ʻana me ka hoʻohana ʻana i ka molecular dynamics modeling.ʻO ka hoʻohālike ʻana o ka conformational dynamics o ka protein complex i hōʻike ʻia i kekahi mau pūnaewele pili e hōʻike ana i nā pilina i ʻike ʻia ma nā ʻike CLMS.Ke hōʻike pū nei mākou i kahi noiʻi hoʻokele o CLMS e ʻike i nā paʻakikī hōʻailona hou i hoʻokomo ʻia e ka interferon, a ke kakali nei i ka hoʻohana nui ʻana o CLMS e ʻike i nā dynamics hou o nā pilina protein i loko o ka microenvironment tumor.
Ma mua o ka hoʻomaka ʻana o kahi pane kūlohelohe adaptive, ua kau ka ʻōnaehana pale kūlohelohe o ka mea hoʻokipa i kahi pane antimicrobial i hoʻopili ʻia e kahi ʻohana o nā cytokine alpha-helical huna i kapa ʻia interferons (IFNs).ʻO nā papa IFN ʻano IFNα a me IFNβ e hoʻāla i nā pane kelepona, me ka antiviral, proapoptotic, proinflammatory, a me nā mokuʻāina antiproliferative.I loko o nā kānaka, ʻike ʻia nā subtypes 13 o IFNα, i hui pū ʻia ma ka chromosome 91. ʻO ka mea kupanaha, ʻo IFNα2 wale nō i aʻo ʻia no ka hoʻohana lāʻau lapaʻau.I kēia mau lā, ua hāʻawi ʻia ka nānā kūikawā i ka noiʻi ʻana i nā ʻano subtype ʻē aʻe o IFNα.Ua hōʻike ʻia kahi noiʻi hou ʻo IFNα14 kekahi o nā isoforms maikaʻi loa i ka hoʻopaʻa ʻana i ka HBV2 a me ka HIV-13,4 replication i hoʻohālikelike ʻia me ka canonical IFNα2 subtype.
Ua hoʻokumu ʻia ʻo ka ʻano I interferon receptor complexes (IFNAR1 a me IFNAR2) e hoʻoulu i kahi hōʻailona transduction cascade i hoʻopili ʻia e Janus kinases TYK2 a me JAK15,6.ʻO kēia Janus kinases phosphorylate signal transducers a me transcriptional protein activators (STAT1 a me STAT2) ma nā koena tyrosine e hoʻomaka i ka SH2 domain-mediated heterodimerization6.A laila, hoʻopaʻa ʻo IRF9 i nā heterodimers STAT e hana i kahi trimeric complex o ka IFN-stimulated factor 3 gene (ISGF3), e unuhi i ka nucleus a hoʻoulu i ka transcription o nā 2000 interferon-stimulated genes (ISGs) 5,6,7,8.
Hoʻokumu ʻia nā ISG i ka iwi kuamoʻo o ka ʻōnaehana pale kino maoli, ʻoi aku hoʻi i ka pane ʻana i ka hoʻouka kaua viral.Ma ke ʻano he lālani mua o ka pale ʻana i ka maʻi maʻi maʻi, hoʻolaha wikiwiki nā cell i nā pilina nui o nā protein cellular me kahi ākea o nā hana olaola.Aia kēia mau protein i nā mea hoʻokae ʻike kumu, nā molekala hōʻailona, ​​​​nā kumu transcription, a me nā protein me nā hana antiviral pololei, a me nā mea hoʻoponopono maikaʻi ʻole o nā pane pale.ʻO ka hapa nui o ka ʻike e pili ana i ka hana ISG mai nā pale hana e hoʻohana ana i nā kiʻi overexpression10,11 a i ʻole nā ​​ʻenehana silencing gene (siRNA, RNAi a me CRISPR)12,13 kahi i hōʻike ʻia ai nā ISG pākahi a hoʻopaʻa ʻia a hoʻāʻo ʻia kā lākou hana ma nā maʻi like ʻole.ʻOiai ua hoʻoholo kēia mau haʻawina i nā waiwai antiviral o kēlā me kēia ISG, ʻaʻole i ʻike nui ʻia nā ʻano molekala kumu o kēlā me kēia ISG.Ua ʻae ʻia ka nui o nā protein e launa pū me hoʻokahi a ʻoi aku paha nā cytokines e hōʻoia i ka hana piha, no laila e pili pololei ana nā ISG a i ʻole kā lākou pilina e hoʻopili ʻia e nā protein cellular.No ka laʻana, ua hōʻike ʻia kahi noiʻi proteomic photocrosslinked hou ʻo ATPase VCP/p97 ma ke ʻano he hoa pili IFITM3 nui, nona ka inhibition e alakaʻi i nā hemahema i ka lysosomal sorting, turnover, a me ka cotransport o IFITM3 me nā ʻāpana viral 14.Me ka hoʻohana ʻana i ka immunoprecipitation, ua ʻike mākou i ka VAPA, kahi protein pili i ka vesicle, ma ke ʻano he hoa pili me IFITM1/2/3 e hoʻopili ana i ka cholesterol-mediated viral maturation, a ua hōʻoia ʻia kēia e kahi noiʻi ʻē aʻe me ka hoʻohana ʻana i ka ʻōnaehana yeast two-hybrid system.Kākoʻo ʻepekema 15 , 16 .
ʻO kahi kaʻina hana koʻikoʻi e pili ana i ka hoʻopaʻa ʻana i ka maʻi a me ka hoʻololi ʻino ʻana ʻo ia ka hōʻike antigen, i hoʻopili ʻia e nā molekole histocompatibility complex (MHC).Hoʻokomo ʻia nā peptides (8-12 amino acids lōʻihi) mai nā protein i hoʻopau ʻia, hoʻopau mua ʻia a i ʻole misfolded i loko o ka heterodimer MHC-I (ʻo ia ka MHC-I nā kaulahao kaumaha a māmā, i kapa ʻia ʻo β-2-microglobulin; β2M) 17,18.Lawe ʻia nā trimers paʻa MHC-I i ka ʻili o ke kelepona, kahi e hōʻike ai lākou i nā peptides intracellular i nā cell CD8+ T (cytotoxic T cell)17.ʻIke a hoʻopau nā pūnae T i kēia mau pathogens a me nā cell e lawe ana i kahi antigen kikoʻī kikoʻī.No laila, hoʻopaʻa pinepine nā pathogens a me nā cell tumor i ke kaʻina hana hōʻike antigen e pale i ka nānā ʻana.Eia kekahi, ua hoʻohaʻahaʻa ʻia ka MHC-I i ka 40-90% o nā maʻi maʻi kanaka a pili pinepine ʻia me kahi prognosis ʻilihune19.
Pono e hoʻololi koke nā genes i ka pane ʻana i nā pathogens ma waena o kahi kūlana hoʻomaha a me kahi kūlana o ka transcription ikaika.No laila, ua manaʻo ʻia kekahi mau protein cellular e pili ana i ka pane ʻana i ka koi IFN kiʻekiʻe i nā manawa pōkole, me ka hoʻoponopono hou ʻana a me ka hoʻololi ʻana o ka promoter chromatin 20,21.ʻO ka hapa nui o nā haʻawina i kālele i ka ʻike ʻana o nā hoa pūmua ISG ponoʻī ma ke alo o IFN.Ua wehewehe kekahi mau noiʻi proteomic a me transcriptomic i nā ʻōnaehana cell model i ka hopena o IFN ma ka ʻāina kelepona.Eia nō naʻe, ʻoiai ka ulu ʻana o ka hoʻomaopopo ʻana i ka dynamics i hoʻoulu ʻia e nā interferon, ʻike liʻiliʻi mākou e pili ana i ke komo ʻana o nā ISG.I ka noʻonoʻo ʻana i ka paʻakikī a me ka manawa e pili ana i ka hōʻailona interferon, ʻelua nīnau e ala mai: (i) hiki ke hoʻopaʻa a hoʻopaʻa i nā complexes multiprotein i komo i ka hōʻailona wikiwiki, a (ii) hiki ke hoʻopaʻa ʻia kēia mau pilina i kahi ākea 3D?
No ka hoʻoponopono ʻana i kēia mau pilikia, ua hoʻokō mākou i ka disuccinimide suberate-mediated chemical cross-linking (DSS) i hui pū ʻia me ka mass spectrometry (CLMS) e aʻo i ka IFNα-induced protein interaction network and its dynamics.Hoʻohui ʻo DSS i nā mea paʻa covalent ma waena o nā koena proximal o nā protein a / a i ʻole nā ​​paʻakikī protein i vivo.Hōʻike ka hōʻike ʻana o MS ma hope o nā pūnaewele crosslinking kikoʻī e hōʻike ana i ka pili ākea o nā wahi i loko o kahi pūmua, i kapa ʻia nā loulou kūloko, a i ʻole nā ​​​​subunits i loko o nā pūmua protein, i kapa ʻia ʻo interrelationships.I ka hoʻohana ʻana i kēia ala, ua ʻike mākou i kekahi mau mea paʻakikī protein-protein hou a me nā ʻoihana hoʻopili multiprotein-induced interferon.Ma ka hoʻāʻo hou ʻana i kahi ʻāpana o kēia mau pilina hou, hōʻike mākou he H2BFS (H2B histone-type FS; ma hope aku i kapa ʻia ʻo H2B) a me MDN1 e hana ma ke ʻano he mau hoa paʻa no HLA-A.
