347 igwe anaghị agba nchara tubing kemịkalụ kemịkalụ, Nchọpụta nke akwụkwọ akụkọ interferon na-anabata mmadụ leukocyte antigen-A (HLA-A) protein chaperone na-eji cross-linked mass spectrometry (CLMS)

Daalụ maka ileta Nature.com.Ị na-eji ụdị ihe nchọgharị nwere oke nkwado CSS.Maka ahụmịhe kachasị mma, anyị na-akwado ka ị jiri ihe nchọgharị emelitere (ma ọ bụ gbanyụọ ọnọdụ ndakọrịta na Internet Explorer).Na mgbakwunye, iji hụ na nkwado na-aga n'ihu, anyị na-egosi saịtị na-enweghị ụdị na Javascript.
Ihe mmịfe na-egosi akụkọ atọ kwa slide.Jiri bọtịnụ azụ na nke na-esote ịkwaga na slides, ma ọ bụ bọtịnụ njikwa slide na njedebe ka ịgafe na slide ọ bụla.

Nkọwa ngwaahịa

Igwe anaghị agba nchara 347L eriri igwe, ọkwa ọkwa: SS347L

Usoro nrịbama interferon na-ebute nzaghachi cytokine siri ike maka ọtụtụ ihe mgbaàmà na-akpata ọrịa na-akpata site na gburugburu ebe obibi, na-ebute ntinye nke ntinye nke protin interferon-inducible.Anyị tinyere DSS-mediated cross-link mass spectrometry (CLMS) iji chọpụta mmekọrịta protein-protein ọhụrụ na ngalaba nke protin na-ebute interferon.Na mgbakwunye na protein interferon-inducible a na-atụ anya ya, anyị chọpụtakwara ihe ọhụrụ intermolecular na intramolecular cross-linked adducts nke canonical interferon-inducible proteins dị ka MX1, USP18, OAS3, na STAT1.Anyị lekwasịrị anya na nkwado orthogonal nke usoro netwọkụ protein interferon-inducible nke protein HLA-A hibere (H2BFS-HLA-A-HMGA1) site na iji nkwekọ-immunoprecipitation na ọmụmụ ihe ha na-aga n'ihu site na iji ụdị ihe nlere anya.Nlereanya mgbanwe mgbanwe nke mgbagwoju protein ahụ kpughere ọtụtụ saịtị mmekọrịta nke gosipụtara mmekọrịta ndị achọpụtara na nchọta CLMS.Ọnụ, anyị na-eweta ọmụmụ ihe pilot nke CLMS iji chọpụta ogige mgbaama ọhụrụ nke interferon kpatara, ma na-atụ anya iji CLMS eme ihe iji chọpụta ihe ọhụrụ mmekọrịta protein na microenvironment tumor.
Tupu mmeghachi omume mgbochi na-agbanwe agbanwe amalite, usoro nchebe ebumpụta ụwa nke onye ọbịa na-ebuli nzaghachi antimicrobial nke ezinụlọ nke cytokines alfa-helical zoro ezo nke a na-akpọ interferon (IFNs).Ụdị I IFN klas IFNa na IFNβ na-arụ ọrụ nzaghachi cellular, gụnyere antiviral, proapoptotic, proinflammatory, na antiproliferative states.N'ime ụmụ mmadụ, a maara 13 subtypes nke IFNα, ha niile na-ejikọta na chromosome 91. N'ụzọ dị ịtụnanya, ọ bụ naanị IFNα2 ka a mụrụ maka iji ụlọ ọgwụ.N'oge na-adịbeghị anya, a na-akwụ ụgwọ nlebara anya pụrụ iche na nyocha na ụdị ndị ọzọ nke IFNα.Nnyocha e mere n'oge na-adịbeghị anya gosiri na IFNα14 bụ otu n'ime isoforms kachasị dị irè na igbochi HBV2 na nje HIV-13,4 ma e jiri ya tụnyere ụdị IFNα2 nke canonical.
E guzobela na ụdị arụrụ arụ ọrụ interferon receptor complex (IFNAR1 na IFNAR2) na-akpalite cascade transduction nke Janus kinases TYK2 na JAK15,6 gbara ya.Ndị a Janus kinases phosphorylate transducers mgbaàmà na transcriptional protein activators (STAT1 na STAT2) na tyrosine residues iji malite SH2 ngalaba-mediated heterodimerization6.N'ikpeazụ, IRF9 na-ejikọta STAT heterodimers iji mepụta mgbagwoju anya trimeric nke IFN-stimulated factor 3 gene (ISGF3), nke na-atụgharị gaa na nucleus ma mee ka nsụgharị nke ihe karịrị 2000 interferon-stimulated genes (ISGs) 5,6,7,8.
ISG na-etolite ọkpụkpụ azụ nke usoro ahụ ji alụso ọrịa ọgụ, ọkachasị na nzaghachi ọgụ nje.Dị ka usoro nchebe mbụ megide ọrịa nje, sel na-ebuga ngwa ngwa mmekọrịta dị ukwuu nke protein cellular yana ọtụtụ ihe omume dị ndụ.Ndị na-edozi protein ndị a gụnyere ndị na-anabata ụkpụrụ, ụmụ irighiri ihe nrịbama, ihe odide ederede, na protein ndị nwere ọrụ mgbochi nje, yana ndị na-achịkwa mmeghachi omume mgbochi9.Ọtụtụ ozi gbasara ọrụ ISG na-abịa site na ihuenyo arụ ọrụ site na iji overexpression screens10,11 ma ọ bụ usoro ịgbachi nkịtị (siRNA, RNAi na CRISPR) 12,13 bụ nke egosipụtara ma ọ bụ gbochie ISG n'otu n'otu ma nwalee ọrụ ha na nje dị iche iche.Ọ bụ ezie na ọmụmụ ihe ndị a ekpebiela ihe mgbochi nje nke ISG n'otu n'otu, usoro mkpụrụ ndụ ihe dị n'okpuru nke ISG ọ bụla ka amabeghị nke ukwuu.A na-anabatakarị na ọtụtụ ndị na-edozi ahụ na otu cytokines ma ọ bụ karịa na-emekọ ihe iji hụ na arụ ọrụ zuru oke, yabụ ma ISG na-emekọ ihe ozugbo ma ọ bụ na-edozi mmekọrịta ha site na protein cellular.Dịka ọmụmaatụ, nchọpụta proteomics fotocrosslinked na nso nso a chọpụtara ATPase VCP / p97 dị ka onye isi mmekọrịta mmekọrịta IFITM3, nke mgbochi ya na-eduga na ntụpọ na nhazi lysosomal, ntụgharị, na cotransport nke IFITM3 na nje nje 14.N'iji immunoprecipitation, anyị chọpụtara VAPA, protein metụtara vesicle, dị ka onye na-emekọrịta ihe na IFITM1 / 2/3 nke na-eme ka ọ dị elu nke na-eme ka cholesterol na-eme ka ọ dị elu, na nke a kwadoro site n'ọmụmụ ihe ọzọ site na iji yist abụọ-hybrid system.Nkwado sayensị 15, 16 .
Usoro usoro ndu dị mkpa nke na-etinye aka na nkwụsị nke ọrịa na mgbanwe ọjọọ bụ ihe ngosi antigen, nke a na-ebute site na nnukwu ihe mgbagwoju anya histocompatibility complex (MHC).A na-etinye Peptides (8-12 amino acids ogologo) sitere na ndị gbawara agbawa, ndị na-agwụcha ma ọ bụ ndị na-edozi ahụ na-atụgharị n'ime MHC-I heterodimer (nke nwere MHC-I arọ na ụdọ dị arọ, nke a na-akpọ β-2-microglobulin; β2M) 17,18 .MHC-I trimers kwụsiri ike na-esi na ya pụta na-ebuga n'elu cell, ebe ha na-ebute peptides intracellular na sel CD8+ T (sel cytotoxic T) 17.Mkpụrụ ndụ T na-amata ma bibie nje ndị a na sel ndị na-ebu antigen akọwapụtara kpọmkwem etuto ahụ.N'ihi ya, ọrịa nje na mkpụrụ ndụ tumor na-egbochikarị usoro ngosi antigen iji zere nleba anya nchebe.Na mgbakwunye, MHC-I na-agbadata na 40-90% nke etuto mmadụ ma na-ejikọta ya na prognosis dara ogbenye19.
