347 ibyuma bidafite ingese bifatanyijemo ibikoresho bya chimique, Kumenyekanisha udushya twa interferon-yitabira abantu leukocyte antigen-A (HLA-A) proteine ​​za chaperone ukoresheje sprometrike ihuza abantu (CLMS)

Urakoze gusura Kamere.com.Ukoresha verisiyo ya mushakisha hamwe na CSS igarukira.Kuburambe bwiza, turagusaba ko ukoresha mushakisha ivuguruye (cyangwa ugahagarika uburyo bwo guhuza uburyo muri Internet Explorer).Mubyongeyeho, kugirango tumenye inkunga ihoraho, twerekana urubuga rudafite imiterere na JavaScript.
Abashoferi berekana ingingo eshatu kuri slide.Koresha inyuma na buto ikurikira kugirango unyuze kumurongo, cyangwa utubuto twa slide mugenzuzi kugirango urangire unyuze kuri buri slide.

Ibisobanuro ku bicuruzwa

Icyuma kitagira umuyonga 347L Igiceri, Igipimo cyicyuma: SS347L

Sisitemu yerekana ibimenyetso bya interferon itera cytokine ikomeye muburyo butandukanye bwibimenyetso byindwara ziterwa nindwara ziterwa nibidukikije, bikavamo kwinjiza uduce duto twa poroteyine za interferon.Twakoresheje DSS-yunganirwa ihuza abantu benshi (CLMS) kugirango tumenye imikoranire mishya ya poroteyine na poroteyine mu rwego rwa poroteyine ziterwa na interferon.Usibye poroteyine ziteganijwe ziteganijwe, twanagaragaje udushya twinshi hagati ya intermolecular na intramolecular ihuza imiyoboro ya poroteyine za Canonical interferon-zidashoboka nka MX1, USP18, OAS3, na STAT1.Twibanze ku kwemeza imiterere yimikorere ya poroteyine ya interferon-idashobora kwinjizwa na poroteyine ya HLA-A (H2BFS-HLA-A-HMGA1) dukoresheje co-immunoprecipitation hamwe nubushakashatsi bwabo bwifashishije uburyo bwa molekile dinamike.Kwerekana imiterere ihuza imbaraga za poroteyine zagaragaje imbuga nyinshi zikorana zigaragaza imikoranire yagaragaye mubisubizo bya CLMS.Hamwe na hamwe, turerekana ubushakashatsi bwikigereranyo bwa CLMS kugirango tumenye ibimenyetso bishya byerekana ibimenyetso biterwa na interferon, kandi dutegerezanyije amatsiko gukoresha CLMS kugirango tumenye imbaraga nshya zikorana na poroteyine muri microen ibidukikije.
Mbere yuko ubudahangarwa bw'umubiri butangira, sisitemu yo kwirwanaho yavukanye itanga igisubizo cya mikorobe yunganirwa n'umuryango wa alpha-helical cytokine rwihishwa bita interferons (IFNs).Ubwoko I IFN ibyiciro IFNα na IFNβ bikora ibisubizo bya selile, harimo antiviral, proapoptotic, proinflammatory, na leta zirwanya antiproliferative.Mu bantu, ubwoko 13 bwa IFNα burazwi, byose byegeranye kuri chromosome 91. Igitangaje, ni uko IFNα2 yonyine yakozwe kugirango ikoreshwe kwa muganga.Vuba aha, hitabwa cyane kubushakashatsi kubindi bisobanuro bya IFNα.Ubushakashatsi buherutse gukorwa bwerekanye ko IFNα14 ari imwe mu isoforms ikora neza mu kugabanya HBV2 na virusi itera SIDA-13,4 ugereranije n’ubwoko bwa IFNα2.
Byagaragaye ko ibikorwa byubwoko bwa I interferon reseptor complexes (IFNAR1 na IFNAR2) bitera casque transduction cascade yahujwe na Janus kinase TYK2 na JAK15,6.Izi Janus kinase fosifora yerekana ibimenyetso hamwe na poroteyine zikora (STAT1 na STAT2) ku bisigazwa bya tirozine kugirango utangire SH2 domaine-yunganirwa na heterodimerisation6.Ibikurikira, IRF9 ihuza heterodimers ya STAT kugirango ibe igizwe ningingo ya trimeric ya IFN-iterwa ningingo ya 3 gene (ISGF3), ihinduranya nucleus kandi itera kwandukura genes zirenga 2000 ziterwa na interferon (ISGs) 5,6,7,8.
ISGs ni urufatiro rwimikorere yubudahangarwa bw'umubiri, cyane cyane mugusubiza virusi.Nkumurongo wambere wo kwirinda kwandura virusi, selile zikoresha byihuse imikoranire ya poroteyine ngengabuzima hamwe nibikorwa byinshi byibinyabuzima.Izi poroteyine zirimo kwakirwa byerekana imiterere, ibimenyetso byerekana molekile, ibintu byandikirwa, hamwe na poroteyine zifite imikorere ya virusi itaziguye, kimwe n’ibintu bibi bigabanya ubudahangarwa bw'umubiri9.Amenshi mu makuru ajyanye nibikorwa bya ISG aturuka kuri ecran ikora ikoresheje ecran ikabije ya 10,11 cyangwa tekinike yo gucecekesha gene (siRNA, RNAi na CRISPR) 12,13 aho ISGs zigaragarira cyangwa zikabuzwa kandi ibikorwa byazo bikageragezwa kuri virusi zitandukanye.Nubwo ubu bushakashatsi bwerekanye imiterere ya virusi ya ISGs, uburyo bwimikorere ya molekile ya buri ISG buracyamenyekana.Muri rusange biremewe ko poroteyine nyinshi zikorana na cytokine imwe cyangwa nyinshi kugirango ibikorwa byuzuye, bityo ISGs zikorana mu buryo butaziguye cyangwa imikoranire yazo ikaba ihuzwa na poroteyine ngengabuzima.Kurugero, ubushakashatsi bwa proteomics buherutse gufotora bwerekanye ko ATPase VCP / p97 nkumufatanyabikorwa ukomeye wa IFITM3, kubuza kwabo kuganisha ku nenge zo gutondeka lysosomal, kuzenguruka, hamwe na cotransport ya IFITM3 hamwe na virusi 14.Twifashishije immunoprecipitation, twasanze VAPA, poroteyine ifitanye isano na viticle, nk'umufatanyabikorwa wa IFITM1 / 2/3 uhuza no gukura kwa virusi ya cholesterol, kandi ibi byashimangiwe n'ubundi bushakashatsi dukoresheje umusemburo wa Hybrid ebyiri.Inkunga ya siyansi 15, 16.
Uburyo bwibanze bwibinyabuzima bugira uruhare mukurwanya kwandura no guhinduka nabi ni kwerekana antigen, igahuzwa na molekile nini ya histocompatibilité (MHC).Peptide (8-12 aminide acide) iva muri poroteyine zifunze, zarangiye imburagihe cyangwa zidahwitse zashyizwe muri heterodimer ya MHC-I (igizwe n'iminyururu iremereye kandi yoroheje ya MHC-I, yitwa β-2-microglobuline; β2M) 17,18.Imyitozo ihamye ya MHC-I ijyanwa hejuru ya selile, aho igaragaza peptide yo mu nda kuri selile CD8 + T (selile cytotoxic T) 17.T selile izi kandi zisenya izo virusi na selile zitwara antigen yihariye.Kubera iyo mpamvu, virusi na selile yibibyimba akenshi bihagarika uburyo bwo kwerekana antigen kugirango birinde gukurikiranwa.Byongeye kandi, MHC-I igabanywa muri 40-90% yibibyimba byabantu kandi akenshi bifitanye isano no guhanura nabi19.