ʻO nā pūnaewele Flo-1 kekahi o nā hiʻohiʻona in vitro maikaʻi loa o ka esophageal adenocarcinoma ʻoiai lākou e hoʻohālike i nā hiʻohiʻona koʻikoʻi o nā maʻi maʻi esophageal22,23.Eia naʻe, ʻaʻole he immunogenic nā maʻi maʻi a pau, a no ka hoʻoholo inā pane ʻo Flo-1 i ka mālama interferon, mālama mākou i nā cell Flo-1 me 10 ng/ml IFNα no 72 mau hola.Hōʻike nā pūnaewele Flo-1 i ka hoʻokomo mua ʻana o pSTAT1 a me IRF1, e hoʻomaka ana i nā hola 2 ma hope o ka mālama ʻana a hoʻomau no nā hola 72, me ka emi ʻana o ka manawa i nā pae paʻa o IRF1 (Figure 1A).ISGs (MX1, IFITM1, OAS1 / 2, a me ISG15) ua ʻike ʻia ua hoʻoikaika ikaika ʻia ma hope o 6 mau hola, e hoʻohālike ana i nā pane o ka waena waena a me ka hopena hope i IFNα (Figure 1A).Hoʻohui pū ʻia kēia mau ʻikepili e hiki ke hoʻohana ʻia kēia hoʻohālike kelepona e aʻo i nā pane interferon.
ʻO nā pane hōʻike protein ʻokoʻa i nā cell Flo-1 ma hope o ka mālama ʻana IFNα.(A) ʻO ka'ōlelo protein i loko o nā pūnaewele Flo-1 i mālamaʻia me 10 ng/ml IFNα no 2, 6, 24, 48 a me 72 mau hola i kālailai ʻia e immunoblot me ka hoʻohana ʻana i nā antibodies ISG i hōʻike ʻia.(B) Coomassie blue stained SDS-PAGE gels of whole cell extracts after cross-linking with DSS no nā manawa i kuhikuhi ʻia.(C) Ua nānā ʻia ka ʻelele immunoblot me ka p53(DO-1) antibody mai nā laʻana like e loiloi i ke kiʻekiʻe o ka hoʻopili ʻana i ka protein.
No ka hopu ʻana i ka ʻāina hoʻopili protein in situ, ua hoʻohana mākou i ka DSS, kahi mea hoʻohana nui ʻia ma muli o kona hikiwawe membrane kiʻekiʻe a me ka manawa pōkole.ʻO ka manawa pōkole pōkole e kōkua i ka pale ʻana i ka hoʻokumu ʻana o nā hui nui o nā protein crosslinked, a laila mālama i ka paʻa o ka crosslinker.No ka hoʻoholoʻana i ka manaʻo DSS maikaʻi loa a pale i ka over-crosslinking, ua hōʻike mua mākou i nā pūnaewele i 5, 2.5, a me 1 mM DSS no 5, 10, 5, a me 30 mau minuke, a nānā i nā lysates e Coomassie-stained SDS-PAGE (ʻaʻole hōʻike ʻia ka ʻikepili).ʻIke ʻia nā lysates cell i pili nui ʻia i ka haʻahaʻa haʻahaʻa a i ka manawa pōkole loa.No laila, ua titrated DSS i 1, 0.5, a me 0.1 mM ma luna o 5 minuke (Figure 1B).Ua ʻike ʻia ka crosslinking maikaʻi loa me 0.5 mM DSS no 5 mau minuke, a ua koho ʻia kēia mau kūlana no nā cell i mālama ʻia me IFNα.Eia kekahi, hōʻike ka Figure 1C i kahi blot Western i hana ʻia me ka p53 (DO-1) antibody e loiloi i ke kiʻekiʻe o ka hoʻopili ʻana i ka protein.
Ua mālama ʻia nā cell Flo-1 me 10 ng/ml IFNα no 24 mau hola ma mua o ka hoʻohui ʻana i ka crosslinker.Ua hoʻopiliʻia nā pūnaewele cross-linked e ka proteolysisʻelua-step a ua hanaʻia nā proteins e FASP (Fig. 2) 24,25.Hoʻopili ʻia nā peptides tryptic i hoʻopaʻa ʻia e ka mass spectrometry (Fig. 2).Hoʻohālikelike ʻia ka spectra MS/MS i ke kaʻina protein a helu ʻia me MaxQuant26,27.Ua ʻike ʻia nā peptides cross-linked mai ka spectra i loaʻa me ka hoʻohana ʻana i ka polokalamu SIM-XL, a ua hui pū ʻia nā pūhui pākahi i loko o kahi pūnaewele paʻakikī me ka hoʻohana ʻana i ka xQuest28 a me SIM-XL29 open source computing software pipelines (Fig. 2).Hoʻomaopopo ʻo SIM-XL i nā pilina protein-protein, nā kaulahao kūloko a me nā kaulahao pākahi i nā hui pūmua maʻalahi a paʻakikī paha a hāʻawi i nā palapala no ka ʻike ʻana i nā pilina ma nā hale protein.Eia kekahi, hoʻonohonoho ia i kēlā me kēia cross-reference ma ke ʻano he helu ID e like me ka maikaʻi spectrum MS/MS29.Ua ʻike ʻia kekahi mau pilina protein-protein a me nā paʻakikī, a ua noiʻi hou ʻia kahi hui hou o ka pilina me ka hoʻohana ʻana i ka co-immunoprecipitation a me nā hoʻololi conformational o nā paʻakikī e hoʻohana ana i ka molecular dynamics (MD) modeling (Fig. 2) 30, 31.
ʻO ka manaʻo hoʻolālā o ke ʻano CLMS.Ua mālama ʻia nā pūnaewele Flo-1 me 10 ng/ml IFNα no nā hola 24 a ukali ʻia e in situ protein cross-linking me ka hoʻohana ʻana i ka DSS a ukali ʻia e ka cell lysis a me ka trypsinization.Hoʻopili ʻia nā laʻana i hoʻopili ʻia me ka hoʻohana ʻana i kahi spectrometer nui Orbitrap a hoʻohālikelike hou ʻia no ka ʻāpana o nā peptide precursors i ka wā LC-MS/MS.Ua ʻike ʻia ʻelua peptides i hoʻopili ʻia mai ka spectra i loaʻa me ka polokalamu Spectrum Recognition Machine o ka Crosslinked Peptides (SIM-XL), a ua hui pū ʻia nā pūhui āpau i loko o kahi pūnaewele paʻakikī me ka hoʻohana ʻana i nā pipeline computational.E kānana i nā pilina hilinaʻi haʻahaʻa e pili ana i nā helu helu kuhi hewa (FDR).Ua hoʻokūpaʻa hou ʻia kekahi mau pilina protein-protein kiʻekiʻe me ka hoʻohana ʻana i ka co-immunoprecipitation, a ua nānā ʻia nā loli conformational i nā complexes me ka hoʻohana ʻana i ka molecular dynamics (MD).
Uaʻikeʻia ka nui o ~ 30,500 a me ~ 28,500 peptides me ka hoʻohanaʻana i MaxQuant i nā hōʻailona IFNα unstimulated a me ka hoʻouluʻana, i kēlā me kēia (Supplementary Table S1, Fig. 3A).ʻO ka hāʻawiʻana i ka lōʻihi o ka peptide i nā hihiaʻelua i hōʻikeʻia i kahi kiʻekiʻe o nā peptides nui aʻe, e hōʻike ana i ka heleʻana o nā peptides i hoʻohuiʻia (Fig. 3B, C).Eia hou, aia ka hapa nui o nā peptides nui ma ka 40-55 i loko o nā hōʻailona IFNα-treated (Fig 3C).Ua hōʻike ʻia ka palapala ʻāina protein e kūʻē i ka ikaika log2 i ka nui o nā protein interferon-stimulated maʻamau i hoʻohālikelike ʻia me nā laʻana i mālama ʻole ʻia, me MX1, IFIT1/3, OAS2/3, DDX58, a me HLA-F (Figure 3D).ʻO ka nānā ʻana i nā ala no nā protein i ʻoi aku ma mua o ʻekolu mau manawa i hoʻonui ʻia i ka pane ʻana i ka mālama ʻana iā IFNα me ka hoʻohana ʻana i ka waihona ala ala Reactome i hōʻike ʻia ʻo MHC-I-mediated antigen hōʻike a me ka hana ʻana ʻo ia ke ala nui loa (Figure 3E).E like me nā hōʻike mua, nā pane antiviral i hoʻopili ʻia e OAS a me ISG15 a me IFNα / β a me ka cytokine hōʻailona ma waena o nā ala i hoʻāla ʻia.Hoʻohui ʻia, ua ʻike ʻia nā lysine- a me serine-specific protein cross-links mai ka MS / MS spectra i loaʻa mua me ka SIM-XL.Ua hōʻike ʻia kahi noiʻi hou he 104 ISG e holo ana i 20 mau maʻi mai 9 mau papa virus ma o ka meta-analysis o kēlā me kēia ISG overexpression haʻawina ma 5 cell type9.Eia naʻe, no ka lanakila ʻana i nā palena helu helu o ka nānā ʻana i nā ʻikepili nui, ua hoʻomaka mākou me kahi ʻikepili liʻiliʻi e ʻimi i nā pilina hiki ke hiki ma waena o ka papa inoa o nā genes IRDS i hōʻike ʻia e Padaria et al., ʻo ka hapa nui o lākou he ISG.
ʻO ka ʻike ʻana i nā protein i hoʻopili ʻia ma ke ʻano he pane i ka IFNα (ʻike i loaʻa mai MaxQuant).(A) Venn diagram e hōʻike ana i ka helu o nā peptides maʻamau a kūʻokoʻa i ʻike ʻia ma IFNα14 i mālama ʻia a mālama ʻole ʻia ʻo Flo-1.Ka māhele ʻana o ka lōʻihi peptide o nā laʻana i hoʻopili ʻole ʻia (B) a me IFNα i mālama ʻia (C).(D) Heat map e hōʻike ana i ka log2 (LFQ intensity) ma waena o ka mālama ʻole ʻia a me ka IFNα14 i mālama ʻia i nā cell Flo-1.Hōʻike ka ʻaoʻao hema i nā protein i hoʻoikaika nui ʻia ma ke alo o IFNα.(E) Histogram e hōʻike ana i nā ala hoʻonui nui 20 ma hope o ka mālama ʻana iā IFNα.Ua kālailai ʻia ka ʻikepili ala ala Reactome ma mua o ʻehā mau hoʻololi i nā pūmua pane pane IFNα.