Mkpụrụ ndụ ihe nketa na-etinye aka na ịzaghachi nje nje ga-agbanwe ngwa ngwa n'etiti ọnọdụ izu ike na ọnọdụ nke ndebanye aha.Ya mere, ọtụtụ protein cellular na-eche na ha ga-etinye aka na nzaghachi maka nnukwu ọchịchọ IFN n'ime oge dị mkpirikpi, gụnyere nhazigharị na mgbanwe nke chromatin 20,21 na-akwalite.Ọtụtụ ọmụmụ lekwasịrị anya na njirimara nke ndị mmekọ protein ISG n'otu n'otu na ọnụnọ IFN.Ọtụtụ ihe ọmụmụ proteomic na transcriptomic na sistemu mkpụrụ ndụ ihe atụ akọwapụtala mmetụta IFN na mbara ala cellular.Otú ọ dị, n'agbanyeghị nghọta na-eto eto banyere ihe ndị interferon na-akpata, anyị ka maara ntakịrị banyere itinye aka nke ISG.Mgbe ị na-atụle mgbagwoju anya na mgbagwoju anya na-adabere na oge nke mgbaàmà interferon, ajụjụ abụọ na-ebilite: (i) ọ ga-ekwe omume ịkwado na ọnyà mgbagwoju anya multiprotein na-etinye aka na akara ngwa ngwa, na (ii) enwere ike ịmekọrịta mmekọrịta ndị a na oghere 3D?
Iji dozie okwu ndị a, anyị mebere disuccinimide suberate-mediated chemical cross-linking (DSS) tinyere mass spectrometry (CLMS) iji mụọ netwọk mmekọrịta protein na-ebute IFNa na ike ya.DSS na-agbakwụnye njikọ dị n'etiti proximal foduru nke protein na/ma ọ bụ ogige protein na vivo.Nyocha MS na-esote na-ekpughe saịtị ndị na-agafe agafe nke na-egosipụta ịdị nso nke mpaghara dị n'ime otu protein, nke a na-akpọ njikọ dị n'ime, ma ọ bụ ihe ndị dị na protein protein, nke a na-akpọ mmekọrịta.N'iji usoro a, anyị achọpụtala ọtụtụ ụlọ ọrụ protein-protein ọhụrụ yana netwọk mmekọrịta multiprotein na-ebute interferon.Site n'ịkwado nsonye nke mmekọrịta ọhụrụ a, anyị na-egosi na H2BFS (H2B histone-ụdị FS; nke a na-akpọ H2B) na MDN1 na-eme dị ka ndị mmekọ maka HLA-A.
Mkpụrụ ndụ Flo-1 bụ otu n'ime ụdị vitro a kacha mara amara nke adenocarcinoma esophageal ka ha na-eṅomi akụkụ ndị bụ isi nke etuto esophageal22,23.Otú ọ dị, ọ bụghị ụbụrụ niile bụ immunogenic, na iji chọpụta ma ọ bụrụ na mkpụrụ ndụ Flo-1 na-anabata ọgwụgwọ interferon, anyị na-emeso mkpụrụ ndụ Flo-1 na 10 ng / ml IFNa maka awa 72.Mkpụrụ ndụ Flo-1 gosipụtara mmalite mmalite nke pSTAT1 na IRF1, na-amalite 2 awa mgbe ọgwụgwọ gasịrị ma na-aga n'ihu maka awa 72, na-adabere na oge na-adabere na ọkwa nke IRF1 (Nyocha 1A).Achọpụtara ISGs (MX1, IFITM1, OAS1/2, na ISG15) ka a na-akpali ya mgbe awa 6 gachara, na-eṅomi nzaghachi nke etiti na mbubreyo na IFNα (Nyocha 1A).Ọnụ, data ndị a na-egosi na enwere ike iji ụdị cellular a mụọ nzaghachi interferon.
Nzaghachi okwu okwu protein dị iche na sel Flo-1 mgbe ọgwụgwọ IFNa gasịrị.(A) A na-enyocha okwu protein na mkpụrụ ndụ Flo-1 na 10 ng/ml IFNα maka awa 2, 6, 24, 48 na 72 site na immunoblot site na iji ọgwụ mgbochi ISG egosipụtara.(B) Coomassie acha anụnụ anụnụ nwere SDS-PAGE gels nke mkpụrụ ndụ sel niile ka emechara njikọ ya na DSS maka oge egosipụtara na mkpokọta.(C) Onye nnọchi anya immunoblot enyocha ya na mgbochi p53 (DO-1) site na otu ihe nlele ahụ iji chọpụta ogo njikọ njikọ protein.
Iji weghara odida obodo mmekọrịta protein dị n'ọnọdụ ahụ, anyị jiri DSS, onye na-emekọrịta ihe n'ọtụtụ ebe n'ihi oke akpụkpọ ahụ ya na oge mmeghachi omume dị mkpụmkpụ.Oge mmeghachi omume dị mkpụmkpụ na-enyere aka igbochi nguzobe nke nnukwu mkpokọta protein ndị jikọtara ọnụ, si otú a na-ejigide nkwụsi ike nke crosslinker.Iji chọpụta ezigbo itinye uche DSS ma zere ịgafefefefefe, anyị buru ụzọ kpughee sel ndị ahụ na 5, 2.5, na 1 mM DSS maka nkeji 5, 10, 5 na 30, n'otu n'otu, wee nyochaa lysates site na Coomassie-stained SDS-PAGE (data egosighi).Cell lysates na-egosi na a na-ejikọta ya nke ọma na ntinye kachasị dị ala yana n'oge kacha nso.Ya mere, a tụrụ DSS ka ọ bụrụ 1, 0.5, na 0.1 mM karịa nkeji 5 (Foto 1B).A na-ahụ njikọ njikọ kacha mma na 0.5 mM DSS maka nkeji 5, a na-ahọrọ ọnọdụ ndị a maka mkpụrụ ndụ ejiri IFNα na-emeso ya.Na mgbakwunye, eserese 1C na-egosi ntụpọ ọdịda anyanwụ emere site na iji mgbochi p53 (DO-1) iji chọpụta ogo njikọ njikọ protein.
A na-emeso mkpụrụ ndụ Flo-1 na 10 ng / ml IFNα maka awa 24 tupu ịgbakwunye crosslinker.E mechakwara jiri proteolysis nke ụzọ abụọ gbakọrọ mkpụrụ ndụ jikọtara ọnụ na protein ndị FASP na-ahazi ya (Fig 2)24,25.A na-enyocha peptides tryptic nke jikọtara cross site na nnukwu spectrometry (Fig 2).A na-ejikọta ụdịdị MS/MS na usoro protein wee tụọ ya na MaxQuant26,27.Achọpụtara peptides jikọtara cross site na spectra enwetara site na iji mmemme SIM-XL, a na-ejikọta ogige nke onye ọ bụla n'ime netwọk dị mgbagwoju anya site na iji xQuest28 na SIM-XL29 open source computing pipelines (Fig 2).SIM-XL na-achọpụta mmekọrịta protein-protein, agbụ dị n'ime na ụdọ onye ọ bụla na ngwakọta protein dị mfe ma ọ bụ dị mgbagwoju anya ma na-enye script maka ịhụ mmekọrịta dị na nhazi protein.Na mgbakwunye, ọ na-edobe nrụtụ aka n'ofe ọ bụla dị ka akara NJ dịka ogo ụdịdị MS/MS29 siri dị.Achọpụtala ọtụtụ mmekọrịta protein-protein nke a pụrụ ịdabere na ya na mgbagwoju anya, a na-enyochakwa usoro mmekọrịta ọhụrụ site na iji mgbochi mgbochi na mgbanwe mgbanwe nke mgbagwoju anya site na iji ihe ngosi molecular dynamics (MD) modeling (Fig. 2) 30, 31.
Nchịkọta atụmatụ nke usoro CLMS.A na-emeso mkpụrụ ndụ Flo-1 na 10 ng / ml IFNα maka awa 24 na-esote njikọ njikọ protein situ site na iji DSS sochiri lysis cell na trypsinization.A na-enyocha ihe nlere anya jikọtara ọnụ site na iji Orbitrap mass spectrometer wee wepụtakwa ya maka mwepu nke peptide precursors n'oge LC-MS/MS.Achọpụtara peptides abụọ jikọtara ọnụ site na ihe ngosi a nwetara site na iji Spectrum Recognition Machine nke Crosslinked Peptides (SIM-XL), na ngwakọta niile jikọtara n'ime netwọk mgbagwoju anya site na iji pipeline mgbakọ.Wepu mmekọrịta ntụkwasị obi dị ala dabere na akara ọnụego ụgha ụgha (FDR).Ọtụtụ mmekọrịta protein-protein nwere ntụkwasị obi ọhụrụ ka akwadoro site na iji ọgwụ mgbochi ọnụ, na mgbanwe mgbanwe n'ime ogige ndị ahụ ka a na-enyocha site na iji ihe nlegharị anya molecular dynamics (MD).