Ingirabuzima fatizo zigira uruhare mu gusubiza indwara ziterwa na virusi zigomba guhinduka vuba hagati yuburuhukiro na leta yo kwandukura.Kubera iyo mpamvu, poroteyine nyinshi za selile zigereranywa kugira uruhare mu gusubiza icyifuzo cya IFN mu gihe gito, harimo kuvugurura no guhindura chromatine ya porotokoro 20,21.Ubushakashatsi bwinshi bwibanze ku kumenyekanisha abafatanyabikorwa ba poroteyine ISG ku giti cyabo imbere ya IFN.Ubushakashatsi butandukanye bwa proteomic na transcriptomic muri sisitemu yicyitegererezo ya selile bwasobanuye ingaruka za IFN kumiterere ya selire.Nubwo, nubwo turushijeho gusobanukirwa ningaruka ziterwa na interferons, turacyari tuzi bike kubyerekeye uruhare rwa ISGs.Iyo urebye ibintu bigoye kandi biterwa nigihe cyo kwerekana ibimenyetso bya interferon, havuka ibibazo bibiri: (i) birashoboka guhagarika no gutega imitego ya Multrotein igira uruhare mukumenyekanisha byihuse, kandi (ii) ubwo bufatanye bushobora gushirwa mumwanya wa 3D?
Kugira ngo dukemure ibyo bibazo, twashyize mu bikorwa disuccinimide suberate-yunganirwa n’imiti ihuza imiti (DSS) ifatanije na mass spectrometrie (CLMS) kugira ngo twige imiyoboro ya poroteyine iterwa na IFNα iterwa ningaruka zayo.DSS yongeraho isano ihuza ibisigisigi byegeranye bya poroteyine na / cyangwa poroteyine muri vivo.Isesengura rya MS ryakurikiyeho ryerekana imbuga zihuza zigaragaza aho uturere turi hagati ya poroteyine runaka, bita guhuza imbere, cyangwa subunits mu nganda za poroteyine, bita isano.Twifashishije ubu buryo, twabonye ibintu byinshi bishya bya poroteyine-protein kimwe n’imikoranire ya interferon-iterwa na Multrotein.Mugukomeza kugerageza igice cyimikoranire mishya, twerekana ko H2BFS (H2B yerekana ubwoko bwa FS; nyuma yiswe H2B) na MDN1 bakora nkabafatanyabikorwa ba HLA-A.
Ingirabuzimafatizo za Flo-1 nimwe mu zizwi cyane muri vitro moderi ya esophageal adenocarcinoma kuko yigana ibintu by'ingenzi biranga ibibyimba byo mu bwoko bwa 22,23.Nyamara, ibibyimba byose ntabwo ari immunogeneque, no kumenya niba selile Flo-1 yitabira kuvura interferon, twakoresheje selile Flo-1 hamwe na 10 ng / ml IFNα mumasaha 72.Ingirabuzimafatizo za Flo-1 zerekanye kwinjiza hakiri kare pSTAT1 na IRF1, guhera amasaha 2 nyuma yo kuvurwa no gukomeza amasaha 72, hamwe no kugabanuka guterwa nigihe cyo kugabanuka kurwego rwa IRF1 (Ishusho 1A).ISGs (MX1, IFITM1, OAS1 / 2, na ISG15) wasangaga zatewe imbaraga nyuma yamasaha 6, bigana ibisubizo byakera hagati na nyuma yicyiciro kuri IFNα (Ishusho 1A).Hamwe na hamwe, aya makuru yerekana ko iyi moderi ya selile ishobora gukoreshwa mukwiga ibisubizo bya interferon.
Itandukaniro rya poroteyine itandukanye isubiza muri selile Flo-1 nyuma yo kuvura IFNα....
Kugirango dufate imiterere ya poroteyine yimiterere, twakoresheje DSS, ikoreshwa cyane muguhuza ibikorwa bitewe na membrane yo hejuru cyane kandi mugihe gito cyo kuyitwara.Igihe kigufi cyo kubyitwaramo gifasha mukurinda ishyirwaho ryinshi rya poroteyine zihujwe, bityo bikagumya guhagarara neza.Kugirango tumenye neza DSS yibanze kandi twirinde guhuza cyane, twabanje kwerekana selile kuri 5, 2.5, na 1 mM DSS muminota 5, 10, 5, na 30, hanyuma tunasesengura lysates na SDS-PAGE ya Coomassie. (amakuru aterekanwa).Lysates ya selile isa nkaho ihujwe cyane kurwego rwo hasi kandi mugihe gito.Kubwibyo, DSS yitiriwe 1, 0.5, na 0.1 mM mu minota 5 (Ishusho 1B).Guhuza neza kwagaragaye hamwe na 0.5 mM DSS muminota 5, kandi ibi bintu byatoranijwe kuri selile zavuwe na IFNα.Mubyongeyeho, Ishusho 1C yerekana blot yo muburengerazuba ikorwa hakoreshejwe antibody ya p53 (DO-1) kugirango isuzume urugero rwa poroteyine ihuza.
Flo-1 selile zavuwe hamwe na 10 ng / ml IFNα mumasaha 24 mbere yo kongeramo.Ingirabuzimafatizo zahujwe nyuma zatewe na proteolysis zintambwe ebyiri kandi proteyine zitunganywa na FASP (Ishusho 2) 24,25.Peptide ya peptide ihujwe yasesenguwe na mass spectrometrie (Ishusho 2).MS / MS yerekanwe noneho ihuzwa na poroteyine ikurikirana kandi igereranywa na MaxQuant26,27.Peptide ihujwe yamenyekanye muri spekure yabonetse ikoresheje porogaramu ya SIM-XL, kandi ibice bitandukanye byahurijwe hamwe mu muyoboro utoroshye ukoresheje xQuest28 na SIM-XL29 bifungura imiyoboro ya porogaramu ya mudasobwa (Ishusho 2).SIM-XL igaragaza imikoranire ya poroteyine-proteyine, iminyururu y'imbere n'iminyururu ya buri muntu mu mvange ya poroteyine yoroshye cyangwa igoye kandi itanga inyandiko zerekana amashusho mu mikorere ya poroteyine.Mubyongeyeho, itondekanya buri kinyuranyo nkamanota ndangamuntu ukurikije ubuziranenge bwa MS / MS29.Hamenyekanye imikoranire myinshi ya poroteyine-protein hamwe n’ibigo, kandi hashyizweho ubushakashatsi bushya bw’imikoreshereze ikoresheje immunoprecipitation hamwe n’imihindagurikire y’imiterere ikoresheje icyerekezo cya molekile (MD) (Ishusho 2) 30, 31.
Igishushanyo mbonera cyuburyo bwa CLMS.Ingirabuzimafatizo za Flo-1 zavuwe hamwe na 10 ng / ml IFNα mu masaha 24 ikurikirwa na protein ihuza imiterere ikoresheje DSS ikurikirwa na lysis selile na trypsinisation.Ingero zahujwe zasesenguwe hakoreshejwe Orbitrap mass spectrometer hanyuma irushaho gukorerwa ibice byo gutandukanya peptide yabanjirije mugihe LC-MS / MS.Peptide ebyiri zahujwe zamenyekanye muri spekure zabonetse hakoreshejwe imashini ya Spectrum Recognition Machine ya porogaramu ya Crosslinked Peptides (SIM-XL), kandi ibice byose byahujwe numuyoboro utoroshye ukoresheje imiyoboro yo kubara.Shungura ikizere gike gishingiye kumanota meza (FDR).Imikoranire mishya myinshi ya protein-protein yizewe yongeye gushimangirwa hakoreshejwe co-immunoprecipitation, kandi impinduka zifatika muri ibyo bigo zarasuzumwe hifashishijwe uburyo bwa molekile dinamike (MD).
Peptide zose hamwe ~ 30.500 na ~ 28.500 zagaragaye hakoreshejwe MaxQuant mu ngero za IFNα zidashushanijwe kandi zishishikaje (Imbonerahamwe yinyongera S1, Ishusho 3A).Ikwirakwizwa rya peptide muri ibyo bihe byombi ryerekanye umubare munini wa peptide nini, byerekana ko hariho peptide ihuza (Ishusho 3B, C).Byongeye kandi, umubare munini wa peptide nini wari uhari muri 40-55 murwego rwa IFNα rwavuwe (Ishusho 3C).Gushushanya poroteyine kurwanya ubukana bwa log2 byerekanaga ko poroteyine zisanzwe ziterwa na interferon ari nyinshi cyane ugereranije n’intangarugero zitavuwe, harimo MX1, IFIT1 / 3, OAS2 / 3, DDX58, na HLA-F (Ishusho 3D).Isesengura ryinzira za poroteyine zirenze inshuro eshatu zikungahaye mu gusubiza imiti ya IFNα ukoresheje ububiko bw’inzira nyabagendwa bwerekanye ko MHC-I-yunganirwa na antigen yo kwerekana no kuyitunganya ari yo nzira yiganje cyane (Ishusho 3E).Bihuye na raporo zabanjirije iyi, ibisubizo bya virusi byahujwe na OAS na ISG15 kimwe na IFNα / β hamwe na signal ya cytokine byari munzira zikora.Byongeye kandi, lysine- na serine yihariye ya poroteyine yambukiranya imipaka yamenyekanye kuva mbere ya MS / MS yaguzwe hakoreshejwe SIM-XL.Ubushakashatsi buherutse gukorwa bwerekanye ko ISG 104 zikoresha virusi 20 zo mu byiciro 9 bya virusi hakoreshejwe meta-gusesengura ubushakashatsi bwakozwe na ISG ku buryo bukabije mu bwoko 5.Ariko, kugirango tuneshe imipaka yo kubara yo gusuzuma imibare minini, twatangiranye na dataset ntoya kugirango tumenye imikoranire ishoboka hagati yurutonde rwa gen IRDS yatangajwe na Padaria nabandi, inyinshi muri zo ni ISG.
