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Enzymatique yegeranye yerekana uburyo bushingiye kuri ester ikora cyangwa fenokisi radicals ikoreshwa cyane mugushushanya proteomes subcellular proteines hamwe na poroteyine zikorana na selile nzima.Nyamara, ester ikora ntigikora cyane, bikavamo radiyo yagutse, kandi radicals ya phenoxy iterwa no kuvura peroxide irashobora kubangamira inzira ya redox.Hano turatanga raporo yegeranye yerekana uburyo bushingiye kumafoto (PDPL) bwakozwe muburyo bwo guhuza poroteyine ya miniSOG ya miniSOG na proteine yinyungu.Bitewe numucyo wubururu kandi bigenzurwa nigihe cyo kwerekana, umwuka wa ogisijeni ukorwa hanyuma ugahita ukemurwa kuranga ibisigisigi bya histidine na aniline probe iragerwaho.Turerekana ubudahemuka bwayo binyuze muri mape ya proteome yihariye.Kugereranya kuruhande rwa PDPL na TurboID yerekana uburyo bwihariye kandi bwuzuye bwa proteomic gukwirakwiza PDPL.Ubukurikira, twashyizeho PDPL kuri coactivator ya BRD4 na E3 Parkin ligase ifitanye isano n'indwara dusanga imikoranire itazwi.Mugusuzuma bikabije, ibice bibiri bitazwi, Ssu72 na SNW1, byamenyekanye kuri Parkin, iyangirika ryayo rikaba ryarahujwe ninzira ya proteasome.
Kuranga neza imiyoboro ya poroteyine ishingiye kubikorwa byinshi byingenzi.Kubwibyo, gushushanya neza spatiotemporal mapping ya proteine bizatanga umusingi wa molekuline yo gusobanura inzira yibinyabuzima, indwara y’indwara, no guhagarika imikoranire hagamijwe kuvura.Kugirango bigerweho, uburyo bushobora kumenya imikoranire yigihe gito muri selile nzima cyangwa ingirangingo zirifuzwa cyane.Gukwirakwiza Mass Mass Spectrometry (AP-MS) byakoreshejwe mumateka kugirango hamenyekane abafatanyabikorwa ba poroteyine zinyungu (POIs).Hamwe niterambere ryuburyo bwa proteomics, Bioplex3.0 yarakozwe, ububiko bunini bwimiyoboro ya poroteyine ishingiye kuri AP-MS.Nubwo AP-MS ifite imbaraga nyinshi, lysis ya selile nintambwe yo kugabanuka mubikorwa byogukora kubogamye kubikorwa byintege nke kandi byigihe gito kandi bigashyiraho ibihangano nyuma ya lysis nkibintu byombi bitagira aho bihurira mbere ya lysis.
Kugira ngo ibyo bibazo bikemuke, aside amine idasanzwe (UAA) hamwe nitsinda rihuza hamwe na enzymatique hafi ya labels (PL) (urugero: APEX na BioID) 5 byateguwe.Nubwo uburyo bwa UAA bwakoreshejwe neza mubintu byinshi kandi butanga amakuru kubyerekeranye na poroteyine itaziguye, biracyakenewe ko hajyaho urubuga rwa UAA.Icy'ingenzi cyane, ni uburyo bwo kuranga stoichiometric butagira catalitike ihindagurika yibirango byabaye.Ibinyuranye, uburyo bwa PL enzymatique, nkuburyo bwa BioID, buhuza ligase ya biotin yakozwe na POI7, hanyuma igakora biotine kugirango ikore biotinyl-AMP ester hagati.Enzyme rero itera kandi ikarekura biotine ikora "igicu" yanditseho ibisigazwa bya lysine byegeranye.Ariko, BioID isaba amasaha arenga 12 kugirango ibone ibimenyetso bihagije byanditseho, ibuza ikoreshwa hamwe nicyemezo cyigihe gito.Ukoresheje ubwihindurize bushingiye ku kwerekana umusemburo, TurboID yateguwe hashingiwe kuri BioID kugirango ikore neza, itume ikirango gikoreshwa neza na biotine mu minota 10, bituma inzira zigenda zikorwa.Kuberako TurboID ikora cyane kandi urwego rwa biotine ya endogenous irahagije kugirango urwego rwo hasi rumenyekanishe, kuranga inyuma biba ikibazo gishobora kuba ikibazo mugihe byongerewe igihe kandi byateganijwe bisabwa hiyongereyeho biotine ya exogenous.Byongeye kandi, ester ikora ntabwo ikora neza (t1 / 2 ~ 5 min), ishobora kuganisha kuri radiyo nini yo kuranga, cyane cyane nyuma yo kuzuza poroteyine zituranye na biotine 5. Mu bundi buryo, guhuza genetike ya asorbate peroxidase yakozwe (ni ukuvuga biotine- fenol radicals kandi yemerera proteine kuranga mumunota umwe 9,10. APEX ikoreshwa cyane mukumenya proteomes zo mu bwoko bwa selile, membrane proteine complexe, hamwe na cytosolike yerekana protein 11,12. inzira ya selile.