Achọpụtara mkpokọta ~ 30,500 na ~ 28,500 peptides site na iji MaxQuant na ihe atụ IFNa na-enweghị mkpali na mkpali, n'otu n'otu (Mgbakwunye S1, Fig. 3A).Nkesa ogologo peptide n'ọnọdụ abụọ ahụ gosipụtara ọnụ ọgụgụ dị elu nke peptides buru ibu, na-egosi ọnụnọ nke peptides jikọtara ọnụ (Fig 3B, C).Tụkwasị na nke ahụ, ọnụ ọgụgụ ka ukwuu nke peptides buru ibu dị na 40-55 dị na IFNa-emeso samples (Fig. 3C).Maapụ protein megide ike log2 gosiri na protein ndị na-akpali akpali nke interferon bụ ndị kacha baa ụba ma e jiri ya tụnyere ihe nlele anaghị agwọta, gụnyere MX1, IFIT1/3, OAS2/3, DDX58, na HLA-F (Nyocha 3D).Nyocha nke ụzọ maka ndị na-edozi ihe karịrị okpukpu atọ na-emeziwanye nzaghachi na ọgwụgwọ IFNα site na iji nchekwa data ụzọ Reactome gosiri na MHC-I-mediated antigen ngosi na nhazi bụ ụzọ kasị mma (Nyocha 3E).N'ikwekọ na akụkọ ndị mbụ, nzaghachi antiviral nke OAS na ISG15 kwadoro yana IFNα / β na akara cytokine so na ụzọ ndị a na-arụ ọrụ.Na mgbakwunye, achọpụtara njikọ cross-protein lysine- na serine-kpọmkwem site na MS/MS spectra enwetara mbụ site na iji SIM-XL.Nnyocha e mere n'oge na-adịbeghị anya kọrọ 104 ISG na-agbasa nje 20 sitere na klaasị nje 9 site na meta-nyocha nke ọmụmụ ihe karịrị ISG n'otu n'otu na ụdị cell 59.Otú ọ dị, iji merie njedebe mgbakọ na mwepụ nke nyocha nnukwu datasets, anyị malitere site na obere dataset iji chọpụta mmekọrịta dị n'etiti ndepụta nke mkpụrụ ndụ ihe nketa IRDS nke Padaria et al., ọtụtụ n'ime ha bụ ISG.
Nchọpụta nke protein ndị nwere njikọ chiri anya egosipụtara dị iche iche na nzaghachi IFNα (data enwetara na MaxQuant).(A) Eserese Venn na-anọchite anya ọnụọgụ peptides nkịtị na nke pụrụ iche achọpụtara na IFNα14 na-agwọta na nlele Flo-1 na-adịghị agwọ ya.Nkesa ogologo peptide na-adịghị agwọ ọrịa (B) na IFNα na-agwọta (C) samples crosslinked.(D) Maapụ okpomọkụ na-anọchi anya log2 (LFQ ike) n'etiti mkpụrụ ndụ Flo-1 na-agwọghị ya na IFNα14.Ogwe aka ekpe na-egosi protein ndị a na-arụsi ọrụ ike n'ihu IFNα.(E) Histogram na-anọchite anya ụzọ 20 isi nkwalite mgbe ọgwụgwọ IFNa gasịrị.Ebe nchekwa data ụzọ Reactome nyochara ihe karịrị mgbanwe okpukpu anọ na protein ndị na-anabata IFNa.
A na-edekọ mkpali ISG nke interferon na-emekọrịta ihe nke ọma, mana n'ọkwa molekul aghọtachaghị etu protein ndị a si ejedebe n'ọtụtụ ọrụ ndu.Anyị ji oke ntụkwasị obi nyocha mmekọrịta protein n'etiti ISG ndị ama ama.N'ụzọ na-akpali mmasị, anyị chọpụtara netwọk gụnyere MX1, USP18, ROBO1, OAS3, na protein STAT1 nke na-emepụta nnukwu mgbagwoju anya na nzaghachi ọgwụgwọ IFNα (Figure 4, Table S2) 32,33,34.Nke kachasị mkpa, a chọtara mmekọrịta ndị a na atọ niile a na-emeso ya na IFNa ma achọtaghị ya na ihe ndị a na-emeghị eme, na-atụ aro na e guzobere ya kpọmkwem na nzaghachi ọgwụgwọ IFNa.A maara na STAT1 na-achịkwa okwu nke ISG ndị a n'ụzọ ederede, mana amụbeghị mmekọrịta ya na ISG na ọkwa protein.Ọdịdị kristal nke STAT1 gosiri na mpaghara helical ya (CCD) adịghị etinye aka na mmekọrịta ya na DNA ma ọ bụ ndị na-emepụta ihe n'oge nhazi nke dimers35.Ndị a α-helices na-etolite nhazi helix helical nke na-enye ebe dị elu nke hydrophilic maka mmekọrịta na-eme 35.Na data CLMS anyị, anyị hụrụ na ọtụtụ n'ime mmekọrịta ya na STAT1 mere na ngalaba SH2 na-ebute CCD, ngalaba njikọ, ma ọ bụ ọdụ C-terminal (ihe fọdụrụ 700-708) (Nyocha 4A).Nnyocha gara aga kwuru na USP18 na-ejikọta na CCD na DNA-binding domain (DBD) nke STAT2 ma na-eweghachi ya na mpaghara nke ụdị I interferon receptor IFNAR2 iji dozie mgbochi nke ụdị I interferon na-egosi 24.Data anyị gosikwara na ngalaba USP18 catalytic na-emekọrịta ihe na STAT1 DBD (Nyocha 4A,D), na-atụ aro na ma STAT1 na STAT2 nwere ike rụọ ọrụ na-adọta USP18 na IFNAR2.
Achọpụtara netwọk ISG protein-protein n'ime sel ejikọtara na IFNa.(A) Mkparịta ụka 2D na-egosi mmekọrịta protein-protein (emepụtara na mmemme SIM-XL), yana ahịrị na-anọchite anya mmekọrịta intermolecular (crosslink cutoff at to 3.5).Akara ngalaba nke njirimara dị iche iche bụ agba32: ngalaba MX1, Dynamin_N (73-249), Dynamin_M (259-547), na GED (569-660).OAS3 ngalaba: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), na OAS1_C (903-108).Ngalaba 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) na fn3 (777–864).Oghere STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), na STAT1_TAZ2bind (715–739).(B) Onye na-ekiri okirikiri nke protein jikọtara ọnụ (MX1, UBP18, OAS3, ROBO1, na STAT1) yana mmekọrịta na mmekọrịta akpọrọ na-acha anụnụ anụnụ na uhie, n'otu n'otu.Edobere oke njikọ obe na 3.5.Ntụpọ ntụpọ na-egosi saịtị mmekọrịta STAT1 nwere MX1 (C), USP18 (D), ROBO1 (E), na OAS3 (F), yana saịtị mmekọrịta K ma ọ bụ S n'etiti peptides abụọ ahụ.N'ọnụọgụ a, edobere akara akara cross-link ka ọ bụrụ 3.0.(G) Ebe mmekọrịta dị iche iche dị n'etiti ngalaba STAT1 na OAS3 DI na-adabere na nhazi protein ha na PyMol (PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) na OAS3 (pdb id: 4s3n34).) mmemme.
A kọwapụtara isoforms abụọ nke USP18 n'ime ụmụ mmadụ, protein zuru oke nke dị na nucleus, na isoform na-enweghị ngalaba N-terminal, USP18-sf, nke a na-ekesa na cytoplasm na nucleus 36.Na mgbakwunye, e buru amụma na N-terminus agaghị ahazi ya na ọ chọghị ọrụ isopeptidase ma ọ bụ njikọ ISG1537.Ọtụtụ n'ime mmekọrịta ndị a chọpụtara n'ọmụmụ ihe anyị dị na N-terminus nke protein, na-atụ aro na mmekọrịta ndị a na-agụnye USP18 zuru oke (Nyocha 4A, D) ma si otú a na-eme na oghere.Ọzọkwa, data anyị na-egosikwa na N-terminus bụ ọkachamara maka mmekọrịta protein-na-protein.Ebe a na-ejikọta IFNAR2 dị n'etiti ihe fọdụrụ 312-368, ma karịsịa, ọ dịghị otu n'ime protein ndị dị na mgbagwoju anya na mpaghara a (Fig 4A) 37,38.Data ndị a jikọtara ọnụ na-egosi na ọ bụ naanị protein nnabata na-eji ngalaba njikọ IFNAR2.Na mgbakwunye, ọ bụ naanị OAS3 na ROBO1 ka achọtara na ha jikọtara ya na ngalaba dị elu nke saịtị njikọ N-terminus na IFNAR2 (Njirimara 4A).
ROBO1 bụ nke immunoglobulin (Ig) superfamily nke transmembrane akara ụmụ irighiri ihe na mejupụtara ngalaba Ig ise na ngalaba fibronectin (Fn) atọ na mpaghara extracellular.Ngalaba ndị a na-esote ya na mpaghara akpụkpọ anụ-proximal na otu helix transmembrane 39. Mpaghara intracellular na-enweghị nhazi dị na C-terminus ma nwee usoro usoro echekwara nke na-emekọrịta ihe na-eme ka protein binding39.Mpaghara sitere na amino acid ~ 1100 ruo 1600 na-enwekarị nsogbu.Anyị chọpụtara na MX1 na-emekọ ihe na ROBO1 site na Ig, Fn, na ngalaba intracellular, ebe ọtụtụ mmekọrịta na STAT1 na-eme n'etiti CCD ya, ngalaba njikọ, na C-terminus nke ROBO1 (Fig. 4A, E).N'aka nke ọzọ, a na-ekesa mmekọrịta na mpaghara DI, DIII, na OAS3 njikọ na protein ROBO1 (Fig. 4A).