Hoʻomaopopo maikaʻi ʻia ka hoʻoulu ʻana o Interferon-mediated ISG, akā ma ka pae molekala ʻaʻole maopopo i ke ʻano o ka hopena o kēia mau protein i kahi ākea o nā hana biological.Ua noiʻi mākou i nā pilina protein me ka hilinaʻi kiʻekiʻe ma waena o nā ISG i ʻike ʻia.ʻO ka mea mahalo, ua ʻike mākou i kahi pūnaewele me MX1, USP18, ROBO1, OAS3, a me STAT1 protein e hana i kahi paʻakikī nui i ka pane ʻana i ka lāʻau IFNα (Figure 4, Table S2) 32,33,34.ʻO ka mea nui loa, ua loaʻa kēia mau pilina ma nā triplicates āpau i mālama ʻia me IFNα a ʻaʻole i loaʻa i nā laʻana i mālama ʻole ʻia, e manaʻo ana ua hoʻokumu ʻia lākou i ka pane ʻana i ka mālama ʻana IFNα.Ua ʻike ʻia ʻo STAT1 transcriptionally e hoʻoponopono i ka hōʻike ʻana o kēia mau ISG, akā ʻaʻole i aʻo ʻia kāna pilina me ISG ma ka pae protein.Ua hōʻike ʻia ke ʻano aniani o STAT1 ʻaʻole pili kāna domain helical (CCD) i ka pilina me DNA a i ʻole protomers i ka wā o ka hoʻokumu ʻana o dimers35.Hoʻokumu kēia mau α-helices i kahi ʻano helical helix e hāʻawi ana i kahi ʻāpana hydrophilic nui no ka hoʻopili ʻana 35 .Ma kā mākou ʻikepili CLMS, ua ʻike mākou i ka hapa nui o nā pilina me STAT1 i hana ʻia ma ka SH2 domain ma mua o ka CCD, ka domain linker, a i ʻole ka huelo C-terminal (koena 700-708) (Figure 4A).Ua hōʻike ʻia kahi noiʻi mua e pili ana ʻo USP18 i ka CCD a me ka DNA-binding domain (DBD) o STAT2 a ua hoʻopaʻa ʻia i ka subunit o ke ʻano I interferon receptor IFNAR2 e hoʻopili i ka inhibition o ke ʻano I interferon hōʻailona 24.Ua hōʻike pū ʻia kā mākou ʻikepili e pili ana ka USP18 catalytic domain me STAT1 DBD (Figure 4A, D), e hōʻike ana e hiki ke pāʻani ʻo STAT1 a me STAT2 i ka huki ʻana iā USP18 i IFNAR2.
ʻIke ʻia ka ʻupena ISG protein-protein i loko o nā keʻena pili keʻa i mālama ʻia me IFNα.(A) 2D pāʻani pāʻani e hōʻike ana i nā pilina protein-protein (i hana ʻia ma ka papahana SIM-XL), me nā laina e hōʻike ana i nā pilina intermolecular (crosslink cutoff set to 3.5).Hōʻailona ʻia nā kāʻei o nā ʻike like ʻole e ko lākou kala32: MX1 domain, Dynamin_N (73–249), Dynamin_M (259–547), a me GED (569–660).Nā kāʻei kapu OAS3: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), a me 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) a me fn3 (777–864).Nā kahua STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), a me STAT1_TAZ2bind (715–739).(B) He mea nānā i ka pōʻai o nā protein cross-linked (MX1, UBP18, OAS3, ROBO1, a me STAT1) me nā pilina a me nā pilina i hōʻailona ʻia me ka uliuli a me ka ʻulaʻula.Ua hoʻonoho ʻia ka paepae loulou cross ma 3.5.Hōʻike nā ʻāpana kiko i nā kahua pānaʻi STAT1 me MX1 (C), USP18 (D), ROBO1 (E), a me OAS3 (F), a me nā pūnaewele pili K a S ma waena o nā peptides ʻelua.Ma ke kiʻi, ua hoʻonohonoho ʻia ka paepae helu cross-link i 3.0.(G) Nā kahua pilina like ʻole ma waena o STAT1 a me OAS3 DI mau kāʻei kapu i hoʻopaʻa ʻia ma luna o kā lākou mau pūmua i PyMol (PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) a me OAS3 (pdb id: 4s3n34).) papahana.
Ua wehewehe ʻia ʻelua isoforms o USP18 i loko o ke kanaka, kahi protein piha piha i ka nui o ka nucleus, a me kahi isoform me ka ʻole o kahi kikowaena N-terminal, USP18-sf, i māhele like ʻia i ka cytoplasm a me ka nucleus 36.Eia kekahi, ua wānana ʻia ka N-terminus ʻaʻole i hoʻonohonoho ʻia a ʻaʻole koi i ka hana isopeptidase a i ʻole ISG1537 paʻa.ʻO ka hapa nui o nā pilina i ʻike ʻia i kā mākou noiʻi aia ma ka N-terminus o ka protein, e hōʻike ana e pili ana kēia mau pilina i ka USP18 piha piha (Figure 4A, D) a pēlā paha e ulu ai i loko o ka nucleus.Eia kekahi, hōʻike pū kā mākou ʻikepili he mea kūikawā ka N-terminus no ka pilina protein-to-protein.Aia ka pūnaewele hoʻopaʻa IFNAR2 ma waena o nā koena 312-368, a me ka mea nui, ʻaʻohe o nā protein i loko o ka paʻa paʻa i kēia māhele (Fig. 4A) 37,38.ʻO kēia mau ʻikepili i hui pū ʻia e hōʻike ana i ka hoʻohana wale ʻia ʻana o ka domain binding IFNAR2 e ka protein receptor.Eia kekahi, ʻo OAS3 a me ROBO1 wale nō i ʻike ʻia e pili ana me nā kāʻei kua i luna o ka N-terminus a me IFNAR2 paʻa pūnaewele (Figure 4A).
Aia ʻo ROBO1 i ka immunoglobulin (Ig) superfamily o nā molekele hōʻailona transmembrane a loaʻa iā ʻelima mau kikowaena Ig a me ʻekolu mau kikowaena fibronectin (Fn) i ka ʻāina extracellular.Hoʻopili ʻia kēia mau kikowaena extracellular e kahi ʻāpana membrane-proximal a me kahi helix transmembrane hoʻokahi 39. Aia kahi ʻāpana intracellular ʻaʻole i kūkulu ʻia ma ka C-terminus a loaʻa i nā motif kaʻina i mālama ʻia e hoʻopaʻa i ka paʻa ʻana i ka protein effector39.ʻO ka ʻāpana mai nā amino acids ~ 1100 a hiki i 1600 ka hapa nui.Ua ʻike mākou ua pili ʻo MX1 me ROBO1 ma o Ig, Fn, a me nā kikowaena intracellular, ʻoiai ʻo ka hapa nui o nā pilina me STAT1 e hana ʻia ma waena o kāna CCD, linker domain, a me ka C-terminus o ROBO1 (Fig. 4A,E).Ma kekahiʻaoʻao, ua māheleʻia nā pilina me DI, DIII, a me OAS3 linker regions a puni ka protein ROBO1 (Fig. 4A).
ʻO ka ʻohana protein oligoadenylate synthase (OAS) e ʻae a hoʻopaʻa i ka RNA (dsRNA) i loko o ka intracellular, e hana i nā loli conformational, a synthesizes 2′,5′-linked oligoadenylates (2-5 As) 40.Ua ʻike ʻia ma waena o nā OAS ʻekolu, hōʻike ʻo OAS3 i ka pilina kiʻekiʻe loa no ka dsRNA a synthesizes ka liʻiliʻi o 2-5 As, hiki ke hoʻāla iā RNase L a no laila e kaupalena i ka hoʻopiʻi viral 41.ʻO ka ʻohana OAS he polymerase beta (pol-β)-like nucleotide transferase domains.Ua hōʻike ʻia nā noiʻi mua e pili ana ka hana catalytic o ka C-terminal domain (DIII) i ka dsRNA-binding domain (DI), i koi ʻia no ka hoʻāla ʻana o OAS342.Ua ʻike mākou i ka hui ʻana o nā kāʻei DI a me DII o OAS3 me CCD a me kahi ʻāpana liʻiliʻi ma waena o SH2 a me STAT1 TAD (Figure 4A, F).ʻO ka uhi ʻana i nā wahi crosslinking like ʻole ma ka ʻōnaehana protein i hōʻike ʻia i kahi pilina ma waena o ka β-sheet a me DBD STAT1 loop a me kahi ʻeke hāmama a i ʻole ka lua i hana ʻia e nā koena 60-75 ma ka domain DI o OAS3 (Fig. 4G).Ua hōʻike pū ʻia ka hoʻonohonoho ʻana o nā protein i loko o ka paʻakikī ʻaʻole kekahi o nā pilina me OAS3 i hoʻopilikia i ka hiki ke hoʻopaʻa ʻia DNA o kāna domain DI (Fig. S1A).Eia kekahi, pili nui ka N-terminal domain o GTPase MX1 me nā kāʻei DI a me DIII o OAS3 (Fig. 4A).Ua ʻike pū mākou i kahi pilina ma waena o OAS1 a me MX1 i ʻekolu mau hana hou ʻana o ka IFNα, kahi i hui pū ʻia ai kahi domain OAS1 (ʻo catalytically active) me nā kikowaena MX1 ʻekolu (Figure S2A,B).