Kumenyekanisha poroteyine zitandukanye zerekanwa zitandukanye mugusubiza IFNα (amakuru yakuwe muri MaxQuant).(A) Igishushanyo cya Venn cyerekana umubare wa peptide isanzwe kandi yihariye yagaragaye muri IFNα14 yavuwe kandi itavuwe Flo-1.Peptide ndende yo gukwirakwiza itavuwe (B) na IFNα ivurwa (C) ingero zifatanije.(D) Gushyushya ikarita yerekana log2 (ubukana bwa LFQ) hagati itavuwe na IFNα14 ivura selile Flo-1.Umwanya wibumoso werekana poroteyine zikora cyane imbere ya IFNα.(E) Histogramu yerekana inzira 20 zingenzi zo gukungahaza nyuma yo kuvura IFNα.Ububiko bwinzira nyabagendwa bwasesenguye impinduka zirenze enye muri poroteyine ziteganijwe IFNα.
Interferon-yunganirwa na ISG itera inkunga yanditse neza, ariko kurwego rwa molekile ntabwo byumvikana neza uburyo izo poroteyine zirangirira mubikorwa byinshi byibinyabuzima.Twakoze iperereza ku mikoranire ya poroteyine hamwe n’icyizere cyo hejuru hagati ya ISGs izwi.Igishimishije, twabonye umuyoboro urimo MX1, USP18, ROBO1, OAS3, na STAT1 za poroteyine zigize urwego runini rusubiza imiti ya IFNα (Ishusho 4, Imbonerahamwe S2) 32,33,34.Icy'ingenzi cyane, iyi mikoranire yabonetse muri bitatu byavuwe hamwe na IFNα kandi ntibyabonetse mubitegererezo bitavuwe, byerekana ko byakozwe muburyo bwo kuvura IFNα.Birazwi ko STAT1 igenga imvugo igenga imvugo ya ISGs, ariko imikoranire yayo na ISGs kurwego rwa poroteyine ntabwo yigeze yigwa.Imiterere ya kristu ya STAT1 yerekanaga ko domaine yayo (CCD) itagira uruhare mubikorwa bya ADN cyangwa protomers mugihe cyo gukora dimers35.Izi α-helices zigize imiterere ya helix itanga igice kinini cyubutaka bwa hydrophilique kugirango imikoranire ibe 35.Mu makuru yacu ya CLMS, twabonye ko imikoranire myinshi na STAT1 yabereye muri domeni SH2 ibanziriza CCD, umuhuza, cyangwa umurizo wa C-terminal (ibisigara 700-708) (Ishusho 4A).Ubushakashatsi bwabanje bwerekanye ko USP18 ihuza na CCD na ADN ihuza ADN (DBD) ya STAT2 kandi ikinjizwa muri subunit yubwoko bwa I interferon reseptor IFNAR2 kugirango ihuze kubuza ubwoko bwa I interferon yerekana 24.Amakuru yacu yerekanaga kandi ko indangarugero ya USP18 ikorana na STAT1 DBD (Igicapo 4A, D), byerekana ko STAT1 na STAT2 byombi bishobora kugira uruhare mukureshya USP18 kuri IFNAR2.
Poroteyine-protein ISG umuyoboro wagaragaye mu ngirabuzimafatizo zihujwe na IFNα..Indangamuntu zindangamuntu zitandukanye zirangwa nibara ryabo32: indangarugero ya MX1, Dynamin_N (73–249), Dynamin_M (259–547), na GED (569–660).Imiyoboro ya OAS3: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), na OAS1_C (903-108).Domisiyo ROBO1, Ig_3 (67-151), I-shiraho (170-258), I-shiraho (262–347), Ig_3 (350–432), Ig_3 (454–529), fn3 (562–646), fn3 (678–758) na fn3 (777–864).Imirima ya STAT1: STAT_int (2–120), STAT_alpha (143–309), STAT_bind (321–458), SH2 (573–657), na STAT1_TAZ2bind (715–739)..Imipaka ihuza imipaka yashyizwe kuri 3.5.Utudomo duto twerekana imbuga za STAT1 zikorana na MX1 (C), USP18 (D), ROBO1 (E), na OAS3 (F), hamwe na K cyangwa S imikoranire hagati ya peptide zombi.Igishushanyo, amanota yambukiranya amanota yashyizwe kuri 3.0..STAT1 (pdb id: 1bf533) na OAS3 (pdb id: 4s3n34).) gahunda.
Isoforms ebyiri za USP18 zasobanuwe mubantu, proteine ​​yuzuye yuzuye yiganjemo nucleus, na isoform idafite N-terminal, USP18-sf, ikwirakwizwa muri cytoplazme na nucleus 36.Byongeye kandi, N-terminus yari yarahanuwe ko itubatswe kandi ntisaba ibikorwa bya isopeptidase cyangwa guhuza ISG1537.Byinshi mubikorwa byagaragaye mubushakashatsi bwacu byari kuri N-terminus ya poroteyine, byerekana ko iyo mikoranire irimo USP18 ndende (Ishusho 4A, D) bityo bikaba bishoboka ko bibera muri nucleus.Byongeye kandi, amakuru yacu yerekana kandi ko N-terminus yihariye imikoranire ya poroteyine na poroteyine.Ikibanza cya IFNAR2 gihuza kiri hagati y ibisigisigi 312-368, kandi cyane cyane, ntanimwe muri poroteyine ziri murwego ruhuza kariya karere (Ishusho 4A) 37,38.Aya makuru yafatiwe hamwe yerekana ko IFNAR2 ihuza indangarugero ikoreshwa gusa na protein yakira.Mubyongeyeho, gusa OAS3 na ROBO1 wasangaga bifitanye isano na domaine hejuru ya N-terminus na IFNAR2 ihuza urubuga (Ishusho 4A).
ROBO1 ni iy'ubudahangarwa bwa immunoglobuline (Ig) burenze urugero bwa transembrane yerekana molekile kandi igizwe na domaine eshanu Ig na domaine eshatu za fibronectine (Fn) mukarere kidasanzwe.Izi domeni zidasanzwe zikurikirwa na membrane-proximal region hamwe na transembrane helix 39. Agace k’imitsi kavukire katubatswe gaherereye kuri C-terminus kandi karimo ibintu byabitswe bikurikirana bihuza poroteyine ihuza 39.Agace kava kuri aside amine ~ 1100 kugeza 1600 ahanini karangiritse.Twabonye ko MX1 ikorana na ROBO1 ikoresheje Ig, Fn, hamwe na domaine zidasanzwe, mugihe imikoranire myinshi na STAT1 ibaho hagati ya CCD yayo, indangarubuga ihuza, na C-terminus ya ROBO1 (Ishusho 4A, E).Ku rundi ruhande, imikoranire n’uturere duhuza DI, DIII, na OAS3 yatanzwe muri poroteyine ya ROBO1 (Ishusho 4A).