Ezinụlọ protein oligoadenylate synthase (OAS) na-anabata ma na-ejikọta RNA (dsRNA) nke intracellular nke nwere eriri abụọ, na-enwe mgbanwe mgbanwe, na synthesizes 2′,5′-linked oligoadenylates (2-5 As) 40.Achọpụtara na n'etiti OAS atọ ahụ, OAS3 na-egosipụta mmekọrịta kachasị elu maka dsRNA ma na-ejikọta ọnụ ọgụgụ kasị nta nke 2-5 Dị ka, nke nwere ike ime ka RNase L rụọ ọrụ ma si otú ahụ na-amachi nje virus 41.Ezinụlọ OAS nwere ngalaba polymerase beta (pol-β) dịka nucleotide transferase ngalaba.Nnyocha gara aga egosila na ọrụ catalytic nke ngalaba C-terminal (DIII) dabere na ngalaba dsRNA-binding (DI), nke achọrọ maka ịgbalite OAS342.Anyị hụrụ na ngalaba DI na DII nke OAS3 na-emekọrịta ihe na CCD na obere mpaghara nkwụsị n'etiti SH2 na STAT1 TAD (Nyocha 4A, F).Ikpuchi saịtị dị iche iche na-agafe na nhazi protein gosipụtara mmekọrịta dị n'etiti β-sheet na DBD STAT1 loop na akpa ma ọ bụ oghere mepere emepe nke 60-75 fọdụrụ na ngalaba DI nke OAS3 (Fig 4G).Ntuzi aka nke ndị na-edozi ahụ dị na mgbagwoju anya gosikwara na ọ dịghị nke ọ bụla n'ime mmekọrịta ya na OAS3 na-egbochi ike DNA-njikọ nke ngalaba DI ya (Fig. S1A).Na mgbakwunye, ngalaba N-terminal nke GTPase MX1 na-emekọrịta ihe na ngalaba DI na DIII nke OAS3 (Fig. 4A).Anyị hụkwara mmekọrịta dị n'etiti OAS1 na MX1 n'ime IFNa atọ a na-eme ugboro ugboro, ebe otu ngalaba OAS1 (na-arụkwa ọrụ ike) na-emekọrịta ihe na ngalaba MX1 atọ niile (Ọnyocha S2A, B).
Ndị na-edozi MX bụ akụkụ nke nnukwu ezinụlọ nke dynein-dị ka GTPases nke nwere ngalaba N-terminal GTPase nke na-ejikọta na hydrolyzes GTP, ngalaba etiti nke na-emekọrịta onwe ya, yana zipa leucine C-terminal nke na-arụ ọrụ dị ka GTPase (LZ). ).ngalaba mmetụta ngalaba25,43.MX1 na-ejikọta na mpaghara nke polymerases viral iji gbochie ndenye nke gene43 viral.Ihuenyo ngwakọ abụọ nke yist a na-akọ na mbụ gosiri na MX1 metụtara PIAS1 na-egbochi mmalite mmalite nke STAT1 site na igbochi ọrụ njikọ DNA ma nwee SUMO E344,45 ligase ọrụ.N'ebe a, anyị na-egosi na MX1 na-ejikọta na STAT1 (Nyocha 4C, D), Otú ọ dị ka mmekọrịta a si emetụta STAT1-mediated gene activation na nzaghachi IFNa chọrọ ọmụmụ ihe ọzọ.Na mgbakwunye, anyị chọpụtakwara na MX1 na-emekọrịta ihe na IFIT3 na DDX60 na atọ IFNa-emeso ugboro ugboro (Fig. S2C).
DDX60 bụ helicase cytoplasmic na-ebute IFN nke akọgoro na mbụ na-arụ ọrụ na RIG-I-nnwere onwe mbibi nke viral RNA46.Ọ na-emekọrịta ihe na RIG-I ma mee ka akara ngosi ya dị na ligand-kpọmkwem n'ụzọ 46. DDX60 mejupụtara DEXD / H-Box helicase ngalaba na C-terminal helicase ngalaba nke na-ejikọta viral RNA na DNA47.Ọtụtụ n'ime mmekọrịta ya na MX1 na IFIT3 na-eme n'ime ogologo mpaghara N- na C-terminal na-enweghị ngalaba canonical ma ọ bụ motifs (Fig. S2E, F).Otú ọ dị, MX1 na-ejikọta ya na DEXD/H-Box helicase domain (Fig. S2E).Protein nke ezinụlọ IFIT nwere mbipụta tandem nke ihe pụrụ iche nke helix-turn-helix motif a na-akpọ tetrapeptide repeat (TPR).Achọpụtara IFIT3 ka ọ bụrụ ezigbo modulator nke RIG-I na-egosi na ya mere akụkụ nke mgbagwoju MAVS.Ejikọtara ọnụ, data anyị na-atụ aro na IFIT3 na DDX60 na-emekọrịta ihe na mpaghara n'etiti TPR 3-6 nke IFIT3 ma nwee ike ịrụ ọrụ na akara ngosi RIG-I / MAVS (Fig. S2F).
Nyere na nyocha nke proteome niile na-agbakọ ọnụ, anyị enyochala nchekwa data UniProt mmadụ niile maka ọnụnọ nke otu n'ime IFNa-emeso ugboro ugboro.Na oyiri a, anyị hụrụ ọtụtụ netwọk mmekọrịta ndị a pụrụ ịdabere na ya maka HLA-A.Nyocha nke ụzọ protein nke MS/MS spectra chọpụtara gosiri na nhazi na ngosi antigen dabeere na MHC-I bụ ụzọ bụ isi nke interferon kpatara (Fig 3D).Ya mere, anyị lekwasịrị anya n'ịmụta mmekọrịta protein nke ụmụ irighiri ihe MHC-I nwere ntụkwasị obi dị elu na ihe atụ niile jikọtara ọnụ.HLA nwere ngalaba α1, α2 na α3 na agbụ ọkụ, yana microglobulin β2 (β2m) bụ protein chaperone na-adịgide adịgide.Ozugbo agbakọtara na endoplasmic reticulum, HLA adịghị eguzosi ike na enweghị peptide ligands50.A na-emepụta oghere peptide na-ejikọta ya site na ngalaba α1 na α2 nke a na-ahazighị nke ọma na nke na-abụghị peptide na ngalaba α351 polymorphic dị ntakịrị.N'ihu IFNα, anyị chọpụtara ụlọ abụọ HLA-A: otu na-emekọ ihe na HMGA1 na H2B (Nyocha 5, Tebụl S3) na nke ọzọ na-emekọ ihe na MDN1, LRCH4 na H2B (Nyocha 6).
IFNα na-ebute netwọk mmekọrịta HLA-A na H2B (H2BFS) na HMGA1.(A) 2D nkata (emepụtara na SIM-XL software) na-egosipụta ụdị mmekọrịta dị iche iche na mgbagwoju H2B-HLA-A-HMGA1: interlink (acha anụnụ anụnụ), interlink (red) na otu njikọ (oji)..Ngalaba nke njirimara dị iche iche bụ agba codeed32: H2B (histone; 2–102) na MHC-I (MHC_1; 25–203, otu C1; 210–290 na MHC_I_C; 337–364).Edobere oke njikọ obe na 3.5.Ntụpọ ntụpọ na-egosi saịtị mmekọrịta HLA-A nwere H2B (B) na HMGA1 (C), yana saịtị mmekọrịta K ma ọ bụ S n'etiti peptides abụọ ahụ.N'ọnụọgụ a, edobere akara akara cross-link ka ọ bụrụ 3.0.(D) Mmekọrịta dị n'etiti protein ndị egosiri na nhazi nke protein H2B, HLA-A na HMGA1 na mmemme PyMOL.Ejiri ihe nkesa Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) na usoro ndebiri maka protein H2B, HLA-A na HMGA1 bụ 1kx552, 1kj349 na 2eze55, n'otu n'otu.
IFNα na-ebute netwọk mmekọrịta HLA-A na H2B (H2BFS), MDN1 na LRCH4.(A) Intramolecular (red) na intermolecular (acha anụnụ anụnụ) crosslinks gosipụtara na maapụ mmekọrịta 2D (emepụtara na ngwanrọ SIM-XL) nke MDN1 nọchiri anya dị ka okirikiri.Edobere oke njikọ obe na 3.5.Ngalaba nke njirimara dị iche iche bụ agba codeed32: H2B (histone; 2-102), MHC-I (MHC_1; 25-203, otu C1; 210-290 na MHC_I_C; 337-364) na LRCH4 (LRR_8 (68-126), LRR_8 (137–194) na CH (535–641)).(B) Mmekọrịta dị n'etiti protein ndị egosiri na nhazi nke protein H2B, HLA-A, LRCH4 na MDN1 na mmemme PyMOL.Emebere ihe owuwu ndị a site na iji ihe nkesa Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) nwere usoro ndebiri 1kx552, 1kj349, 6hlu62 na 6i2665 maka protein H2B, HLA-A, LRCH4 na MDN1, n'otu n'otu.Ntụpọ ntụpọ na-egosi saịtị mmekọrịta K ma ọ bụ S maka HLA-A nwere H2B (C), LRCH4 (D), na MDN1 (E).Maka nkata, atọrọ oke akara akara cross-link ka ọ bụrụ 3.0.