ʻO nā proteins MX he ʻāpana o ka ʻohana nui o nā GTPases e like me dynein i loaʻa kahi kikowaena N-terminal GTPase e hoʻopaʻa a hydrolyzes GTP, kahi kikowaena waena e hoʻopili ana i ka hui ʻana iho, a me kahi pahu leucine C-terminal e hana ma ke ʻano he GTPase (LZ). ).domain effector domain25,43.Hoʻopaʻa ʻo MX1 i nā ʻāpana o nā polymerases viral e pale i ka unuhi ʻana o ka gene viral43.Ua hōʻike mua ʻia kahi hū hū ʻelua-hybrid pale i ka PIAS1-pili MX1 ke kāohi i ka STAT1-mediated gene activation ma ke kāohi ʻana i ka hana DNA-binding a loaʻa pū kekahi SUMO E344,45 ligase hana.Maʻaneʻi, hōʻike mākou e pili ana ka MX1 iā STAT1 (Figure 4C, D), akā naʻe pehea e pili ai kēia pilina i ka STAT1-mediated gene activation ma ka pane ʻana iā IFNα pono e aʻo hou.Eia kekahi, ua ʻike pū mākou ua launa pū ʻo MX1 me IFIT3 a me DDX60 i nā hana hou ʻekolu IFNα-treated (Fig. S2C).
ʻO DDX60 kahi helicase cytoplasmic i hoʻokomo ʻia e IFN i hōʻike mua ʻia e pāʻani i kahi kuleana ma RIG-I-kūʻokoʻa degradation o viral RNA46.Hoʻopiliʻo ia me RIG-I a hoʻoulu i kāna hōʻailona ma keʻano ligand-specific 46. Aia ka DDX60 i kahi domain helicase DEXD / H-Box a me kahi domain helicase C-terminal e hoʻopaʻa i ka RNA viral a me DNA47.ʻO ka hapa nui o kāna mau pilina me MX1 a me IFIT3 e loaʻa ana i loko o nā ʻāpana N- a me C-terminal lōʻihi me ka ʻole o ka canonical domain a motifs (Fig. S2E,F).Eia naʻe, pili pū ka MX1 me ka DEXD / H-Box helicase domain (Fig. S2E).Loaʻa i nā protein o ka ʻohana IFIT nā kope like ʻole o kahi kumu helix-turn-helix ʻokoʻa i kapa ʻia ʻo ka tetrapeptide repeat (TPR).Ua ʻike ʻia ʻo IFIT3 he modulator maikaʻi o ka hōʻailona RIG-I a no laila he ʻāpana o ka complex MAVS.Hoʻohui pūʻia, hōʻike kā mākouʻikepili i ka pilina o IFIT3 a me DDX60 ma ka māhele ma waena o TPR 3-6 o IFIT3 a hiki ke hoʻokani i kahi hana i ka hōʻailona RIG-I / MAVS (Fig. S2F).
Hāʻawi ʻia i ka nānā ʻana i ka proteome holoʻokoʻa i ka helu ʻana, a laila nānā mākou i ka waihona UniProt kanaka holoʻokoʻa no ka loaʻa ʻana o kekahi o nā hana hou i mālama ʻia e IFNα.I loko o kēia kope, ua loaʻa iā mākou kekahi mau pūnaewele pili pono loa no HLA-A.ʻO ka nānāʻana i nā ala protein iʻikeʻia e MS / MS spectra i hōʻike i ka MHC-I-based antigen processing a me ka hōʻikeʻana i ke ala nui i hoʻokomoʻia e ka interferon (Fig. 3D).No laila, ua kālele mākou i ke aʻo ʻana i ka pilina pūmua o nā molekala MHC-I me ka hilinaʻi kiʻekiʻe i nā laʻana pili kea.Aia ka HLA i nā domain α1, α2 a me α3 a me nā kaulahao māmā, a ʻo ka microglobulin β2 (β2m) he protein chaperone mau.I ka manawa e ʻākoakoa ai i loko o ka endoplasmic reticulum, ʻaʻole paʻa ka HLA i ka loaʻa ʻole o nā peptide ligands50.Hoʻokumu ʻia ka peptide-binding groove e ka polymorphic kiʻekiʻe a me ka unstructured α1 a me α2 domain i loko o ke ʻano non-peptide a me ka liʻiliʻi polymorphic α351 domain.Ma ke alo o IFNα, ua ʻike mākou i ʻelua HLA-A complexes: pili kekahi me HMGA1 a me H2B (Figure 5, Table S3) a pili pū kekahi me MDN1, LRCH4 a me H2B (Figure 6).
Hoʻokomo ʻo IFNα i kahi pūnaewele pili HLA-A me H2B (H2BFS) a me HMGA1.(A) 2D plot (hana ʻia ma SIM-XL lako polokalamu) e hōʻike ana i nā ʻano like ʻole o ka pilina ma ka H2B-HLA-A-HMGA1 complex: interlink (blue), interlink (ulaʻula) a me ka loulou hoʻokahi (ʻeleʻele)..ʻO nā kāʻei kapu o nā ʻike like ʻole he coded32: H2B (histone; 2–102) a me MHC-I (MHC_1; 25–203, hui C1; 210–290 a me MHC_I_C; 337–364).Ua hoʻonoho ʻia ka paepae loulou cross ma 3.5.Hōʻike nā ʻāpana kiko i nā kahua pānaʻi HLA-A me H2B (B) a me HMGA1 (C), a me nā kahua pili K a S ma waena o nā peptides ʻelua.Ma ke kiʻi, ua hoʻonohonoho ʻia ka paepae helu cross-link i 3.0.(D) Nā pilina ma waena o nā protein i hōʻike ʻia ma nā hale o nā protein H2B, HLA-A, a me HMGA1 i ka papahana PyMOL.Ua hoʻohālikelike ʻia kēia mau hale me ka hoʻohana ʻana i ka server Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) a ʻo nā ʻano hoʻohālike no nā protein H2B, HLA-A a me HMGA1 he 1kx552, 1kj349 a me 2eze55, kēlā me kēia.
Hoʻokomo ʻo IFNα i kahi ʻoihana pili HLA-A me H2B (H2BFS), MDN1 a me LRCH4.(A) Intramolecular (ʻulaʻula) a me intermolecular (uliuli) i hōʻike ʻia ma kahi palapala 2D interactive (i hana ʻia ma ka polokalamu SIM-XL) me MDN1 i hōʻike ʻia ma ke ʻano he pōʻai.Ua hoʻonoho ʻia ka paepae loulou cross ma 3.5.ʻO nā kāʻei kapu o nā ʻike like ʻole he coded32: H2B (histone; 2–102), MHC-I (MHC_1; 25–203, hui C1; 210–290 a me MHC_I_C; 337–364) a me LRCH4 (LRR_8 (68–126), LRR_8 (137–194) a me CH (535–641)).(B) Nā pilina ma waena o nā protein i hōʻike ʻia i loko o nā hale o nā protein H2B, HLA-A, LRCH4, a me MDN1 i ka papahana PyMOL.Hoʻohālikelike ʻia kēia mau hale me ka hoʻohana ʻana i ka server Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) me nā hoʻolālā template 1kx552, 1kj349, 6hlu62 a me 6i2665 no nā protein H2B, HLA-A, LRCH4 a me MDN1, pakahi.Nā kiko kiko e hōʻike ana i nā kahua pānaʻi K a i ʻole S no HLA-A me H2B (C), LRCH4 (D), a me MDN1 (E).No nā ʻāpana, ua hoʻonohonoho ʻia ka paepae helu cross-link i 3.0.
Ma waho aʻe o ka mālama ʻana i ka pono o ka genome, pili pū ka histone H2B i ka hoʻoponopono ʻana i ka transcription.Aia ka protein H2B i kahi kikowaena histone kikowaena (HFD) i hana ʻia e ʻekolu α-helice i hoʻokaʻawale ʻia e nā puka lou a me kahi huelo C-terminal 41,52.Loaʻa ka hapa nui o ka pilina me H2B i ka α1 helix, e hāʻawi ana i ka trimerization me ka HFD heterodimer (Fig. 5A,B).ʻOiai ua komo nā lysins i ka hoʻopaʻa ʻana i ka DNA, ʻo kekahi mau lysins kekahi mau wahi acetylation a i ʻole methylation.No ka laʻana, ʻaʻole i komo nā koena K43, K46, a me K57 mai H2B i ka hoʻopaʻa ʻana i ka DNA pololei, akā he mau pahuhopu ia o nā hoʻololi post-transcriptional53.Pēlā nō, hiki i nā koena K44, K47, a me K57 i ka H2B ke pāʻani i kahi hana ʻē aʻe i ke alo o IFNα, me nā pilina me nā protein ʻē aʻe (Fig. 5A, B).Eia kekahi, ho'āla ka extrachromosomal histone H2B i ka pane immune ma nā ʻano cell like ʻole, e hana ana ma ke ʻano he cytosolic sensor e ʻike ai i nā ʻāpana DNA (dsDNA) pālua ʻelua i loaʻa mai nā mea maʻi a i ʻole nā ​​​​pūnaewele pōʻino54.Ma ke alo o nā maʻi maʻi DNA, hoʻopau ka H2B i ka hana IFN-β a me STAT154 phosphorylation.ʻIke ʻia ʻo H2B e neʻe i loko a i waho o ka nucleus ma mua o nā histones kumu ʻē aʻe54.Ua nānā pū ʻia nā pilina H2B me MDN1 a me LRCH4 i nā laʻana i koho ʻole ʻia.Ua ʻike mākou ua launa pū ʻo HLA-A me H2B i nā laʻana ʻekolu i mālama ʻia e IFNα a i hoʻokahi laʻana hana hou ʻole.Hōʻike kēia mau ʻikepili i ke kuleana o H2B i kahi hana physiological ʻē aʻe kūʻokoʻa i ka hoʻoponopono transcriptional.