Synthase ya oligoadenylate (OAS) umuryango wa poroteyine wemera kandi ugahuza RNA (dsRNA) ingirangingo ebyiri, igahinduka, kandi igahuza 2 ′, 5′ ihuza oligoadenylates (2-5 As) 40.Byagaragaye ko muri OAS eshatu, OAS3 yerekana isano iri hejuru ya dsRNA kandi ikomatanya umubare muto wa 2-5 As, ishobora gukora RNase L bityo bikagabanya kwandura virusi 41.Umuryango wa OAS ugizwe na polymerase beta (pol-β) isa na nucleotide yohereza.Ubushakashatsi bwibanze bwerekanye ko ibikorwa bya catalitiki ya C-terminal (DIII) biterwa na dsRNA-ihuza indangarubuga (DI), isabwa kugirango ibikorwa bya OAS342 bikorwe.Twabonye ko domeni ya DI na DII ya OAS3 ikorana na CCD n'akarere gato gahuza SH2 na STAT1 TAD (Ishusho 4A, F).Gupfukirana imbuga zinyuranye zihuza imiterere ya poroteyine byagaragaje imikoranire hagati ya et-urupapuro na DBD STAT1 izenguruka hamwe nu mufuka ufunguye cyangwa umwobo wakozwe n ibisigazwa 60-75 muri DI domaine ya OAS3 (Ishusho 4G).Icyerekezo cya poroteyine ziri muri urwo ruganda cyerekanaga kandi ko nta mikoranire na OAS3 yabangamiye ubushobozi bwa ADN bwo guhuza imbaraga za domeni ya DI (Ishusho S1A).Mubyongeyeho, N-terminal ya GTPase MX1 ikorana cyane na DI na DIII ya OAS3 (Ishusho 4A).Twabonye kandi imikoranire hagati ya OAS1 na MX1 muburyo butatu bwa IFNα bwakorewe inshuro, aho indangarubuga imwe ya OAS1 (nayo ikora catalitiki) yakoranye na domaine zose uko ari eshatu MX1 (Igicapo S2A, B).
Intungamubiri za MX ziri mu muryango mugari wa Dynein imeze nka GTPase irimo N-terminal GTPase ya NTP ihuza na hydrolyzes GTP, urwego rwagateganyo ruhuza kwishyira hamwe, hamwe na C-terminal leucine zipper ikora nka GTPase (LZ ).indangarubuga ya domaine25,43.MX1 ihuza subunits ya polymerase ya virusi kugirango ibuze kwanduza virusi ya virusi43.Imisemburo yavuzwe mbere yerekana ibice bibiri-bivangavanze yerekanaga ko PIAS1 ifitanye isano na MX1 ibuza gukora gene ya STAT1 yunganirwa no guhagarika ibikorwa bya ADN ihuza kandi ifite ibikorwa bya SUMO E344,45.Hano, twerekana ko MX1 ihuza na STAT1 (Igicapo 4C, D), icyakora uburyo iyi mikoranire igira ingaruka kumikorere ya gene-STAT1 yunganirwa mugusubiza IFNα ikeneye ubushakashatsi bwimbitse.Twongeyeho, twasanze kandi MX1 yarakoranye na IFIT3 na DDX60 muburyo butatu bwa IFNα bwakorewe (Isanamu S2C).
DDX60 ni IFN iterwa na cytoplasmeque helicase yavuzwe mbere igira uruhare mukwangirika kwa RIG-I kwigenga kwa virusi RNA46.Ihuza na RIG-I kandi ikora ibimenyetso byayo muburyo bwihariye bwa ligand 46. DDX60 igizwe numuyoboro wa DEXD / H-Box hamwe na C-terminal ya helicase ihuza virusi RNA na ADN47.Byinshi mubikorwa byayo na MX1 na IFIT3 bibera muntara ndende ya N- na C-terminal idafite domaine yemewe cyangwa motif (Ishusho S2E, F).Ariko, MX1 nayo ifitanye isano na DEXD / H-Agasanduku ka helicase (Ishusho S2E).Poroteyine z'umuryango wa IFIT zifite kopi ya tandem ya motif yihariye ya helix-ihinduka-helix motif yitwa tetrapeptide gusubiramo (TPR).IFIT3 wasangaga ari modulator nziza ya RIG-I amarenga bityo rero igice cyikigo cya MAVS.Dufatiye hamwe, amakuru yacu yerekana ko IFIT3 na DDX60 zikorana cyane cyane mukarere kari hagati ya TPR 3–6 ya IFIT3 kandi zishobora kugira uruhare mukumenyesha RIG-I / MAVS (Ishusho S2F).
Urebye ko gusuzuma proteome yose yibara cyane, twagenzuye data base yumuntu UniProt kugirango habeho imwe muri IFNα ivurwa.Muri iyi kopi, twasanze imiyoboro myinshi yizewe cyane ya HLA-A.Isesengura ryinzira za poroteyine zagaragajwe na MS / MS yerekanwe ko MHC-I ishingiye kuri antigen gutunganya no kwerekana ari inzira nyamukuru iterwa na interferon (Ishusho 3D).Kubwibyo, twibanze ku kwiga imikoranire ya poroteyine ya molekile ya MHC-I hamwe n’icyizere cyo hejuru mu byitegererezo byose bifitanye isano.HLA igizwe na α1, α2 na α3 domaine n'iminyururu yoroheje, na microglobuline β2 (β2m) ni proteine ​​ihoraho ya chaperone49.Iyo HLA imaze guteranyirizwa muri retopulum ya endoplasme, HLA ntigihinduka mugihe habuze peptide ligands50.Peptide-binding groove ikorwa na polymorphique cyane kandi itubatswe α1 na α2 domaine muburyo butari peptide hamwe na polymorphique α351 ugereranije.Imbere ya IFNα, twabonye ibigo bibiri bya HLA-A: imwe ikorana na HMGA1 na H2B (Ishusho 5, Imbonerahamwe S3) indi ikorana na MDN1, LRCH4 na H2B (Ishusho 6).
IFNα itera umuyoboro wa HLA-A ukorana na H2B (H2BFS) na HMGA1...Imiterere yindangamuntu zitandukanye ni amabara yanditseho32: H2B (histone; 2-102) na MHC-I (MHC_1; 25–203, itsinda C1; 210-2290 na MHC_I_C; 337–364).Imipaka ihuza imipaka yashyizwe kuri 3.5.Ibibanza byerekana utudomo byerekana HLA-A imbuga za H2B (B) na HMGA1 (C), hamwe na K cyangwa S imikoranire hagati ya peptide zombi.Igishushanyo, amanota yambukiranya amanota yashyizwe kuri 3.0.(D) Isano iri hagati ya poroteyine zerekanwa mu miterere ya poroteyine H2B, HLA-A, na HMGA1 muri gahunda ya PyMOL.Izi nyubako zagereranijwe hifashishijwe seriveri ya Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) naho inyandikorugero ya poroteyine ya H2B, HLA-A na HMGA1 yari 1kx552, 1kj349 na 2eze55.
IFNα itera umuyoboro wa HLA-A imikoranire na H2B (H2BFS), MDN1 na LRCH4..Imipaka ihuza imipaka yashyizwe kuri 3.5.Imiterere yindangamuntu zitandukanye ni amabara yanditseho32: H2B (histone; 2-102), MHC-I (MHC_1; 25–203, itsinda C1; 210-2290 na MHC_I_C; 337–364) na LRCH4 (LRR_8 (68–126), LRR_8 (137–194) na CH (535–641)).(B) Isano iri hagati ya poroteyine zerekanwa mu miterere ya poroteyine H2B, HLA-A, LRCH4, na MDN1 muri gahunda ya PyMOL.Izi nyubako zagereranijwe hifashishijwe seriveri ya Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) ifite inyandikorugero 1kx552, 1kj349, 6hlu62 na 6i2665 kuri poroteyine za H2B, HLA-A, LRCH4 na MDN1, bikurikiranye.Utudomo twerekana K cyangwa S imbuga zikorana na HLA-A hamwe na H2B (C), LRCH4 (D), na MDN1 (E).Kubibanza, amanota yambukiranya imipaka yashyizwe kuri 3.0.