Na mgbakwunye na ịnọgide na-eguzosi ike n'ezi ihe nke genome, histone H2B na-etinyekwa aka na nhazi nke ederede.Protein H2B nwere ngalaba histone Central (HFD) nke kpụrụ atọ α-helices kewapụrụ site na loops na ọdụ C-terminal 41,52.Ọtụtụ n'ime mmekọrịta ya na H2B na-eme na α1 helix, nke na-enye trimerization na HFD heterodimer (Fig 5A, B).Ọ bụ ezie na lysins na-etinye aka na njikọ DNA, ụfọdụ lysins bụkwa acetylation ọzọ ma ọ bụ saịtị methylation.Dịka ọmụmaatụ, ihe fọdụrụ K43, K46, na K57 sitere na H2B adịghị etinye aka na njikọ DNA kpọmkwem, kama ọ bụ ebumnuche nke mgbanwe mgbanwe dị iche iche gafesịrị53.N'otu aka ahụ, ihe fọdụrụ K44, K47, na K57 na H2B nwere ike ịrụ ọrụ ọzọ na ọnụnọ IFNα, gụnyere mmekọrịta ya na protein ndị ọzọ (Fig 5A, B).Na mgbakwunye, extrachromosomal histone H2B na-arụ ọrụ nzaghachi mgbochi ọrịa n'ụdị sel dị iche iche, na-eme ihe dị ka ihe mmetụta cytosolic iji chọpụta iberibe DNA nwere eriri abụọ (dsDNA) sitere na nje na-efe efe ma ọ bụ mkpụrụ ndụ mebiri emebi54.N'ebe nje virus DNA nọ, nkwụsị H2B gbochiri mmepụta IFN-β na STAT154 phosphorylation.A makwaara H2B ka ọ na-abanye na n'ime oghere ngwa ngwa karịa isi histones54.A hụkwara mmekọrịta H2B na MDN1 na LRCH4 n'ụdị nlele anaghị agwọta.Anyị chọpụtara na HLA-A na H2B na-emekọrịta ihe n'ime ihe nlele atọ a na-agwọ IFNa yana n'otu nlele na-emegharịghị ya.Ihe data ndị a na-egosipụta ọrụ H2B na ọrụ physiological ọzọ na-adabereghị na nhazi ederede.
HMGA1 (otu nnukwu ngagharị AT-Hook 1), obere nucleoprotein bara ụba na amino acid na-akwalite ọrịa, ka achọpụtara na njikọ ya na HLA-A.O nwere ọdụ C-terminal acidic na DBD atọ dị iche iche akpọrọ AT hooks n'ihi na ha na-ejikọta na obere oghere nke mpaghara AT bara ụba na dsDNA55,56.Njikọ a na-eme ka DNA gbagọọ ma ọ bụ gbatịa, na-ekwe ka ihe odide ederede nwee ike ịnweta usoro nkwenye ya.Ekwenyere na ọdụ C-terminal na-etinye aka na mmekọrịta protein-protein yana iwere ihe ndị na-edegharị ihe, ebe ọ bụ na mutant ihichapụ C-terminal enweghị ike ibido transcript57.Ọzọkwa, ngalaba a nwere ọtụtụ saịtị phosphorylation echekwara echekwabara bụ ndị amaara mkpụrụ maka kinases 58 .Anyị hụrụ mmekọrịta HLA-A na H2B na HMGA1 n'èzí ngalaba C-terminal, na-atụ aro na ngalaba C-terminal bụ nke a na-ejikarị maka njide ihe odide ederede (Fig 5A, C).Protein HMGA na-asọ mpi histone H1 maka ijikọ na DNA nkwụnye, si otú ahụ na-abawanye ohere57.N'otu aka ahụ, ọ dị ka HMGA na-emekọ ihe na histone H2B tinyere DNA njikọ na asọmpi na histone H1.HMGB1 na-ebute okwu nke HLA-A, -B, na -C na mkpụrụ ndụ dendritic, na-eduga na ọrụ ha59, mana mmekọrịta dị n'etiti HMG na HLA ka akọpụtabeghị mbụ.Anyị chọpụtara na HMGA1 na-emekọrịta ihe na ngalaba α1 na α3 nke HLA-A, yana ọtụtụ n'ime mmekọrịta na-abụghị nke 3 DBD ya (Nyocha 5A,C).N'aka anyị, a chọtara HLA-A ka ọ dị na nucleus (data egosighi), ma nyekwa na H2B na HMGA1 dịkwa na nucleus, mmekọrịta a nwere ike ime na nucleus.Egosipụtara ihe mgbakwasị ụkwụ akọwapụtara n'etiti H2B, HLA-A na HMGA1 na eserese 5D.
Imirikiti mmekọrịta nke HLA-A na protein ndị ọzọ na-eme n'ime mpaghara α1 na α2 na ngalaba C-terminal mebiri emebi (Fig 6).N'otu n'ime ihe atụ ndị a, anyị chọpụtara na HLA-A na-emekọrịta ihe na ọdụ ọdụ N-terminal nke LRCH4 (Foto 6A,D).LRCH4 na-achịkwa ịgbalite TLR4 yana ntinye cytokine LPS, si otú a na-emegharị nzaghachi mgbochi ebumpụta ụwa60,61.Ọ bụ protein akpụkpọ anụ nwere leucine-rich repeats (LRRs) na calmodulin (CH) homology motif na ectodomain ya, na-esote ngalaba transmembrane (TMD) 60, 62.A kọwo ngalaba CH ka ọ na-emekọrịta mmekọrịta protein-protein 60.Mgbatị nke ihe dị ka amino acid 300 dị n'etiti ngalaba LRR na CH bụ nke a na-enweta nke ọma mana enweghị nsogbu.Dabere na ọrụ nke mpaghara ọgba aghara dị ka ndị ogbugbo nke netwọk protein-protein na njem vesicular 63, anyị chọpụtara na ọtụtụ mmekọrịta protein na-eme na mpaghara ndị nwere nsogbu.A na-ekesa mmekọrịta ya na MDN1 n'ogologo ogologo protein ahụ, gụnyere ngalaba LRR1, LRR6, CH, na mpaghara enweghị usoro, ebe H2B na-ejikọta na ngalaba CH (Fig. 6A, B).N'ụzọ doro anya, ọ dịghị nke ọ bụla n'ime mmekọrịta ndị ahụ gụnyere TMJ, na-atụ aro kpọmkwem usoro CLMS (Figure 6A, B).
Achọpụtakwara MDN1 dị ka akụkụ nke netwọkụ protein HLA-A (Figure 6A).Ọ bụ nke ezinụlọ AAA nke protein (ATPases metụtara ọrụ dị iche iche).Nke a bụ otu ngalaba N-terminal AAA na-ahazi n'ime mgbanaka hexameric ma wepụ ihe mgbakọ na 60S 64 ribosomal subunit.yiri ka ọ dị ka dynein64,65,66.Na mgbakwunye, mpaghara Asp/Glu bara ụba na-esote ngalaba MIDAS (saịtị ion dabere na metal).N'ihi nnukwu nha nke MDN1 (ihe dị ka 5600 amino acids) na nsoro ya nwere oke na-edozi ahụ nke ọma, amachaghị maka nhazi ya na ọrụ ya n'ime mmadụ.Anyị chọpụtara HLA-A, H2B, na LRCH4 dị ka ndị mmekọ njikọ MDN1 ma kpughee nhazi ha dị ka ogige protein na PyMol (Fig. 6A, B).Protein atọ a na-emekọrịta ihe na ngalaba AAA, ngalaba njikọ njikọ dynein, yana ikekwe ngalaba MIDAS MDN1.N'ime akụkọ gara aga, ịdị ọcha nke protein bait chọpụtara MDN1 dị ka protein jikọtara ya na histone H2B67.Na mgbakwunye, nnyocha e mere n'oge na-adịbeghị anya kọkwara mmekọrịta dị n'etiti MDN na HLA-B na sel HCT116 site na iji spectrometry dị ọcha dị ọcha, na-akwado nchọpụta anyị68.Nchọpụta nke mgbagwoju a na ihe nlele IFNa-emeso na-egosi ọrụ maka MDN1 na akara interferon.