Ua ʻike ʻia ʻo HMGA1 (hui mobility kiʻekiʻe AT-Hook 1), kahi nucleoprotein liʻiliʻi i waiwai i nā waikawa amino hoʻoikaika i ka maʻi, i hui pū me HLA-A.Loaʻa iā ia kahi huelo C-terminal acid a me ʻekolu mau DBD ʻokoʻa i kapa ʻia ʻo AT hooks no ka mea ua nakinaki lākou i ke awāwa liʻiliʻi o ka ʻāina waiwai AT ma dsDNA55,56.ʻO kēia hoʻopaʻa ʻana e hoʻopololei a hoʻopololei paha ka DNA, e ʻae ana i nā kumu transcription canonical e komo i kāna kaʻina consensus.Manaʻo ʻia ka huelo C-terminal e pili ana i ka pilina protein-protein a me ka hoʻopaʻa ʻana i nā kumu transcription, ʻoiai ʻaʻole hiki i nā mutants holoi C-terminal ke hoʻomaka i ka transcription57.Eia kekahi, aia kekahi mau kahua phosphorylation i mālama ʻia i ʻike ʻia nā substrate no kinases 58.Ua nānā mākou i ka pilina o HLA-A a me H2B me HMGA1 ma waho o ke kikowaena C-terminal, e hōʻike ana e hoʻohana nui ʻia ke kikowaena C-terminal no ka transcription factor binding (Fig. 5A, C).Hoʻokūkū nā protein HMGA me ka histone H1 no ka hoʻopaʻa ʻana i ka DNA adapter, a laila e hoʻonui ai i ka hiki57.Pēlā nō, me he mea lā e pili ana ka HMGA me ka histone H2B ma ka DNA linker i ka hoʻokūkū me ka histone H1.Hoʻokomo ʻo HMGB1 i ka hōʻike o HLA-A, -B, a me -C i nā cell dendritic, e alakaʻi ana i kā lākou activation59, akā ʻaʻole i hōʻike mua ʻia kahi pilina ma waena o HMG a me HLA.Ua ʻike mākou ua pili ʻo HMGA1 me nā kāʻei kapu α1 a me α3 o HLA-A, me ka hapa nui o nā pilina ma waho o kāna 3 DBD (Figure 5A,C).Ma ko mākou mau lima, ua ʻike ʻia ʻo HLA-A i loko o ka nucleus (ʻaʻole i hōʻike ʻia ka ʻikepili), a hāʻawi ʻia ʻo H2B a me HMGA1 i loko o ka nucleus, hiki ke hana ʻia kēia pilina ma ka nucleus.Hōʻike ʻia nā hoʻohui kikoʻī i ana ʻia ma waena o H2B, HLA-A, a me HMGA1 ma ke Kiʻi 5D.
ʻO ka hapa nui o nā pilina o HLA-A me nā protein ʻē aʻe e loaʻa i loko o kāna mau kikowaena α1 a me α2 a me ka domain C-terminal disordered (Fig. 6).Ma kekahi o kēia mau hiʻohiʻona, ua ʻike mākou ua hui pū ʻo HLA-A me ka huelo N-terminal o LRCH4 (Figure 6A, D).Hoʻoponopono ʻo LRCH4 i ka hoʻoulu ʻana o TLR4 a me ka LPS cytokine induction, a laila hoʻololi i ka pane kūlohelohe kūlohelohe60,61.He pūmua membrane me ʻeiwa mau leucine-rich repeats (LRRs) a me kahi motif homology calmodulin (CH) i kona ectodomain, a ukali ʻia e kahi transmembrane domain (TMD) 60, 62.Ua hōʻike ʻia nā kāʻei kapu CH e hoʻopili i ka pilina protein-protein 60.Hiki ke loaʻa ma kahi o 300 mau amino acids ma waena o nā kāʻei kapu LRR a me CH akā pilikia.Ma muli o ka hana o nā wahi i hoʻokaʻawale ʻia ma ke ʻano he mediators o nā pūnaewele protein-protein a me nā vesicular transport 63, ua ʻike mākou i ka hapa nui o ka pilina o ka protein i nā ʻāpana pilikia.Hoʻokaʻawale ʻia nā pilina me MDN1 a puni ka lōʻihi o ka protein, me ka LRR1, LRR6, CH domains, a me nā ʻāpana random, ʻoiai ʻo H2B i hoʻopaʻa ʻia i ka domain CH (Fig. 6A, B).ʻO ka mea nui, ʻaʻohe o nā pilina i hoʻokomo i ka TMJ, e hōʻike ana i ka kikoʻī o ke ala CLMS (Figure 6A, B).
Ua ʻike ʻia ʻo MDN1 ma ke ʻano he ʻāpana o ka pūnaewele protein HLA-A (Figure 6A).No ka ʻohana AAA o nā protein (ATPases pili me nā hana like ʻole).ʻO kēia ka N-terminal AAA domain e hoʻonohonoho i loko o kahi apo hexameric a wehe i ka helu hui mai ka 60S 64 ribosomal subunit.ua like ia me dynein64,65,66.Eia kekahi, hahai ʻia ka ʻāina waiwai ʻo Asp/Glu e ka MIDAS domain (metal ion dependent site).Ma muli o ka nui o ka MDN1 (ma kahi o 5600 amino acids) a me kāna homology liʻiliʻi me nā protein i aʻo maikaʻi ʻia, liʻiliʻi ka ʻike e pili ana i kona ʻano a me kāna hana i loko o ke kanaka.Ua ʻike mākou iā HLA-A, H2B, a me LRCH4 ma ke ʻano he mau hoa paʻa MDN1 a hōʻike i ko lākou ʻano ʻano he mau protein complexes ma PyMol (Fig. 6A,B).Hoʻopili kēia mau protein ʻekolu me ka domain AAA, ka dynein-like linker domain, a me ka MIDAS MDN1 domain.Ma kahi hōʻike mua, hoʻomaʻemaʻe hoʻomaʻemaʻe i nā protein maunu i ʻike ʻia ʻo MDN1 ma ke ʻano he protein pili me ka histone H2B67.Eia kekahi, ua hōʻike pū kekahi noiʻi hou i kahi pilina ma waena o MDN a me HLA-B i nā cell HCT116 me ka hoʻohana ʻana i ka affinity-purified mass spectrometry, e kākoʻo ana i kā mākou ʻike68.ʻO ka ʻike ʻana o kēia paʻakikī i nā laʻana i mālama ʻia e IFNα e hōʻike ana i kahi kuleana no MDN1 i ka hōʻailona interferon.
No ka mea he polymorphic loa nā genes HLA, ua unuhi mākou i ka heluhelu ʻana ma ka palapala ʻana i ka HLA-A, -B, a me -C mai ka ʻikepili sequencing RNA o nā cell Flo-1 (ʻaʻole i hōʻike ʻia ka ʻikepili).Ua hōʻike ʻia nā ʻokoʻa koʻikoʻi ma waena o HLA-A, -B, a me -C ma nā wahi i loaʻa ai nā peptides cross-linked ma HLA-A (Figure S3).Eia kekahi, ʻaʻole mākou i ʻike i ka hoʻopili ʻana i ka protein-to-protein cross-linking o nā molekala HLA-B/C me nā protein H2B/HMGA1/MDN1/LRCH4.Hōʻike kēia i ka pilina pūmua i loaʻa ma waena o HLA-A, MDN1, LRCH1 a me HMGA1 he HLA-A kikoʻī.Eia hou, ua hōʻike ʻia ka loiloi proteomic o nā laʻana non-crosslinked (Table S4) i ʻoi aku ka nui o ka HLA-A i ke kaʻina o ka hoʻohālikelike ʻana me HLA-B a i ʻole HLA-C.ʻO nā peptides i ʻike ʻia no ka HLA-A he kiʻekiʻe i ka ikaika ma nā ʻāpana ʻelua i mālama ʻia e IFNα a i mālama ʻole ʻia.
No ka hōʻoia ʻana ʻaʻole ma muli o ka hoʻopili ʻole ʻana o nā protein ʻelua ma kahi kokoke, ua hōʻoia hou mākou i ʻelua mau mea pili HLA-A hou ma o ka hana ʻana i ka co-immunoprecipitation assays.Uaʻikeʻia nā pilina HLA-A me ka MDN1 a me ka H2B endogenous i loko o nā pūnaewele Flo-1 i hoʻoponoponoʻoleʻia e IFNα (Figure 7, Figure S4).Ua hōʻoia mākou ua hopu ʻia ʻo HLA-A e H2B i nā immunoprecipitates a ʻo kēia hui ʻana ma muli o ka mālama ʻana o IFNα mai ka wā i hala ʻole ai ʻo HLA-A i nā sample immunoprecipitate mai nā cell i mālama ʻole ʻia (Figure 7A).Eia naʻe, hōʻike kā mākou ʻikepili e hoʻoponopono ʻokoʻa ʻo IFNα i ka HLA-A e hoʻopaʻa ana iā H2B a me MDN1.Hoʻokomo ʻo IFNα i ka hui ʻana ma waena o H2B a me HLA-A, akā hoʻemi i kona hui pū me MDN1.Ua ʻike mākou ua pili ʻo MDN1 me HLA-A i nā mana, a ʻo ka hoʻohui ʻana o IFNα i hōʻemi i kēia pilina kūʻokoʻa i ka hoʻokomo ʻana o MDN1 e IFNα (Figure 7B, C).Eia kekahi, hopu ka HLA-A immunoprecipitation i ka H2B ma nā pūnaewele A549 (Fig. S4), e hōʻike ana he kūʻokoʻa kēia pilina ma ke ʻano cell.Hoʻohui pū ʻia, kākoʻo kēia mau hopena i nā pilina interferon-mediated o HLA-A me H2B a me MDN1.