Usibye gukomeza ubusugire bwa genome, histone H2B nayo igira uruhare mugutunganya inyandiko.Poroteyine ya H2B igizwe na domeni yo hagati (HFD) igizwe na α-helices eshatu zitandukanijwe nu muzingo n'umurizo wa C-terminal 41,52.Byinshi mubikorwa na H2B biboneka muri α1 helix, itanga trimerisation hamwe na heterodimer ya HFD (Ishusho 5A, B).Nubwo lysine igira uruhare muguhuza ADN, lysine zimwe na zimwe nizindi mbuga za acetylation cyangwa methylation.Kurugero, ibisigisigi K43, K46, na K57 kuva H2B ntabwo bigira uruhare muguhuza ADN itaziguye, ahubwo ni intego yibyahinduwe nyuma yinyandiko mvugo53.Muri ubwo buryo, ibisigisigi K44, K47, na K57 muri H2B birashobora kugira urundi ruhare imbere ya IFNα, harimo imikoranire nizindi poroteyine (Ishusho 5A, B).Byongeye kandi, extrachromosomal histone H2B itangiza ubudahangarwa bw'umubiri mubwoko butandukanye, ikora nka sensor ya cytosolike kugirango imenye ibice bibiri bya ADN (dsDNA) biva mubice byanduye cyangwa selile byangiritse 54.Imbere ya virusi ya ADN, kugabanuka kwa H2B kwabujije umusaruro IFN-β na fosifori ya STAT154.H2B izwi kandi kwimuka no gusohoka muri nucleus byihuse kuruta andi mateka yibanze 54.Imikoranire ya H2B na MDN1 na LRCH4 yagaragaye no mubitegererezo byatoranijwe bitavuwe.Twabonye ko HLA-A yakoranye na H2B muri sample zose uko ari eshatu zavuwe na IFNα no murugero rumwe rutavuwe.Aya makuru yerekana uruhare rwa H2B mubindi bikorwa bya physiologique bitagendeye kumyandikire.
HMGA1 (itsinda rinini cyane AT-Hook 1), nucleoproteine ​​ntoya ikungahaye kuri aside amine itera indwara, yamenyekanye ifatanije na HLA-A.Ifite umurizo C-terminal umurizo hamwe na DBD eshatu zitandukanye zitwa AT hook kuko zihuza umwobo muto wo mukarere gakungahaye kuri AT muri dsDNA55,56.Uku guhambira gutuma ADN yunama cyangwa igororotse, ituma ibintu byandikirwa byemewe byinjira muburyo bwumvikanyweho.Umurizo wa C-terminal wizera ko ugira uruhare mubikorwa bya poroteyine-proteyine no kwinjiza ibintu byandikirwa, kubera ko ihinduka rya C-terminal ridashobora gutangiza transcription57.Byongeye kandi, iyi domeni ikubiyemo imbuga nyinshi za fosifora zabitswe zizwiho insimburangingo ya kinase 58.Twabonye imikoranire ya HLA-A na H2B na HMGA1 hanze ya C-terminal, byerekana ko indangarugero ya C ikoreshwa cyane cyane muguhuza inyandiko (Ishusho 5A, C).Poroteyine za HMGA zirushanwa na histone H1 kugirango zihuze na ADN ya adapt, bityo byongerwe kuboneka57.Muri ubwo buryo, birasa nkaho HMGA ikorana na histone H2B ihuza ADN ihuza na histone H1.HMGB1 itera imvugo ya HLA-A, -B, na -C mu ngirabuzimafatizo ya dendritic, bigatuma bakora 59, ariko imikoranire hagati ya HMG na HLA ntabwo yigeze itangazwa.Twasanze HMGA1 ikorana na α1 na α3 domaine ya HLA-A, hamwe nimikoranire myinshi hanze ya DBD yayo 3 (Ishusho 5A, C).Mu biganza byacu, HLA-A wasangaga iherereye muri nucleus (amakuru aterekanwa), kandi urebye ko H2B na HMGA1 nabo bahari muri nucleus, iyi mikoranire ishobora kuba muri nucleus.Ibyongeweho byapimwe hagati ya H2B, HLA-A, na HMGA1 byerekanwe mubishusho 5D.
Imikoranire myinshi ya HLA-A hamwe nizindi poroteyine ziboneka muri domaine zayo α1 na α2 hamwe na C-terminal idahwitse (Ishusho 6).Muri rumwe muri izo ngero, twasanze HLA-A ikorana n'umurizo wa N-terminal utameze neza wa LRCH4 (Ishusho 6A, D).LRCH4 igenga ibikorwa bya TLR4 hamwe na LPS cytokine, bityo igahindura ubudahangarwa bw'umubiri 60,61.Ni poroteyine ya membrane ifite icyenda ikungahaye kuri leucine (LRRs) hamwe na motif ya homoduline (CH) ya homologiya muri ectodomain yayo, ikurikirwa na transembrane (TMD) 60, 62.Imiyoboro ya CH yavuzweho guhuza poroteyine-proteyine 60.Kurambura aminide acide igera kuri 300 hagati ya LRR na CH birashoboka cyane ariko birangiritse.Dushingiye ku mikorere y'uturere duhungabanye nk'abunzi b'imiyoboro ya poroteyine-protein hamwe no gutwara imitsi ya 63, twasanze imikoranire ya poroteyine ibera mu turere dufite ibibazo.Imikoranire na MDN1 yatanzwe mu burebure bwa poroteyine, harimo LRR1, LRR6, CH domaine, hamwe n’uturere tutabishaka, mu gihe H2B ihuza cyane na domeni ya CH (Ishusho 6A, B).Ikigaragara ni uko, nta nimwe mu mikoranire yarimo TMJ, yerekana umwihariko w'uburyo bwa CLMS (Ishusho 6A, B).
MDN1 yamenyekanye kandi nk'urwego rwa poroteyine ya HLA-A (Ishusho 6A).Ni iyumuryango wa AAA ya proteine ​​(ATPase ijyanye nibikorwa bitandukanye).Nuburyo bumwe N-terminal AAA indangarugero itegura impeta ya hexameric kandi ikuraho ibintu byo guterana muri 60S 64 ribosomal subunit.bigaragara ko isa na dynein 64,65,66.Mubyongeyeho, akarere gakize ka Asp / Glu gakurikirwa na MIDAS (urubuga rwa ion rushingiye).Bitewe nubunini bwa MDN1 (hafi 5600 aminide acide) hamwe na homologiya ntoya hamwe na poroteyine zize neza, bike bizwi ku miterere n'imikorere yabantu.Twabonye HLA-A, H2B, na LRCH4 nkabafatanyabikorwa ba MDN1 kandi twerekanye icyerekezo cyabo nkibintu bya poroteyine muri PyMol (Ishusho 6A, B).Izi poroteyine eshatu zikorana na domaine ya AAA, indangarubuga isa na dynein, hamwe na MIDAS MDN1.Muri raporo yabanjirije iyi, kweza intungamubiri za poroteyine byerekanaga MDN1 nka poroteyine ifitanye isano na histone H2B67.Byongeye kandi, ubushakashatsi buherutse kandi bwagaragaje imikoranire hagati ya MDN na HLA-B mu tugari twa HCT116 hakoreshejwe uburyo bwa sprometrometrike yanduye, bishyigikira ibyo twabonye68.Kumenyekanisha uru ruganda muri IFNα zavuwe byerekana urugero rwa MDN1 mukumenyesha interferon.
Kuberako gen ya HLA ifite polymorphique cyane, twakuyemo urutonde rusoma ikarita ya HLA-A, -B, na -C uhereye kuri RNA ikurikirana ya selile Flo-1 (amakuru aterekanwa).Urutonde rwa peptide rujyanye no gusoma rukurikiranye rwerekanye itandukaniro rikomeye hagati ya HLA-A, -B, na -C mu turere aho peptide ihuza peptide yari i HLA-A (Ishusho S3).Twongeyeho, ntitwabonye poroteyine-kuri-poroteyine ihuza molekile ya HLA-B / C na poroteyine H2B / HMGA1 / MDN1 / LRCH4.Ibi birerekana ko imikoranire ya poroteyine iboneka hagati ya HLA-A, MDN1, LRCH1 na HMGA1 ari HLA-A yihariye.Byongeye kandi, isesengura rya proteomic ryerekana ingero zidahujwe (Imbonerahamwe S4) ryerekanye ko HLA-A ifite urwego rukurikiranye ugereranije na HLA-B cyangwa HLA-C.Peptide yagaragaye kuri HLA-A yari ifite ubukana bwinshi muri IFNα ivurwa kandi itavuwe.