N'ihi na mkpụrụ ndụ ihe nketa HLA bụ polymorphic nke ukwuu, anyị weputara usoro nke na-agụ maapụ HLA-A, -B na -C site na data usoro RNA nke mkpụrụ ndụ Flo-1 (data egosighi).Usoro Peptide kwekọrọ n'ịgụ usoro n'usoro gosipụtara nnukwu ọdịiche dị n'etiti HLA-A, -B, na -C na mpaghara ebe peptides jikọtara cross dị na HLA-A (Njirimara S3).Na mgbakwunye, anyị ahụghị njikọ njikọ protein-na-protein nke mkpụrụ ndụ HLA-B/C na protein H2B/HMGA1/MDN1/LRCH4.Nke a na-egosi na mmekọrịta protein dị n'etiti HLA-A, MDN1, LRCH1 na HMGA1 bụ kpọmkwem HLA-A.Tụkwasị na nke ahụ, nyocha nke proteomic nke ihe atụ ndị na-abụghị ndị na-ejikọtaghị ọnụ (Table S4) gosiri na HLA-A nwere mkpuchi usoro dị elu ma e jiri ya tụnyere HLA-B ma ọ bụ HLA-C.Ihe peptides ndị a chọpụtara maka HLA-A dị elu n'ime ma IFNa-gwọọ na nlele adịghị agwọ ya.
Iji hụ na mmekọrịta ndị a chọpụtara ebe a abụghị n'ihi njikọ na-abụghị nke a na-ejikọtaghị ọnụ nke protein abụọ dị nso na nso nso a, anyị kwadoro ihe abụọ HLA-A ọhụrụ na-emekọrịta ihe site n'ịme nyocha nke imekọ ihe ọnụ.Achọpụtara mmekọrịta HLA-A na endogenous MDN1 na H2B n'ime ma IFNa-agwọ ọrịa na sel Flo-1 a na-agwọghị (Nyocha 7, Figure S4).Anyị kwadoro na H2B weghaara HLA-A na immunoprecipitates nakwa na mkpakọrịta a bụ n'ihi ọgwụgwọ IFNα ebe ọ bụ na HLA-A na-anọghị na ihe nlele immunoprecipitate sitere na sel ndị a na-adịghị agwọ ọrịa (Figure 7A).Otú ọ dị, data anyị na-atụ aro na IFNa dị iche iche na-achịkwa njikọ HLA-A na H2B na MDN1.IFNa na-ebute mkpakọrịta n'etiti H2B na HLA-A, mana ọ na-ebelata njikọ ya na MDN1.Anyị chọpụtara na MDN1 jikọtara ya na HLA-A na njikwa, na mgbakwunye nke IFNa belatara mmekọrịta a na-adabere na ntinye MDN1 site na IFNα (Nyocha 7B, C).Na mgbakwunye, HLA-A immunoprecipitation weghaara H2B na sel A549 (Fig. S4), na-atụ aro na mmekọrịta a na-adabere na ụdị cell.N'ịchịkọta ọnụ, nsonaazụ ndị a na-akwado mmekọrịta nke interferon-mediated nke HLA-A na H2B na MDN1.
HLA-A na-emekọrịta ihe H2B na MDN1.Ndị nnọchi anya endogenous H2B (A) na MDN1 (B) immunoblots bụ immunoprecipitated site na IFNa-agwọta sel Flo-1 wee nyocha maka ọgwụ mgbochi egosipụtara.Eji òké na oke bekee IgG mee ihe dị ka njikwa na-adịghị mma.(C) A na-egosipụta ọnụọgụ (ntinye) nke antigens dị iche iche site na immunoblots nyochara megide ọgwụ mgbochi egosipụtara, β-actin ejiri mee ihe dị ka njikwa nbudata.
Achọpụtara ihe arụrụ arụ nke otu n'ime interferon butere nnukwu netwọlụ ejikọtara ọnụ, H2B-HLA-A-HMGA1.Anyị na-eji ihe nlegharị anya nke molecular dynamics dị ka ụzọ ọzọ iji ghọta mgbanwe mgbanwe nke protein ndị na-etinye aka na mgbagwoju anya a (Nyocha 8).Ntụnye sitere na data CLMS na-atụ aro na enwere ike ịnwe ngbanwe dị iche iche nke protein H2B, HLA-A na HMGA1.Ya mere, a haziri ogige ndị a nwere ike ime ka ọ bụrụ ihe mgbaze: H2B-HLA-A, HMGA1-HLA-A, na H2B-HLA-A-HMGA1.Ihuenyo docking protein-protein mbụ na-eji MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) tụrụ aro enwere ike ime mgbanwe dị iche n'etiti protein ndị a (Fig 8A).Nleta anya nke mgbagwoju protein docking kpughere ọtụtụ mkparịta ụka yana mgbanwe enwere ike (Foto 5A, 8).Ya mere, a na-egosi otu nkwekọ nwere ike ime na eserese 8A (nke ejiri akara cross-links) wee nyochaa ya ọzọ site na iji pipeline modeling MD.Na mgbakwunye, njikọ nke H2B ma ọ bụ HMGA1 na HLA-A na-eme ka njikọ dị elu nke H2B maka HLA-A (Fig 8A).
Usoro mgbanwe nke netwọk enwere ike n'etiti ogige H2B (H2BFS) -HLA-A, HMGA1-HLA-A na H2B-HLA-A-HMGA1.(A) Ogwe aka ekpe bụ maapụ 2D (emepụtara na sọftụwia SIM-XL) nke intramolecular (red) na intermolecular (acha anụnụ anụnụ) crosslinks (crosslink cutoff at to 3.5).Na mgbakwunye, a na-edobe ihe fọdụrụ na-ejikọ ọnụ na nhazi nke protein H2B, HLA-A na HMGA1.E weputara nkwekọ njikọ nke protein ndị a site na iji pipeline docking etinyere na ngwugwu MOE.Ogwe aka ekpe nke dị n'okpuru na-egosi ụdị nhazi dị iche iche nwere ike ime nke H2B-HLA-A na HMGA1-HLA-A nwere njikọ protein-protein dị iche iche (GBVI/WSA dG; kcal/mol).(B) Standard deviation (RMSD) nke atomic ọnọdụ (ewezuga hydrogen atom) maka onye ọ bụla protein Ọdịdị.(C) Intermolecular protein-protein hydrogen njikọ mmekọrịta sitere na ogige simulated dị iche iche na-atụle mkparịta ụka kpọmkwem nke oge ≥ 10 ns.Edebere ebe ị ga-egbu onye inye onyinye-ịnabata h-bond ka ọ bụrụ 3.5 Å, na akụkụ mbepụ onye inyeaka-H-anabata ka edobere ka ọ bụrụ ≥ 160°–180°.(D) Mpempe akwụkwọ ndị akpọrọ aha na-emekọrịta mmekọrịta protein-protein HLA-A na ndị mmekọ ha, na-agbatị ≥ 20 ns, ewepụtara na ogige HLA-A-H2B na HLA-A-HMGA1.Ọdịdị protein na-anọchi anya nhazi nke 100 ns MDS.(E) Mmekọrịta dị n'etiti ogige HLA-A-H2B na HLA-A-HMGA1 tụnyere mmekọrịta nke simulation H2B-HLA na-enyocha ihe karịrị 100 ns dabere na saịtị K ma ọ bụ S n'etiti peptides abụọ ahụ.Mgbagwoju anya /HMGA1-HLA-A/H2B-HLA-A-HMGA1.Edebere uru ọnụ ụzọ maka nleba anya nke njikọ njikọ na 3.0, na mmekọrịta dị iche iche sitere na MDS na-ewere ≥ 10 ns ka etinyere n'uche.A na-ahụta ihe owuwu protein site na iji ngwungwu BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, USA) na Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).
Nkwụsi ike nke mkpụrụ ndụ HLA-A ka oge na-aga (ngbanwe ọkọlọtọ; RMSD ma ọ bụ ụkpụrụ ọkọlọtọ; RMSF) gosipụtara na ọnụnọ nke protein H2B ma ọ bụ HMGA1 na ogige ahụ kwadoro HLA-A (Nyocha 8B, Figure S5).Protein HMGA1 na-ejikọta nke ọma na saịtị B2M nke HLA-A, na-ebute nkwụsi ike nke amino acid HLA-A na mgbagwoju HLA-A-HMGA1 ma ọ bụ H2B-HLA-A-HMGA1 (Ọnọdụ 8B, foto S5).karịsịa, HLA residus ~ 60-90 na ~ 180-210 chọpụtara na ọ dịghị agbanwe agbanwe n'ihu H2B (FIG. 8B).H2B na HMGA1 gosipụtara njikọ dị mma na HLA-A na mgbagwoju H2B-HLA-A-HMGA1 ma e jiri ya tụnyere HLA-A na-ejikọta na H2B ma ọ bụ HMGA1 naanị (Foto 8C,D; Tebụl S5).Ihe ndị fọdụrụ na hydrogen bonding (MD modeled high occupancy ≥ 10 ns) dakọtara na saịtị mmekọrịta CLMS (K ma ọ bụ S residues) na mgbagwoju anya, na-atụ aro na mmekọrịta nke CLMS chọpụtara bụ nke a pụrụ ịdabere na ya.Ntụkwasị obi (Fig 8E).Na CLMS na MD modeling, HLA-A foduru n'etiti ihe dị ka 190-210 na ihe dị ka 200-220 amino asịd ka ahụrụ na-ejikọta H2B na HMGA1, otu (FIG. 8E).