Hoʻomaʻemaʻe pū ʻo HLA-A iā H2B a me MDN1.Ua hoʻopau ʻia nā immunoblots endogenous H2B (A) a me MDN1 (B) mai nā pūnaewele Flo-1 i mālama ʻia e IFNα a nānā ʻia no nā antibodies i hōʻike ʻia.Ua hoʻohana ʻia ka ʻiole a me ka rabbit IgG ma ke ʻano he mana maikaʻi ʻole.(C) Hōʻike ʻia nā helu (hoʻokomo) o nā antigens like ʻole e nā immunoblots i hoʻopaʻa ʻia e kūʻē i nā antibodies i hōʻike ʻia, ua hoʻohana ʻia ka β-actin ma ke ʻano he mana hoʻouka.
Ua noiʻi ʻia nā ʻano hana o kekahi o ka interferon-induced high trust cross-linked network, H2B-HLA-A-HMGA1.Ua hoʻohana mākou i ka molecular dynamics modeling ma ke ʻano he ala ʻē aʻe e hoʻomaopopo i ka conformational dynamics o nā protein i komo i kēia paʻakikī (Figure 8).Hōʻike nā manaʻo mai ka ʻikepili CLMS i ka hiki ke hoʻohālikelike ʻia o nā protein H2B, HLA-A, a me HMGA1.No laila, ua hoʻohālike ʻia nā mea paʻakikī e hiki mai ana i loko o kahi mea hoʻoheheʻe: H2B-HLA-A, HMGA1-HLA-A, a me H2B-HLA-A-HMGA1.Ua hōʻike ʻia kahi pānaʻi docking protein-protein mua me ka MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) i nā conformations hiki ke ʻokoʻa ma waena o kēia mau protein (Fig. 8A).ʻO ka ʻike ʻana i ka paʻakikī protein docking i hōʻike ʻia i kekahi mau pilina a me nā conformations hiki ke loaʻa (Figure 5A, 8).No laila, hōʻike ʻia kahi hoʻohālikelike i hiki ke hōʻike ʻia ma ke Kiʻi 8A (me nā loulou cross-labeled) a ua loiloi hou ʻia me ka hoʻohana ʻana i ka pipeline modeling MD.Eia kekahi, ʻo ka hoʻopaʻa ʻana o H2B a i ʻole HMGA1 iā HLA-A e hōʻike ana i ka pilina kiʻekiʻe o H2B no HLA-A (Fig. 8A).
Nā hoʻololi hoʻohālikelike o nā pūnaewele hiki ke hiki ma waena o nā hui H2B (H2BFS)-HLA-A, HMGA1-HLA-A, a me H2B-HLA-A-HMGA1.(A) He palapala ʻāina 2D ka ʻaoʻao hema (i hana ʻia ma ka polokalamu SIM-XL) o nā loulou intramolecular (ʻulaʻula) a me intermolecular (uliuli) (crosslink cutoff set i 3.5).Hoʻohui ʻia, ua hoʻopaʻa inoa ʻia nā koena hoʻohui cross-linking ma nā hale o nā protein H2B, HLA-A, a me HMGA1.Ua unuhi ʻia nā conformations pili o kēia mau protein me ka hoʻohana ʻana i ka pipeline docking i hoʻokō ʻia ma ka pūʻulu MOE.Hōʻike ka ʻaoʻao hema haʻahaʻa i nā conformations like ʻole o ka H2B-HLA-A a me HMGA1-HLA-A paʻakikī me nā pilina pili pūmua-protein like ʻole (GBVI/WSA dG; kcal/mol).(B) ʻO ka hoʻokaʻawale maʻamau (RMSD) o nā kūlana atomika (koe nā ʻātoma hydrogen) no kēlā me kēia kūkulu protein.(C) Intermolecular protein-protein hydrogen paʻa pilina mai nā ʻano paʻakikī i hoʻohālikelike ʻia e noʻonoʻo ana i nā pilina kikoʻī o ka lōʻihi ≥ 10 ns.Ua hoʻonohonoho ʻia ka mamao o ka ʻokiʻoki hāʻawi h-pono i ka 3.5 Å, a ua hoʻonohonoho ʻia ka ʻoki ʻoki o ka mea hāʻawi-H-acceptor i ≥ 160 °-180 °.(D) Hoʻopaʻa inoa ʻia nā koena e hoʻopili ana i ka pilina protein-protein HLA-A me ko lākou mau hoa pakahi, e ≥ 20 ns, i unuhi ʻia mai nā dummy HLA-A-H2B a me HLA-A-HMGA1 paʻakikī.Hōʻike nā ʻōnaehana protein i kahi ʻano awelika o 100 ns MDS.(E) Nā pilina ma waena o HLA-A-H2B a me HLA-A-HMGA1 paʻakikī i hoʻohālikelike ʻia me nā pilina i ʻike ʻia e ka simulation H2B-HLA ma luna o 100 ns ma muli o ke kahua hoʻopili K a S ma waena o nā peptides ʻelua.Nā mea paʻakikī /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Ua hoʻonohonohoʻia ka helu paepae no ka loiloiʻana i nā loulou i ka 3.0, a ua mālamaʻia nā pilina kūikawā mai MDS e lawe ana i ka ≥ 10 ns.Ua ʻike ʻia nā ʻōnaehana protein me ka hoʻohana ʻana i ka BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) a me ka Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).
ʻO ke kūpaʻa o nā molekala HLA-A i ka manawa (ka hoʻokaʻawale maʻamau; RMSD a i ʻole ka hoʻokaʻawale maʻamau; RMSF) i hōʻike ʻia i ka hiki ʻana o nā protein H2B a i ʻole HMGA1 i loko o nā paʻakikī i hoʻopaʻa i ka HLA-A (Figure 8B, Figure S5).Hoʻopili paʻa ka protein HMGA1 i ka pūnaewele B2M o HLA-A, e hoʻoulu ai i ka paʻa o ka HLA-A amino acids i loko o ka HLA-A-HMGA1 a i ʻole H2B-HLA-A-HMGA1 complex (Figure 8B, Figure S5).ʻo ka mea nui, ua ʻike ʻia nā koena HLA ~ 60-90 a me ~ 180-210 i ka liʻiliʻi i ke alo o H2B (FIG. 8B).Ua hōʻike ʻo H2B a me HMGA1 i ka hoʻopaʻa maikaʻi ʻana iā HLA-A ma ka paʻakikī H2B-HLA-A-HMGA1 i hoʻohālikelike ʻia me HLA-A i hoʻopaʻa ʻia iā H2B a i ʻole HMGA1 wale nō (Figure 8C,D; Papa S5).ʻO nā koena i komo i ka hoʻopaʻa hydrogen (MD modeled high occupancy ≥ 10 ns) coincide with CLMS interaction sites (K or S koena) i loko o ka paʻakikī, e hōʻike ana he hilinaʻi loa nā pilina i ʻike ʻia e CLMS.Pono (Fig. 8E).Ma ka hoʻohālike CLMS a me MD, ua ʻike ʻia nā koena HLA-A ma waena o 190-210 a me 200-220 mau waikawa amino e hoʻopaʻa iā H2B a me HMGA1, i kēlā me kēia (FIG. 8E).
Hoʻokumu ka pilina o ka protein-protein i nā ʻoihana hoʻoikaika ikaika e hoʻopili i ke kamaʻilio intracellular i ka pane ʻana i kekahi mau mea hoʻoulu.Ma muli o ka nui o nā proteomic approaches e ʻike i nā loli i ka pae kūlana paʻa holoʻokoʻa o kahi protein, koi ka dynamics interaction protein-protein i nā mea hana hou e hopu ai i nā pilina paʻa, a ʻo CLMS kekahi mea hana.ʻO ka'ōnaehana hōʻailona interferon he pūnaewele cytokine e hiki ai i nā pūnaewele ke pane i ka laulā o nā hōʻailona pathological pathogenic a me ka intrinsic, e hoʻopau ana i ka hoʻokomoʻana i nā subsets o nā protein interferon-inducible.Ua noi mākou iā CLMS e hoʻoholo inā hiki ke ʻike ʻia nā pilina protein-protein hou ma waena o kahi papa o nā protein i hoʻokomo ʻia i ka interferon.Ua hoʻohana ʻia ka hoʻopaʻa ʻana i ka hoʻopaʻa ʻana i ka hoʻopili ʻana i ka protein honua i loko o kahi kumu hoʻohālike Flo-1 e pane ai i ka interferon no ka hopu ʻana i nā paʻakikī protein.ʻO ka unuhi ʻana o nā peptides tryptic mai nā cell i hoʻopaʻa ʻole ʻia a hoʻopaʻa ʻia e hiki ai ke helu peptide, hoʻonui i ke ala, a me ka puʻunaue peptide lōʻihi me ka ikaika LFQ i wehewehe ʻia.Ua ʻike ʻia nā protein interferon-inducible canonical ma ke ʻano he mana kūloko maikaʻi, ʻoiai ua nānā ʻia nā hoʻohui hou intermolecular a intramolecular cross-linked o nā protein canonical interferon-inducible e like me MX1, UP18, OAS3 a me STAT1.Ua noiʻi ʻia nā hiʻohiʻona like ʻole a me nā pilina ma nā wahi hana.