Kugirango tumenye neza ko imikoranire yagaragaye hano itatewe no guhuza kwihariye kwa poroteyine ebyiri hafi y’ahantu hegereye, twakomeje kwemeza ibintu bibiri bishya bya HLA-A bikorana n’ubushakashatsi bwakozwe na immunoprecipitation.Imikoranire ya HLA-A na MDN1 ya endogenous MDN1 na H2B byagaragaye muri selile zombi za IFNα zivuwe kandi zitavuwe (Ishusho 7, Ishusho S4).Twemeje ko HLA-A yafashwe na H2B muri immunoprecipitates kandi ko iri shyirahamwe ryatewe no kwivuza IFNα kuva HLA-A idahari mu ngero z’ubudahangarwa ziva mu ngirabuzimafatizo zitavuwe (Ishusho 7A).Ariko, amakuru yacu yerekana ko IFNα itandukanya HLA-A ihuza H2B na MDN1.IFNα itera ishyirahamwe hagati ya H2B na HLA-A, ariko igabanya ubufatanye na MDN1.Twasanze MDN1 yari ifitanye isano na HLA-A mugenzuzi, kandi kongeramo IFNα byagabanije imikoranire idashingiye ku kwinjiza MDN1 na IFNα (Ishusho 7B, C).Byongeye kandi, HLA-A immunoprecipitation yafashe H2B muri selile A549 (Igicap. S4), byerekana ko iyi mikoranire idashingiye ku bwoko bwakagari.Ufatiye hamwe, ibisubizo bishyigikira interferon-ihuza imikoranire ya HLA-A na H2B na MDN1.
HLA-A ifatanya H2B na MDN1.Abahagarariye endogenous H2B (A) na MDN1 (B) immunoblots bakingiwe indwara ya selile ya IFNα yavuwe na Flo-1 hanyuma basuzumwa antibodi zerekanwe.Imbeba ninkwavu IgG byakoreshejwe nkigenzura ribi..
Imiterere yimiterere yimwe muma interferon-iterwa na interineti yizewe cyane ihuza imiyoboro ihuza H2B-HLA-A-HMGA1.Twifashishije ingengabihe ya molekulari yerekana ubundi buryo bwo gusobanukirwa imbaraga za poroteyine zigira uruhare muri uru ruganda (Ishusho 8).Imyanzuro yavuye mu makuru ya CLMS yerekana ko bishoboka guhuza poroteyine za H2B, HLA-A, na HMGA1.Kubwibyo, ibigo bikurikira bishobora kugereranywa muburyo bukemuka: H2B-HLA-A, HMGA1-HLA-A, na H2B-HLA-A-HMGA1.Porogaramu ya mbere ya poroteyine-poroteyine ikoresheje MOE (Molecular Operating Environment; Chemical Computing Group Inc, Montreal, Quebec, Kanada) yerekanaga uburyo bushoboka butandukanye hagati ya poroteyine (Ishusho 8A).Kwerekana amashusho ya poroteyine ya docking yerekanaga imikoranire myinshi hamwe nibishoboka (Ishusho 5A, 8).Rero, ihinduka rimwe rishoboka ryerekanwa mu gishushanyo cya 8A (gifite ibimenyetso byambukiranya imipaka) kandi byongeye gusuzumwa hifashishijwe umuyoboro wa moderi wa MD.Byongeye kandi, guhuza H2B cyangwa HMGA1 na HLA-A byerekana isano iri hejuru ya H2B kuri HLA-A (Ishusho 8A).
Imikorere ihuza imiyoboro ishoboka hagati ya H2B (H2BFS) -HLA-A, HMGA1-HLA-A, na H2B-HLA-A-HMGA1..Byongeye kandi, ibisigisigi bihuza ibisigara byamenyekanye byanditse ku miterere ya poroteyine H2B, HLA-A, na HMGA1.Ihuriro rifitanye isano na poroteyine ryakuwe hifashishijwe umuyoboro wa docking washyizwe muri MOE.Umwanya wibumoso wo hepfo werekana uburyo butandukanye bushoboka bwa H2B-HLA-A na HMGA1-HLA-A hamwe nibintu bitandukanye bya poroteyine-protein bihuza (GBVI / WSA dG; kcal / mol).(B) Gutandukana bisanzwe (RMSD) byimyanya ya atome (ukuyemo atome ya hydrogen) kuri buri poroteyine..Intera ya h-bond itanga-intera yagabanijwe yashyizwe kuri 3.5 Å, naho umuterankunga-H-yakira impande zose zashyizwe kuri ≥ 160 ° –180 °..Imiterere ya poroteyine yerekana impuzandengo ya 100 ns MDS..Ibigo / HMGA1-HLA-A / H2B-HLA-A-HMGA1.Agaciro ntarengwa kugirango isuzumabumenyi ryambukiranya ryashyizwe kuri 3.0, kandi imikoranire yihariye ya MDS ifata ns 10 ns yafashwe.Imiterere ya poroteyine yagaragaye hifashishijwe sitidiyo ya BIOVIA (Dassault Systèmes, BIOVIA Corp., San Diego, CA, Amerika) hamwe n’ibidukikije bikora (MOE; Chemical Computing Group Inc, Montreal, Quebec, Kanada).
Ihungabana rya molekile ya HLA-A mugihe (gutandukana bisanzwe; RMSD cyangwa gutandukana bisanzwe; RMSF) byagaragaje ko kuba proteine ​​za H2B cyangwa HMGA1 mubigo byahagaritse HLA-A (Ishusho 8B, Igicapo S5).Poroteyine ya HMGA1 ihuza cyane na B2M ya HLA-A, bigatuma ituze rya HLA-A amino acide mu kigo cya HLA-A-HMGA1 cyangwa H2B-HLA-A-HMGA1 (Ishusho 8B, Ishusho S5).byumwihariko, ibisigazwa bya HLA ~ 60-90 na ~ 180-210 wasangaga bidahinduka imbere ya H2B (FIG. 8B).H2B na HMGA1 berekanye neza guhuza HLA-A mu kigo cya H2B-HLA-A-HMGA1 ugereranije na HLA-A ihuza H2B cyangwa HMGA1 yonyine (Ishusho 8C, D; Imbonerahamwe S5).Ibisigarira bigira uruhare muguhuza hydrogène (MD yerekana umwanya munini ≥ 10 ns) bihura nimbuga za CLMS (ibisigisigi bya K cyangwa S) murwego, byerekana ko imikoranire yagaragajwe na CLMS yizewe cyane.Kwizerwa (Isanamu 8E).Mu kwerekana imiterere ya CLMS na MD, ibisigazwa bya HLA-A hagati ya 190-210 na aside amine hafi 200-220 wasangaga bihuza H2B na HMGA1, (FIG. 8E).
Imikoranire ya poroteyine na poroteyine ikora imiyoboro ihuza imiterere ihuza itumanaho ridasanzwe mu gusubiza ibintu bimwe na bimwe bitera imbaraga.Kuberako uburyo bwinshi bwa proteomics bwerekana impinduka murwego rusange ruhagaze neza rwa poroteyine, imbaraga za protein-protein zikorana imbaraga zisaba ibikoresho byinyongera byo gufata intera ihuza, kandi CLMS nimwe mubikoresho nkibi.Sisitemu yerekana ibimenyetso bya interferon numuyoboro wa cytokine utuma ingirabuzimafatizo zisubiza ibimenyetso bitandukanye byangiza ibidukikije byangiza ibidukikije, bikarangirira no kwinjiza uduce duto twa poroteyine za interferon.Twakoresheje CLMS kugirango tumenye niba imikoranire ya poroteyine-protein ishobora kumenyekana mu itsinda rya poroteyine ziterwa na interferon.Isesengura rya poroteyine ku isi yose mu isesengura rya interferon-isubiza Flo-1 selile yakoreshejwe mu gufata poroteyine.Gukuramo peptide ya tryptic ivuye mu ngirabuzimafatizo zidahuza kandi zifasha kwemerera kubara peptide, gutunganya inzira, no gukwirakwiza peptide hamwe nubusobanuro bwa LFQ.Intungamubiri za Canonical interferon-inducible proteine ​​zagaragaye nkigenzura ryiza ryimbere, mugihe hagaragaye imiyoboro mishya ya intermolecular na intramolecular ihuza imiyoboro ya poroteyine za kanoniki interferon-zidashoboka nka MX1, UP18, OAS3 na STAT1.Hakozwe iperereza ku bintu bitandukanye byubatswe n’imikoranire mu bice bikora.