Mmekọrịta protein-protein na-etolite netwọk nhazi dị ike nke na-eme ka nkwurịta okwu intracellular na-azaghachi na ụfọdụ mkpali.N'ihi na ọtụtụ ụzọ proteomics na-achọpụta mgbanwe na ọkwa ọkwa protein n'ozuzu ya, mmekọrịta mmekọrịta protein-protein chọrọ ngwaọrụ ndị ọzọ iji weghara njikọ njikọ, na CLMS bụ otu ngwa ọrụ dị otú ahụ.Usoro nrịbama interferon bụ netwọk cytokine nke na-enye ohere maka mkpụrụ ndụ ịzaghachi n'ọtụtụ ihe mgbaàmà na-akpata gburugburu ebe obibi na nke dị n'ime ya, na-ejedebe na ntinye nke ntinye nke protin interferon-inducible.Anyị tinyere CLMS iji chọpụta ma ọ bụrụ na enwere ike ịmata mmekọrịta protein-protein ọhụrụ n'etiti otu panel nke protein interferon butere.A na-eji nyocha njikọ njikọ protein zuru ụwa ọnụ n'ụdị cell Flo-1 na-anabata interferon iji weghara ogige protein.Mwepụta nke peptides tryptic site na sel ndị na-abụghị ndị na-ejikọta ya na cross-linked na-enye ohere maka ịgụta peptide, imewanye ụzọ, na nkesa ogologo peptide na nkọwa LFQ siri ike.Achọpụtara proteins interferon-inducible nke Canonical dị ka njikwa dị mma n'ime, ebe a na-ahụ njikọ intermolecular ọhụrụ na intramolecular cross-linked interferon-inducible proteins dị ka MX1, UP18, OAS3 na STAT1.Achọpụtala atụmatụ nhazi dị iche iche na mmekọrịta na mpaghara arụ ọrụ.
Achọpụtara mmekọrịta dị n'etiti HLA-A, MDN1 na H2B site na immunoblotting na sel Flo-1 na A549 na-agwọghị ya na IFNa.Nsonaazụ anyị gosipụtara na HLA-A nwere H2B n'ụzọ dabere na IFNa.Ọrụ anyị na-anọchite anya ụzọ na-adọrọ mmasị maka nyocha ọzọ nke nhazi nke ogige abụọ a.Ọ ga-abụkwa ihe na-adọrọ mmasị ịgbasa ụzọ CLMS na panel nke ahịrị sel iji chọpụta mmekọrịta protein interferon na-adabere na ụdị cell.N'ikpeazụ, anyị jiri MD modeling dị ka ụzọ ọzọ iji ghọta mgbanwe mgbanwe nke protein na-etinye aka na mgbagwoju H2BFS-HLA-A-HMGA1, bụ nke na-enyocha mkparịta ụka intramolecular na intermolecular.Ntụnye sitere na data CLMS na-atụ aro na enwere ike ịnwe ngbanwe dị iche iche nke protein H2BFS, HLA-A na HMGA1.Nkwekọrịta dị iche iche enwere ike n'etiti ogige protein docking ndị a gosipụtara ọtụtụ mmekọrịta yiri nke ahụ hụrụ na dataset CLMS.Otu n'ime isi ike nke usoro anyị bụ na ọ na-enye ohere njirimara dị mfe nke ịmekọrịta mkpụrụ ndụ ihe nketa polymorphic dị elu dị ka HLA, ya mere ọ ga-adọrọ mmasị ịmụ mmekọrịta nke protein HLA haplotype kpọmkwem nke na-esi ike ịmụ ihe.N'ịchịkọta ọnụ, data anyị na-egosi na enwere ike iji CLMS gbasaa nghọta anyị banyere netwọk mgbaàmà interferon na-ebute ma nye ihe ndabere maka ịmụ usoro intercellular dị mgbagwoju anya na microenvironment tumor.
E nwetara mkpụrụ ndụ Flo-1 site na ATCC ma debe ya na DMEM (Gibco) gbakwunyere na 1% penicillin/streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) ma chekwaa ya na 37 ° C na 5% CO2.Mbubata.A na-etolite mkpụrụ ndụ na 70-80% confluence tupu a gwọọ ya na IFNα14 (nke Edinburgh Protein Production Facility mere).A zụtara kemịkalụ na reagents ndị ọzọ n'aka Sigma Aldrich belụsọ na amataghị ya.
A na-emepụta mkpụrụ ndụ Flo-1 na efere 6-ọma na n'echi ya, a na-emeso mkpụrụ ndụ ahụ na 10 ng / ml IFNα14 maka awa 24 ruo ihe dịka 80% confluence.A na-asacha mkpụrụ ndụ ugboro atọ na PBS ma jikọta ya na DSS dị ọhụrụ (Thermo Fisher Scientific) (gbasara na DMSO) na PBS maka 5 min na 37 ° C. na njedebe ikpeazụ nke 0.5 mM.Ejiri PBS dochie mmeghachi omume crosslinking DSS ma kwụsịchaa DSS site na ịgbakwunye 20 mM Tris (pH 8.0) na PBS maka 15 min na 37°C.A na-anakọta mkpụrụ ndụ site na nchacha ma na-anakọta ya na obere tubes na-ejikọta ya (Axygen).
Ejiri pellet cell jiri 300 µl nke urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) jiri nkeji 30 mee ihe n'ime ụlọ okpomọkụ yana ịma jijiji mgbe ụfọdụ.Emere usoro centrifugation niile na 14,000 xg na 8 Celsius.Centrifuge na lysate maka nkeji 10 ma bufee supernatant na tube ọhụrụ.A na-agbaze ihe ndị ọzọ doro anya na 150 μl nke ihe nchekwa lysis nke abụọ (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) maka nkeji 30 ma ọ bụ karịa ruo mgbe e nwetara ngwọta mmiri na-edozi ahụ.A na-eme ka lysate centrifuged maka nkeji 20 na supernatant jikọtara ya na lysate nwetara na nzọụkwụ gara aga.A tụlere mkpokọta protein site na iji nyocha Micro BCA (Thermo Fisher Scientific) dịka ntuziaka onye nrụpụta maka usoro microplate siri dị.A na-awụsa ihe nlele ahụ ngwa ngwa na nitrogen mmiri mmiri wee chekwaa ya na -80 Celsius.
A na-ahazi ihe dị ka 100 μg nke protein nwere njikọ chiri anya site na iji usoro nkwadebe nlele filtration gbanwetụrụ (FASP) dịka Wisniewski et al kọwara.69 Na nkenke, protein jikọtara ya na 200 µl nke urea buffer (8 M urea na 0.1 M Tris, pH 8.5), vortexed na ọkara.Emere usoro centrifugation niile na 14,000 xg na 25 Celsius.A na-ebufe ọkara mbụ nke protein lysate na-ejikọta ya na 10 kDa Microcon centrifugal filter filter nke nwere akpụkpọ anụ Ultracel-10 (Merck), na-esote centrifugation na nzacha maka nkeji 25.Mgbe ahụ gbakwunye ọkara nke abụọ nke protein na nzacha ma megharịa otu usoro ahụ.Emere mgbake protein site na ịgbakwunye 100 μl nke 17 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) na urea buffer.A kpaliri mgbake na thermomixer na 600 rpm maka 30 min na 37 ° C.Tụkwasị na nke ahụ, a na-agbanye kọlụm ahụ ma na-eme ka protein na-ejikọta ya na-ebelata site na iji 100 μl nke 50 mM iodoacetamide na urea buffer.A na-eme mmeghachi omume alkylation na ụlọ okpomọkụ maka nkeji 20 n'ọchịchịrị.Gbanwee kọlụm ahụ, jiri 100 µl urea buffer sachaa mgbidi kọlụm ahụ ugboro atọ, wee kee centrifuge.A na-arụ otu ọrụ ahụ ugboro 3 site na iji 100 μl nke 100 mm ammonium bicarbonate.Tupu trypsinization, dochie tube nchịkọta na nke ọhụrụ.Tinye ihe nchekwa mgbaze nwere 50 mM ammonium bicarbonate na 1 µl trypsin gbanyere n'ime ihe nchekwa trypsin (Promega).A na-edobe oke nke trypsin na protein n'ihe dịka 1:33, na mmeghachi omume mgbari na-abanyekwa n'otu abalị na 37 Celsius C. n'ime ụlọ iru mmiri.E wepụrụ peptide jikọtara ya na nzacha site na centrifugation maka nkeji 25.Emelitere mgbake Peptide site na ịgbakwunye 50 μl nke 0.5 M NaCl na nzacha, na-esote centrifugation maka nkeji 25.