Ua ʻike ʻia kahi pilina ma waena o HLA-A, MDN1 a me H2B e ka immunoblotting ma Flo-1 a me A549 i mālama ʻia a mālama ʻole ʻia me IFNα.Hōʻike kā mākou hopena i ka HLA-A complexes me H2B ma kahi ʻano hilinaʻi IFNα.ʻO kā mākou hana he ala hoihoi no ka ʻimi hou ʻana i ka hoʻonohonoho like ʻana o kēia mau paʻakikī ʻelua.He mea hoihoi nō hoʻi ka hoʻonui ʻana i ke ala CLMS i kahi papa o nā laina kelepona e ʻike ai i nā pilina protein interferon-mediated cell-type-independent interferon-mediated protein.ʻO ka mea hope loa, ua hoʻohana mākou i ka hoʻohālike MD ma ke ʻano he ala ʻē aʻe e hoʻomaopopo i ka conformational dynamics o nā protein i komo i loko o ka paʻakikī H2BFS-HLA-A-HMGA1, nāna i hahai i nā kūkākūkā intramolecular a me intermolecular cross-talks.Hōʻike nā manaʻo mai ka ʻikepili CLMS i ka hiki ke hoʻohālikelike ʻia o nā protein H2BFS, HLA-A, a me HMGA1.Ua hōʻike ʻia nā ʻano hoʻohālikelike like ʻole ma waena o kēia mau paʻi protein docking i kekahi mau pilina like me nā mea i ʻike ʻia ma ka dataset CLMS.ʻO kekahi o nā ikaika nui o kā mākou ʻano hana, ʻo ia ka mea hiki ke ʻike maʻalahi i ka launa pū ʻana i nā genes polymorphic nui e like me HLA, no laila e hoihoi ke aʻo ʻana i nā pilina o nā protein haplotype-specific HLA i paʻakikī ke aʻo ʻia.Hoʻohui pū ʻia, hōʻike kā mākou ʻikepili hiki ke hoʻohana ʻia ka CLMS e hoʻonui i ko mākou ʻike i nā pūnaewele hōʻailona interferon-induced a hāʻawi i kumu no ke aʻo ʻana i nā ʻōnaehana intercellular paʻakikī i ka microenvironment tumor.
Ua loaʻa nā pūnaewele Flo-1 mai ATCC a mālama ʻia i DMEM (Gibco) i hoʻohui ʻia me 1% penicillin/streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) a mālama ʻia ma 37 ° C a me 5% CO2.Incubation.Ua ulu nā pūnaewele i ka 70-80% confluence ma mua o ka mālama ʻia ʻana me IFNα14 (hana ʻia e Edinburgh Protein Production Facility).Ua kūʻai ʻia nā mea ʻē aʻe a me nā reagents mai Sigma Aldrich ke ʻole i ʻike ʻia.
Hoʻoulu ʻia nā pūnaewele Flo-1 i nā papa 6-well a i ka lā aʻe ua mālama ʻia nā cell me 10 ng/ml IFNα14 no nā hola 24 a hiki i kahi 80% hui.Ua holoiʻia nā pūnaewele iʻekolu manawa me ka PBS a hoʻopaʻaʻia me ka DSS hou i hoʻomākaukauʻia (Thermo Fisher Scientific) (hoʻoheheʻeʻia i DMSO) ma PBS no 5 min ma 37 ° C. i ka hopena hope loa o 0.5 mM.Ua pani ʻia ka hopena crosslinking DSS me ka PBS a ua kinai ʻia ke koena DSS ma ka hoʻohui ʻana i 20 mM Tris (pH 8.0) i PBS no 15 min ma 37°C.Ua hōʻiliʻili ʻia nā kelepona ma ka ʻoki ʻana a hōʻiliʻili ʻia i loko o nā paipu haʻahaʻa haʻahaʻa (Axygen).
Hoʻopili ʻia ka pellet cell me 300 µl o ka urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) no 30 min ma ka lumi wela me ka haʻalulu i kekahi manawa.Ua hana ʻia nā ʻanuʻu centrifugation a pau ma 14,000 xg ma 8 ° C.Centrifuge i ka lysate no 10 mau minuke a hoʻololi i ka supernatant i kahi paipu hou.Ua hoʻoheheʻe ʻia nā ʻāpana akaka i koe ma 150 μl o ka lua lysis buffer (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) no 30 mau minuke a ʻoi aku a hiki i ka loaʻa ʻana o kahi wai wai homogeneous.Hoʻopili ʻia ka lysate no 20 mau minuke a hui pū ʻia ka supernatant me ka lysate i loaʻa i ka pae mua.Ua loiloi ʻia ka nui o ka protein me ka hoʻohana ʻana i ka Micro BCA assay (Thermo Fisher Scientific) e like me nā ʻōlelo a ka mea hana no nā kaʻina microplate.Hoʻopili koke ʻia nā laʻana i loko o ka hau hau a mālama ʻia ma -80°C.
Ma kahi o 100 μg o ka protein soluble cross-linked protein i hana ʻia me ka hoʻololi ʻana i kahi kānana hoʻomākaukau hoʻomākaukau ʻana (FASP) e like me ka wehewehe ʻana e Wisniewski et al.69 I ka pōkole, ua hoʻopili ʻia ka protein me 200 µl o ka urea buffer (8 M urea i 0.1 M Tris, pH 8.5), vortex a ʻoki ʻia.Ua hana ʻia nā ʻanuʻu centrifugation a pau ma 14,000 xg ma 25 ° C.Ua hoʻoili ʻia ka hapa mua o ka lysate protein cross-linked i kahi kānana 10 kDa Microcon centrifugal me kahi membrane Ultracel-10 (Merck), a ukali ʻia e ka centrifugation ma ka kānana no 25 mau minuke.A laila e hoʻohui i ka hapa lua o ka protein i ka kānana a hana hou i nā hana like.Hana ʻia ka hoʻihoʻi ʻana o ka protein ma ka hoʻohui ʻana i 100 μl o 17 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) i loko o ka urea buffer.Hoʻoulu ʻia ka hoʻihoʻi ʻana ma kahi thermomixer ma 600 rpm no 30 min ma 37 ° C.Eia hou, ua centrifuged ke kolamu a ua alkylated ka protein i hoʻemi ʻia i hoʻopili ʻia me ka hoʻohana ʻana i 100 μl o 50 mM iodoacetamide i loko o ka urea buffer.Ua lawe ʻia ka hopena alkylation ma ka lumi wela no 20 mau minuke i ka pōʻeleʻele.E hoʻololi i ke kolamu, holoi i nā paia o ke kolamu 3 manawa me 100 µl urea buffer, a laila centrifuge.Hana ʻia ka hana like i 3 mau manawa me ka hoʻohana ʻana i 100 μl o 100 mM ammonium bicarbonate.Ma mua o ka trypsinization, hoʻololi i ka paipu hōʻiliʻili me kahi hou.E hoʻohui i ka mea hoʻoheheʻe ʻai me 50 mM ammonium bicarbonate a me 1 µl trypsin i hoʻoheheʻe ʻia i loko o ka pahu trypsin (Promega).Ua mālama ʻia ka ratio o trypsin i ka protein ma kahi o 1:33, a ua hoʻokomo ʻia nā hopena digestion i ka pō ma 37 ° C. i loko o kahi keʻena humid.Ua hoʻokuʻu ʻia ka peptide crosslinked mai ka kānana e ka centrifugation no 25 mau minuke.Ua hoʻomaikaʻi ʻia ka hoʻihoʻi ʻana o ka peptide ma ka hoʻohui ʻana i 50 μl o 0.5 M NaCl i ka kānana, a ukali ʻia e ka centrifugation no 25 mau minuke.
Ua hoʻohana ʻia nā kolamu C18 Micro Spin (Harvard Apparatus) no ka hoʻopau ʻana i nā peptides tryptic i hoʻopili ʻia ma hope o ka protocol i wehewehe ʻia e Bouchal et al.70 me nā hoʻololi liʻiliʻi.ʻO ka pōkole, ua hoʻāla ʻia nā kolamu spin C18 me ʻekolu holoi ʻana o 0.1% formic acid (FA) i ka acetonitrile (AcN) (Merck) a me ʻelua holoi o 0.1% FA.Ua hydrated ka kolamu me 0.1% FA no 15 mau minuke.E hoʻouka i nā laʻana i loko o nā kolamu wili a holoi i 3 manawa me 0.1% FA.Ua hoʻokaʻawale ʻia nā peptides desalted me kahi gradient stepwise me ka hoʻohana ʻana i 50%, 80% a me 100% AcN ma 0.1% FA.Hoʻomaloʻo ʻia nā laʻana i loko o kahi concentrator SpeedVac Plus (Eppendorf) a hiki i ka nalo ʻana o ke koena wai.Ua hoʻoheheʻe ʻia nā peptides eluted i ka 100 μl o 0.08% trifluoroacetic acid i 2.5% AcN a ua ana ʻia nā ʻike ma kahi NanoDrop 2000 (Thermo Scientific).Ma kahi o 1 μg o ka peptide crosslinked no kēlā me kēia hāpana i hoʻokomo ʻia i loko o ka ʻōnaehana LC-MS/MS.