Imikoranire hagati ya HLA-A, MDN1 na H2B yagaragaye ikingira immunoblotting muri selile Flo-1 na A549 zavuwe kandi zitavuwe na IFNα.Ibisubizo byacu byerekana ko HLA-A igizwe na H2B muburyo bwa IFNα.Igikorwa cacu kirerekana inzira ishimishije yo gukomeza gushakisha ubufatanye bwahantu hamwe.Byaba byiza kandi kwagura uburyo bwa CLMS kumwanya wumurongo wimikorere kugirango tumenye selile-yigenga-interferon-yunganirwa na poroteyine.Hanyuma, twakoresheje icyitegererezo cya MD nk'ubundi buryo bwo gusobanukirwa imbaraga za poroteyine zigira uruhare mu ruganda rwa H2BFS-HLA-A-HMGA1, rwakurikiranaga ibiganiro hagati y’ibiganiro na intermolecular.Imyanzuro iva mu makuru ya CLMS yerekana ko bishoboka guhuza poroteyine za H2BFS, HLA-A, na HMGA1.Ihinduka rishoboka hagati yibi bikoresho bya poroteyine byerekanaga imikoranire myinshi isa niyagaragaye muri dataset ya CLMS.Imwe mumbaraga zingenzi zuburyo bwacu nuko itanga uburyo bworoshye bwo kumenya imikoranire ya gen polymorphique cyane nka HLA, bityo bizaba bishimishije kwiga imikoranire ya proteine ​​yihariye ya HLA haplotype ubundi bigoye kwiga.Dufatiye hamwe, amakuru yacu yerekana ko CLMS ishobora gukoreshwa mugutahura neza imiyoboro yerekana ibimenyetso biterwa na interferon kandi ikanatanga urufatiro rwo kwiga sisitemu igoye cyane hagati yimitsi yibidukikije.
Ingirabuzimafatizo za Flo-1 zabonetse muri ATCC kandi zibungabungwa muri DMEM (Gibco) hiyongeraho 1% penisiline / streptomycine (Invitrogen), 10% ya serumu y'inda y'inda (Gibco) ibikwa kuri 37 ° C na 5% CO2.Inkubation.Ingirabuzimafatizo zarakuze zigera kuri 70-80% mbere yo kuvurwa na IFNα14 (yakozwe na Edinburgh Protein Production Facility).Indi miti yose hamwe na reagent byaguzwe muri Sigma Aldrich keretse bivuzwe ukundi.
Flo-1 selile yarezwe mumasahani 6-bukeye bukeye ingirabuzimafatizo zavuwe hamwe na 10 ng / ml IFNα14 mumasaha 24 kugeza hafi 80%.Ingirabuzimafatizo zogejwe inshuro eshatu hamwe na PBS hanyuma zomekwa kuri DSS nshya (Thermo Fisher Scientific) (yashonze muri DMSO) muri PBS muminota 5 kuri 37 ° C. kugeza kuri 0.5 mM.Imyitwarire ya DSS yasimbujwe PBS hanyuma DSS isigaye yazimye hongerwaho 20 mM Tris (pH 8.0) muri PBS muminota 15 kuri 37 ° C.Ingirabuzimafatizo zegeranijwe no gusiba hanyuma zegeranijwe mu tubari duto duhuza (Axygene).
Pellet selile yatewe hamwe na 300 µl ya urea lysis buffer (8 M urea, 0.1 M Tris, pH 8.5) muminota 30 mubushyuhe bwicyumba hamwe no kunyeganyega rimwe na rimwe.Intambwe zose za centrifugation zakozwe kuri 14,000 xg kuri 8 ° C.Centrifuge lysate muminota 10 hanyuma wohereze supernatant kumuyoboro mushya.Ibice bisigaye bisigaye byashongeshejwe muri 150 μl ya kabiri ya lysis buffer (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) muminota 30 cyangwa irenga kugeza habonetse igisubizo cyamazi yo mumazi.Lysate yashizwemo iminota 20 hanyuma ndengakamere ivangwa na lysate yabonetse muntambwe ibanza.Intungamubiri za poroteyine zapimwe hifashishijwe Micro BCA (Thermo Fisher Scientific) ukurikije amabwiriza yakozwe na microplate.Ingero zahise zikonjeshwa muri azote yuzuye kandi zibikwa kuri 80 ° C.
Hafi ya 100 μg za poroteyine zishongeshejwe zahujwe zakozwe hifashishijwe porogaramu yahinduwe yo kuyungurura icyitegererezo (FASP) nkuko byasobanuwe na Wisniewski n'abandi.69 Muri make, poroteyine ihujwe na 200 µl ya buffer ya urea (8 M urea muri 0.1 M Tris, pH 8.5), ikazunguruka kandi ikagabanywa kabiri.Intambwe zose za centrifugation zakozwe kuri 14,000 xg kuri 25 ° C.Igice cya mbere cya protein lysate ihujwe yimuriwe muri kDa 10 ya Microcon centrifugal filter igikoresho gifite Ultracel-10 membrane (Merck), hanyuma ikurikirwa na centrifugation kuri filteri muminota 25.Noneho ongeramo igice cya kabiri cya poroteyine muyungurura hanyuma usubiremo intambwe imwe.Kugarura poroteyine byakozwe hongerwaho 100 μl ya 17 mM tris (2-carboxyethyl) fosifine hydrochloride (TCEP) muri buffer ya urea.Isubiranamo ryashyizwe kuri thermomixer kuri 600 rpm kuminota 30 kuri 37 ° C.Mubyongeyeho, inkingi yashizwemo kandi igabanywa rya poroteyine yagabanijwe ihuza alkylated ukoresheje 100 μl ya 50 mM iodoacetamide muri buffer ya urea.Alkylation reaction yakorewe mubushyuhe bwicyumba muminota 20 mwijima.Kuzenguruka inkingi, koza inkuta zinkingi inshuro 3 hamwe na 100 µl urea buffer, hanyuma centrifuge.Igikorwa kimwe cyakozwe inshuro 3 ukoresheje 100 μl ya 100 mM ammonium bicarbonate.Mbere yo kugerageza, simbuza icyegeranyo cyo gukusanya ikindi gishya.Ongeramo buffer igogora irimo 50 mM ammonium bicarbonate na 1 µl trypsin ivangwa muri buffer ya trypsin (Promega).Ikigereranyo cya trypsin na poroteyine cyagumishijwe nka 1:33, kandi igogorwa ryigogorwa ryakozwe ijoro ryose kuri 37 ° C. mucyumba cyuzuye.Peptide ihujwe yakuwe muyungurura na centrifugation muminota 25.Kugarura Peptide byatejwe imbere wongeyeho 50 μl ya 0.5 M NaCl muyungurura, hakurikiraho centrifugation muminota 25.
C18 Inkingi ya Micro Spin (Harvard Apparatus) yakoreshejwe mugukuraho peptide ya tryptic peptide ikurikira protocole yasobanuwe na Bouchal nabandi 70 bahinduye bike.Muri make, C18 spin inkingi yakoreshejwe hamwe no gukaraba bitatu bya 0.1% acide formique (FA) muri acetonitrile (AcN) (Merck) hamwe no gukaraba bibiri bya 0.1% FA.Inkingi yahujwe na 0.1% FA muminota 15.Shira ingero muri spin inkingi hanyuma ukarabe inshuro 3 hamwe na 0.1% FA.Peptide yanduye yakuwe muburyo bukurikiranye buhoro buhoro ukoresheje 50%, 80% na 100% AcN muri 0.1% FA.Ingero zumishijwe mumashanyarazi ya SpeedVac Plus (Eppendorf) kugeza amazi asigaye yabuze burundu.Peptide yanduye yashongeshejwe muri 100 μl ya 0,08% ya acide trifluoroacetic muri 2,5% AcN kandi ibipimo byapimwe kuri NanoDrop 2000 (Thermo Scientific).Hafi ya 1 μg ya peptide ihujwe kuri buri cyitegererezo yatewe muri sisitemu ya LC-MS / MS.