A na-eji kọlụm C18 Micro Spin (Harvard Apparatus) mee ka peptides tryptic jikọtara cross na-agbaso ụkpụrụ nke Bouchal et al.70 kọwara na obere mgbanwe.Na nkenke, ejiri ncha atọ nke 0.1% formic acid (FA) rụọ ọrụ ogidi ogidi C18 na acetonitrile (AcN) (Merck) na ncha abụọ nke 0.1% FA.Ejiri 0.1% FA mee mmiri na kọlụm ahụ maka nkeji iri na ise.Bunye ihe nlele n'ime ogidi ogho wee were 0.1% FA saa ugboro 3.Eji gradient steepụ were were 50%, 80% na 100% AcN were 0.1% FA were gbachaa peptides ahụ agbazere n'usoro.Akpọkọrọ ihe nlele ndị a na SpeedVac Plus concentrator (Eppendorf) ruo mgbe mmiri fọdụrụnụ kwụsịrị kpamkpam.A gbazere peptides elu gbazere na 100 μl nke 0.08% trifluoroacetic acid na 2.5% AcN na a tụlere mkpokọta na NanoDrop 2000 (Thermo Scientific).Ihe dị ka 1 μg nke peptide crosslinked kwa sample ka agbanyere n'ime usoro LC-MS/MS.
E kewapụrụ peptides jikọtara ya na usoro UltiMate 3000 RSLCnano LC (Thermo Scientific) jikọtara na Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Achịkọtara peptides jikọtara ya na ID 300 µm, kọlụm njide 5mm µ tupu kọlụm C18 juru na C18 PepMap100 sorbent na 5 µm PepMap sorbent (Thermo Scientific).Jikwaa mgbapụta mgbapụta ahụ atọrọ na 5 µl/min 0.08% trifluoroacetic acid agbazere na 2.5% AcN.E kewapụrụ peptides jikọtara ya na kọlụm silica fused analytical nwere dayameta dị n'ime nke 75 μm na ogologo 150 mm, jupụtara na 2 μm PepMap sorbent (Thermo Scientific).Usoro mkpanaaka A na B nwere 0.1% FA n'ime mmiri yana 0.1% FA na acetonitrile, n'otu n'otu.gradient na-amalite na 2.5% B wee na-abawanye n'ahịrị ruo 40% B karịa nkeji 90, wee ruo 90% B n'ime nkeji 2 na-esote.A na-edobe nhazi usoro mkpanaka na 90% B maka nkeji 10 wee gbadata n'ahịrị ruo 2.5% B karịa nkeji 2.A haziri kọlụm ahụ na 2.5% B maka nkeji 8 tupu okirikiri ọzọ.peptides nwere njikọ gafere site na kọlụm nyocha bụ ionized na isi iyi nanoelectrospray ionization (NSI) wee gbanye n'ime Exploris 480 mass spectrometer (Thermo Scientific).
Orbitrap Exploris 480 mass spectrometer na-arụ ọrụ na ọnọdụ njikọ data dị mma.Emere nyocha zuru oke na ọnọdụ ngalaba na mkpebi 120,000 nwere ntọala nso site na m/z 350 Th ka m/z 2000 Th.Edebere ebumnuche AGC ahaziri ahazi na 300% yana oge ntinye kacha nke 50ms.Emebela nchọpụta ọnụ ọgụgụ monoisotopic maka peptides.Atọọ oke izu ike mmachi ka ọ bụrụ eziokwu ma ọ bụrụ na achọta ụzọ ole na ole.Edebere ike ionic kacha nta nke onye mbido ka ọ bụrụ 5.0e3 yana ụgwọ izizi ihe ruru +8 etinyere na nnwale ahụ.
Oge okirikiri n'etiti nnukwu nyocha na ọnọdụ njikọ data ka atọrọ ka 2.5 sekọnd.Edebere mwepu oke dị egwu ka ọ bụrụ 20 s ka nkewachara nke mbụ nke ion precursor.Edobere mpio ikewapụ ụzọ ka ọ bụrụ 2 Th.Ụdị ike nkuku a na-ahazi nke ọma nwere ọnọdụ ike nkuku a kapịrị ọnụ ka ahọpụtara na nyocha MS/MS dabere na data.Ike ndakọrịta atọrọ na 30%.Emebere mkpebi Orbitrap ka ọ bụrụ 15,000 yana ebumnuche AGC na 100%.Omenala kacha oge ịgba ntụtụ ka atọrọ ka millisekos 60.
Tupu nyochaa netwọk protein-protein n'ime ihe atụ jikọtara ọnụ, anyị na-eji ngwugwu MaxQuant (ụdị 1.6.12.0) 26,27 hazie faịlụ raw ahụ iji chọpụta peptides / protein ndị nwere ike ịchọta na nlele.Tụkwasị na nke ahụ, a na-eme nyocha proteomic yiri nke ahụ na nlele Flo-1 na-enweghị njikọ na-agwọghị ya na IFNa.Achọtara data MS/MS na nchekwa data mmadụ UniProt (www.uniprot.org) (ebugoro Ọgọst 12, 2020 nwere ndenye 75,093) site na iji igwe nchọta Andromeda27 arụnyere n'ime ya.Emere ọchụchọ ahụ na-egosighị nkọwapụta nke enzyme na mgbanwe dị iche iche nke deamidation (N, Q) na oxidation (M).Edebere nnabata oke ibu ụzọ na 20 ppm yana ion ngwaahịa na 0.02 Da.Edebere ngbanwe nke mbụ na nke kachasị na 10 ppm.Oke kachasị nke peptide ka edobere na 4600 Da na myirịta usoro a ka edobere n'etiti 7 na 25 amino acids (aa).A na-eme nyocha nyocha ọzọ site na iji mmemme Perseus (ụdị 1.6.10.45).A na-agbakọ ọdịnaya protein site na ịhazigharị ike nke protein (LFQ ike; enweghị akara)27 na ụkpụrụ ike gbanwere na Log2.Ejiri ngwugwu pheatmap (v1.0.12) dị na R (v 4.1.2).Emere nyocha nkwalite okporo ụzọ site na iji nchekwa data ụzọ Reactome maka protein ndị IFNa gwọchara nke arụnyere ihe karịrị ugboro anọ ma e jiri ya tụnyere ihe nlele anaghị agwọ ya.
Achọpụtara lysine (K) ma ọ bụ serine (S) kpọmkwem kemịkalụ kemịkalụ nke ogige protein nke LC-MS/MS na-enyocha site na iji igwe njirimara spectroscopic (SIM-XL) maka peptides jikọtara ya (SIM-XL)29.Nke mbụ, enwere ike ịmekọrịta mmekọrịta dị n'etiti interferon-associated (IFN) DNA mmebi mbinye aka mgbochi (IRDS) mkpụrụ ndụ ihe nketa site na iji dataset protein IRDS kọwara na Padariya et al.28.Nyochaa ọnọdụ niile na ikwugharị UniProt mmadụ dum na-akpa ike, yabụ nchekwa data UniProt mmadụ niile (www.uniprot.org) (ebudatara 12 Ọgọst 2020, nwere ndenye 75,093) megidere IFNa-emeso ugboro ugboro.Otu nzacha maka mmekọrịta ntụkwasị obi dị elu.Mmekọrịta ndị a dị oke mkpa enwetara gbasawanye ma nwalee na ugboro ugboro na ọnọdụ niile.
Na SIM-XL, a na-eji DSS mee ihe maka crosslinker (XL) na ngbanwe ịdị arọ XL na mgbanwe mgbanwe dị na 138.06 na 156.07, n'otu n'otu.A na-atụle saịtị mmeghachi omume crosslinking ndị a: KK, KS na KN-TERM, na-enweghị ion onye nta akụkọ.Edobere ma precursor na iberibe ppm ka ọ bụrụ 20 ma atọrọ ụzọ Xrea ka ọ bụrụ 0.15.A na-ewere trypsin dị ka nke akọwapụtara n'ụzọ zuru ezu, na usoro nkewa C-trap (HCD) nwere ike dị elu.Ọnụ ụzọ mbelata DB ike XCorr na ọnụ ọgụgụ kacha nta nke peptides maka mbelata DB ike ka edobere na 2.5 na 2, n'otu n'otu.Akụkụ ndị ọzọ bụ: ihe gbasara puru omume monoisotope na mbelata oke ndakọrịta, opekempe 4 AA residues kwa strand na kacha ụgwọ strand, yana 3 maxima nke nkewa agbaghara.A tụlere maapụ 2D nke esiri na ya (SIM-XL) wee jiri ihe ngosi eserese xQuest28 wuo maapụ 2D ahụ.A na-enye njikọ njikọ protein na nhazi protein na PyMol (PyMOL Molecular Graphics System, ụdị 2.0 Schrödinger, LLC).
Ejiri ihe nkesa Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) 11 na-eji ụkpụrụ nke homology modeling na mmejuputa iwu nke "Zozoo Markov Method".Phyre2 na-emepụta ihe nlere anya dabere na nhazi usoro na usoro protein amaara.Maka H2BFS, HLA-A, HMGA1, LRCH4, na protein MDN1, a na-eji arụ ọrụ 1kx552, 1kj349, 2eze55, 6hlu62, na 6i2665.Na mgbakwunye, a tụlekwara usoro nke AlphaFold71 MX1, UBP18 na ROBO1.Ejiri ngwungwu BIOVIA Discovery Studio Visualizer hụ ihe nhazi protein ahụ (Dassault Systèmes, BIOVIA, San Diego, CA, USA) na ngwugwu gburugburu Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).