Ua hoʻokaʻawale ʻia nā peptides pili keʻa ma kahi ʻōnaehana UltiMate 3000 RSLCnano LC (Thermo Scientific) i hoʻopili ʻia me kahi Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Ua hōʻiliʻili ʻia nā peptides cross-linked ma kahi 300 µm ID, 5 mm lōʻihi µ-pre-column C18 kolamu hopu i hoʻopili ʻia me C18 PepMap100 sorbent a me 5 µm PepMap sorbent (Thermo Scientific).E hoʻouka i ke kahe o ka paila i hoʻonohonoho ʻia ma 5 µl/min 0.08% trifluoroacetic acid i hoʻoheheʻe ʻia i 2.5% AcN.Ua hoʻokaʻawale ʻia nā peptides cross-linked ma kahi kolamu silica fused analytical me ke anawaena o loko o 75 μm a me ka lōʻihi o 150 mm, i hoʻopiha ʻia me kahi sorbent 2 μm PepMap (Thermo Scientific).Loaʻa i nā pae kelepona A a me B he 0.1% FA i ka wai a me 0.1% FA i ka acetonitrile.Hoʻomaka ka gradient ma 2.5% B a piʻi i ka laina laina i 40% B ma luna o 90 mau minuke, a laila i 90% B i nā minuke 2 e hiki mai ana.Ua mālama ʻia ka haku mele paʻa ma 90% B no 10 mau minuke a laila hoʻemi i ka linearly i 2.5% B ma luna o 2 mau minuke.Ua hoʻohālikelike ʻia ke kolamu ma 2.5% B no 8 mau minuke ma mua o ka pōʻai hou.Ua hoʻokuʻu ʻia nā peptides i hoʻopili ʻia mai ke kolamu analytical i kahi kumu nanoelectrospray ionization (NSI) a hoʻokomo ʻia i loko o kahi Exploris 480 mass spectrometer (Thermo Scientific).
Hoʻohana ʻia ka Orbitrap Exploris 480 mass spectrometer ma ke ʻano hoʻoponopono ʻikepili kūpono.Ua hana ʻia kahi scan piha ma ke ʻano ʻāpana ma kahi hoʻonā o 120,000 me nā hoʻonohonoho ākea mai m/z 350 Th a m/z 2000 Th.Ua hoʻonohonoho ʻia ka pahuhopu AGC maʻamau ma 300% me ka manawa hoʻokomo kiʻekiʻe o 50ms.Ua hoʻokumu ʻia ka ʻike kiʻekiʻe monoisotopic no nā peptides.Hoʻonohonoho ʻia ka ʻāpana hoʻomaha hoʻomaha i ka ʻoiaʻiʻo inā he liʻiliʻi nā mea mua i loaʻa.Ua hoʻonohonoho ʻia ka ikaika ionic haʻahaʻa o ka precursor i 5.0e3 a ua hoʻokomo ʻia ka precursor charge states a hiki i +8 i nā hoʻokolohua.
Ua hoʻonohonoho ʻia ka manawa pōʻai ma waena o nā scan nui i ke ʻano hoʻoponopono data i 2.5 kekona.Ua hoʻonohonoho ʻia ka hoʻokuʻu ʻia ʻana o ka dynamic mass i 20 s ma hope o ka ʻāpana mua o ka ion precursor.Ua hoʻonohonoho ʻia ka puka hoʻokaʻawale precursor i 2 Th.Ua koho ʻia ke ʻano o ka ikehu hoʻokuʻi maʻamau me kahi ʻano ikehu hoʻopaʻa paʻa ma kahi scan MS/MS pili i ka ʻikepili.Hoʻonohonoho ʻia ka ikehu collision i 30%.Ua hoʻonohonoho ʻia ka hoʻonā Orbitrap i 15,000 a ʻo ka pahuhopu AGC i 100%.Hoʻonohonoho ʻia ka manawa ʻoi loa maʻamau i 60 milliseconds.
Ma mua o ka hahai ʻana i ka pūnaewele protein-protein i nā laʻana i hoʻopili ʻia, ua hoʻoponopono mākou i nā faila maka me ka hoʻohana ʻana i ka pūʻolo MaxQuant (version 1.6.12.0)26,27 e ʻike i nā peptides / protein i hiki ke loaʻa i nā laʻana.Eia kekahi, ua hana ʻia nā loiloi proteomic like ʻole ma nā ʻano Flo-1 i hoʻopili ʻole ʻia i mālama ʻia a mālama ʻole ʻia me IFNα.Ua ʻimi ʻia ka ʻikepili MS/MS ma ka waihona kanaka UniProt (www.uniprot.org) (i hoʻouka ʻia ʻo ʻAukake 12, 2020, loaʻa nā helu 75,093) me ka hoʻohana ʻana i ka mīkini ʻimi i kūkulu ʻia ʻo Andromeda27.Ua hana ʻia ka ʻimi me ka ʻole o ka hōʻike ʻana i ka kikoʻī o ka enzyme a me nā hoʻololi like ʻole o ka deamidation (N, Q) a me ka oxidation (M).Ua hoʻonohonoho ʻia nā tolerances nuipa precursor ma 20 ppm a me nā ion huahana ma 0.02 Da.Ua hoʻonohonoho ʻia ka hoʻokaʻawale mua a me ka nui i ka 10 ppm.ʻO ka nui o ka nui o ka peptide i hoʻonohonoho ʻia ma 4600 Da a ua hoʻonohonoho ʻia ke kaʻina like ma waena o 7 a me 25 amino acids (aa).Hoʻohana ʻia ka ʻikepili helu hou me ka hoʻohana ʻana i ka papahana Perseus (version 1.6.10.45).Ua helu ʻia ka ʻike o ka protein ma ka hoʻomaʻamaʻa ʻana i ka ikaika spectral o ka protein (LFQ intensity; unlabeled quantification)27 a ua hoʻololi ʻia nā waiwai ikaika i Log2.Ua kūkulu ʻia kahi pūʻulu hierarchical o nā protein i ʻike ʻia e ko lākou ikaika peptide me ka hoʻohana ʻana i ka pheatmap (v1.0.12) pūʻolo ma R (v 4.1.2).Ua hana ʻia ka loiloi hoʻonui ʻana i ke ala me ka hoʻohana ʻana i ka waihona ala ala Reactome no nā protein i mālama ʻia e IFNα i ʻoi aku ma mua o ʻehā mau manawa i hoʻāla ʻia i hoʻohālikelike ʻia me nā laʻana i mālama ʻole ʻia.
Ua hana ʻia ka ʻike ʻana i ka lysine (K) a i ʻole serine (S) i nā loulou kemika kūikawā o nā paʻakikī protein i nānā ʻia e LC-MS/MS me ka mīkini ʻike spectroscopic (SIM-XL) no nā peptides i hoʻopili ʻia (SIM-XL)29.ʻO ka mea mua, ua noiʻi ʻia nā pilina ma waena o interferon-associated (IFN) DNA damage resistance signature (IRDS) genes me ka hoʻohana ʻana i ka dataset protein IRDS i wehewehe ʻia ma Padariya et al.28.ʻO ka nānā ʻana i nā kūlana āpau a me ka hana hou ʻana o ke kanaka holoʻokoʻa UniProt he koʻikoʻi koʻikoʻi, no laila ʻo ka waihona UniProt kanaka holoʻokoʻa (www.uniprot.org) (i hoʻoiho ʻia 12 ʻAukake 2020, loaʻa nā helu 75,093) e kūʻē i ka hana hou ʻana i mālama ʻia e IFNα.ʻO kekahi o nā kānana no nā pilina hilinaʻi kiʻekiʻe.Ua hoʻonui a hoʻāʻo ʻia kēia mau pilina koʻikoʻi i loaʻa i nā hana hou a me nā kūlana.
I ka SIM-XL, ua hoʻohana ʻia ʻo DSS no ka crosslinker (XL) a ua hoʻonohonoho ʻia ka hoʻololi kaumaha XL a me ka hoʻololi kaumaha hoʻololi i 138.06 a me 156.07.Manaʻo ʻia nā pae hoʻololi crosslinking penei: KK, KS a me KN-TERM, me ka ʻole o nā ion reporter.Ua hoʻonohonoho ʻia ka precursor a me ka ʻāpana ppm i 20 a ua hoʻonohonoho ʻia ka paepae Xrea i 0.15.Ua manaʻo ʻia ʻo Trypsin he kikoʻī loa, a ua hoʻokō ʻia kahi ʻano ʻāpana C-trap (HCD) kiʻekiʻe.ʻO ka XCorr dynamic DB hōʻemi paepae a me ka helu liʻiliʻi o nā peptides no ka hoʻohaʻahaʻa DB dynamic i hoʻonohonoho ʻia i 2.5 a me 2.ʻO nā ʻāpana ʻē aʻe: monoisotope probability and peak coincidence cutoff, liʻiliʻi loa he 4 AA koena no ke kaula a me ka uku o ke kaula kiʻekiʻe, a me 3 maxima o nā māhele i hala.Ua kālailai ʻia nā palapala ʻāina 2D i humuhumu ʻia ma (SIM-XL) a ua hoʻohana ʻia ke kiʻi kiʻi xQuest28 e kūkulu i nā palapala 2D.Hāʻawi ʻia nā huila protein ma nā hale pūmua ma PyMol (PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC).
Ua hana ʻia nā ʻōnaehana hoʻohālike protein me ka hoʻohana ʻana i ka server Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)11 me ka hoʻohana ʻana i nā kumu o ka homology modeling a me ka hoʻokō ʻana o ka "Hidden Markov Method".Hoʻokumu ʻo Phyre2 i nā hale hoʻohālike e pili ana i ka hoʻohālikelike ʻana me nā ʻōnaehana protein i ʻike ʻia.No nā protein H2BFS, HLA-A, HMGA1, LRCH4, a me MDN1, ua hoʻohana ʻia nā hana hoʻohālike 1kx552, 1kj349, 2eze55, 6hlu62, a me 6i2665.Eia kekahi, ua noʻonoʻo ʻia ke ʻano o AlphaFold71 MX1, UBP18 a me ROBO1.Ua ʻike ʻia ka ʻōnaehana protein me ka hoʻohana ʻana i ka pūʻolo BIOVIA Discovery Studio Visualizer (Dassault Systèmes, BIOVIA, San Diego, CA, USA) a me ka Molecular Operating Environment package (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).