Peptide ihujwe yatandukanijwe kuri sisitemu ya UltiMate 3000 RSLCnano LC (Thermo Scientific) ihujwe na Orbitrap Exploris 480 ya mass spectrometer (Thermo Scientific).Peptide ihujwe yakusanyirijwe kuri 300 µm ID, mm 5 z'uburebure µ-mbere-inkingi ya C18 ifata inkingi yuzuye C18 PepMap100 sorbent na 5 µm PepMap sorbent (Thermo Scientific).Shyiramo pompe itemba yashyizwe kuri 5 µl / min 0.08% acide trifluoroacetic yashonga muri 2,5% AcN.Peptide ihujwe yatandukanijwe kumurongo wisesenguye wa silika hamwe na diametre yimbere ya 75 mkm nuburebure bwa mm 150, yuzuyemo 2 mμm PepMap sorbent (Thermo Scientific).Icyiciro kigendanwa A na B cyari kigizwe na 0.1% FA mumazi na 0.1% FA muri acetonitrile.Ikigereranyo gitangirira kuri 2,5% B kandi kiyongera kumurongo kugeza kuri 40% B muminota 90, hanyuma ukagera kuri 90% B muminota 2 iri imbere.Igice cya mobile kigizwe na 90% B muminota 10 hanyuma kigabanuka kumurongo kugera kuri 2,5% B muminota 2.Inkingi yagereranijwe kuri 2,5% B muminota 8 mbere yizunguruka.Peptide ihujwe na peptide yavuye mu nkingi zisesenguye zashyizwe mu isoko rya nanoelectrospray ionisation (NSI) hanyuma zinjizwa muri Exploris 480 mass spectrometer (Thermo Scientific).
Orbitrap Exploris 480 mass spectrometer ikora muburyo bwiza bwo guhuza amakuru.Isuzuma ryuzuye ryakozwe muburyo bwigice ku cyemezo cya 120.000 hamwe nurwego ruri hagati ya m / z 350 Th kugeza m / z 2000 Th.Intego isanzwe ya AGC yashyizwe kuri 300% hamwe nigihe kinini cyo kwinjiza 50ms.Monoisotopic peak detection yashyizweho kuri peptide.Imbogamizi yo kuruhuka ibipimo byashyizwe mubyukuri niba bike bibanziriza kuboneka.Imbaraga ntoya ya ionic ya preursor yashyizwe kuri 5.0e3 naho prursor yishyuza leta igera kuri +8 yashyizwe mubushakashatsi.
Igihe cyizunguruka hagati ya scan nini muburyo bwo guhuza amakuru yashyizwe kumasegonda 2.5.Dynamic mass exclusion yashyizwe kuri 20 s nyuma yo gutandukana kwambere kwa ion ibanziriza.Idirishya ryabanjirije kwigunga ryashyizwe kuri 2 Th.Ubwoko bwingufu zisanzwe zo kugongana hamwe nuburyo butajegajega bwo kugongana bwatoranijwe muri data ishingiye kuri scan ya MS / MS.Ingufu zo kugongana zashyizwe kuri 30%.Icyemezo cya Orbitrap cyashyizwe kuri 15.000 naho intego ya AGC igera 100%.Igihe ntarengwa cyo gutera inshinge gishyirwa kuri milisegonda 60.
Mbere yo gukurikirana urusobe rwa poroteyine-poroteyine mu ngero zifitanye isano, twatunganije amadosiye mbisi dukoresheje paki ya MaxQuant (verisiyo 1.6.12.0) 26.27 kugira ngo tumenye peptide / proteyine zishobora gukurikiranwa.Byongeye kandi, isesengura risa na proteomic ryakozwe ku ngero za Flo-1 zidahuye zavuwe kandi zitavuwe hamwe na IFNα.Amakuru ya MS / MS yashakishijwe mu bubiko bw’abantu UniProt (www.uniprot.org) (yashyizwe ku ya 12 Kanama 2020, arimo ibyanditswe 75.093) akoresheje moteri ishakisha Andromeda27.Ubushakashatsi bwakozwe buterekanye umwihariko wa enzyme no guhindura ibintu bitandukanye bya deamidation (N, Q) na okiside (M).Precursor kwihanganira imbaga yashyizwe kuri 20 ppm na ion yibicuruzwa kuri 0.02 Da.Itangiriro kandi ntarengwa ryo gutandukana ryashyizwe kuri 10 ppm.Umubare ntarengwa wa peptide washyizwe kuri 4600 Da naho isano ikurikirana yashyizwe hagati ya acide amine 7 na 25 (aa).Ubundi isesengura mibare ryakozwe hakoreshejwe gahunda ya Perseus (verisiyo 1.6.10.45).Intungamubiri za poroteyine zabazwe mu guhuza ubukana bwa poroteyine (ubukana bwa LFQ; umubare utabigenewe) 27 kandi agaciro k’imbaraga zahinduwe kuri Log2.Ihuriro rya poroteyine zagaragajwe nuburemere bwa peptide zubatswe hifashishijwe pheatmap (v1.0.12) muri R (v 4.1.2).Isesengura ryitunganyirizwa ryinzira ryakozwe hifashishijwe ububiko bwinzira nyabagendwa ya poroteyine zavuwe na IFNα zakozwe inshuro zirenga enye ugereranije n’intangarugero zitavuwe.
Kumenyekanisha lysine (K) cyangwa serine (S) ihuza imiti yihariye ya protein igenzurwa na LC-MS / MS yakozwe hakoreshejwe imashini imenyekanisha spekitroscopique (SIM-XL) kuri peptide ihuza (SIM-XL) 29.Ubwa mbere, imikoranire ishoboka hagati ya interferon ifitanye isano (IFN) ADN yangiza umukono wa IRDS (IRDS) hakoreshejwe iperereza ryakozwe na protein dataset ya IRDS yasobanuwe muri Padariya nabandi.28.Kugenzura ibintu byose no gusubiramo ibya UniProt yabantu byose birabaze cyane, kubwibyo data base yumuntu UniProt (www.uniprot.org) (yakuweho 12 Kanama 2020, ikubiyemo ibyanditswe 75.093) irwanya IFNα ivurwa.Imwe muyunguruzi yo kwizerana gukomeye.Iyi mikoranire-ikomeye cyane yabonetse yaguwe kandi igeragezwa mubisubiramo byose.
Muri SIM-XL, DSS yakoreshejwe kuri crosslinker (XL) naho guhinduranya ibiro XL no guhindura ibiro byashyizwe kuri 138.06 na 156.07.Imbuga zikurikira zifatika zifatwa: KK, KS na KN-TERM, nta banyamakuru ion.Byombi bibanziriza nibice ppm byashyizwe kuri 20 naho urwego Xrea rwashyizwe kuri 0.15.Trypsin yafatwaga nkaho idasanzwe, kandi uburyo bwo gucamo ingufu nyinshi C-trap (HCD) bwashyizwe mubikorwa.XCorr ifite imbaraga zo kugabanya DB igabanya umubare ntarengwa wa peptide yo kugabanya imbaraga za DB zashyizwe kuri 2.5 na 2.Ibindi bipimo ni: monoisotope ibishoboka no guhura nimpanuka yo kugabanuka, byibuze ibisigisigi 4 AA bisigara kumurongo hamwe nubunini ntarengwa, hamwe na 3 maxima yo gutandukana.Ikarita yavuyemo 2D yasesenguwe muri (SIM-XL) hanyuma ishusho ya xQuest28 ikoreshwa mu kubaka amakarita 2D.Guhuza poroteyine ku miterere ya poroteyine bitangwa muri PyMol (PyMOL Molecular Graphics Sisitemu, verisiyo 2.0 Schrödinger, LLC).
Imiterere ya poroteyine yakozwe hakoreshejwe seriveri ya Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)11 ikoresheje amahame yo kwerekana imiterere ya homologiya no gushyira mu bikorwa “Uburyo bwihishe Markov”.Phyre2 itanga imiterere yicyitegererezo ishingiye kumurongo ukurikirana hamwe na poroteyine izwi.Kuri H2BFS, HLA-A, HMGA1, LRCH4, na MDN1 za poroteyine, imiterere yicyitegererezo 1kx552, 1kj349, 2eze55, 6hlu62, na 6i2665.Hiyongereyeho, imiterere ya AlphaFold71 MX1, UBP18 na ROBO1 nayo yasuzumwe.Imiterere ya poroteyine yagaragaye hifashishijwe porogaramu ya BIOVIA Discovery Studio Visualizer (Dassault Systèmes, BIOVIA, San Diego, CA, Amerika) hamwe na Molecular Operating Environment pack (MOE; Chemical Computing Group Inc, Montreal, Quebec, Kanada).