Rero, uburyo bushya bushobora kubyara amoko menshi yanduza-radiyo yo guhagarika amoko afite umwanya muremure kandi wigihe gito utabangamiye cyane inzira za selile bizaba byiyongera kuburyo busanzwe. Mu bwoko butagaragara, ogisijeni imwe rukumbi yadushishikaje kubera igihe gito cyayo na radiyo ikwirakwizwa (t1 / 2 <0,6 µs mu ngirabuzimafatizo) 13. Mu bwoko butagaragara, ogisijeni imwe rukumbi yadushishikaje kubera igihe gito cyayo na radiyo ikwirakwizwa (t1 / 2 <0,6 µs mu ngirabuzimafatizo) 13. С. Muburyo bukora, ogisijeni imwe rukumbi yatwitayeho kubera igihe gito cyayo hamwe na radiyo ikwirakwizwa (t1 / 2 <0,6 µs muri selile) 13.在 活性 物质 中 ， 态 氧 因其 寿命 短 和 扩散 1 1 1 1 1 1 1 1 1/2 <0,6 µs） 而 引起 了 我们 的 注意 13 С. Muburyo bukora, ogisijeni imwe ikurura ibitekerezo byacu kubera igihe gito cyayo hamwe na radiyo ikwirakwizwa (t1 / 2 <0,6 μs muri selile).Umwuka wa ogisijeni wavuzwe ko uhindura methionine, tirozine, histidine na tryptophan ku buryo butemewe, bigatuma iba polar 14,15 kugira ngo ihuze na amine cyangwa thiol ishingiye kuri 16,17.Nubwo ogisijeni imwe rukumbi yakoreshejwe mu kuranga agace ka RNA, ingamba zo gusubiramo ibimenyetso bya POI byegeranye bikomeza kutashakishwa.Hano, turerekana urubuga rwitwa Photoactivation-iterwa na label yegeranye (PDPL), aho dukoresha urumuri rwubururu kugira ngo tumurikire POIs zahujwe na miniSOG yifotora kandi tugatera ingufu za ogisijeni ya ogisijeni kugira ngo ihindure ibisigisigi byegeranye, hanyuma hakurikiraho guhindura amine kugirango ihindure ubushakashatsi bwa chimique muri ingirabuzimafatizo nzima..Twagerageje itsinda ryimiti yimiti kugirango tumenye neza tagi kandi tumenye imbuga zahinduwe dukoresheje proteomics ifunguye.Kugereranya kuruhande rwa PDPL na TurboID yerekana uburyo bwihariye kandi bwuzuye bwa proteomic gukwirakwiza PDPL.Twakoresheje ubu buryo ku bimenyetso byihariye bya poroteyine yo mu bwoko bwa poroteyine yo mu bwoko bwa poroteyine no kumenyekanisha muri rusange poroteyine zerekana abafatanyabikorwa ba poroteyine BRD4 ifitanye isano na kanseri hamwe na E3 ligase Parkin yanduye indwara ya Parkinson, yemeje urusobe ruzwi kandi rutamenyekana rwa poroteyine. imikoranire..Ubushobozi bwa PDPL bwo kumenya insimburangingo ya E3 murwego runini rwa poroteyine byerekana ibihe bisabwa kumenyekana guhuza indirect.Inzira ebyiri zitazwi za parkin zahujwe na ubiquitination-proteasome byemejwe neza.
Ubuvuzi bwa Photodynamic (PDT) 19 hamwe na chromofore ifashwa na laser idakora (CALI) 20, aho imirasire yumucyo hamwe na fotosensiziseri itanga ogisijeni imwe, irashobora gukora poroteyine zigamije cyangwa gutera urupfu.Kubera ko ogisijeni imwe rukumbi ari ikintu cyoroshye cyane hamwe n’ikwirakwizwa rya teoretiki ya intera igera kuri 70 nm, okiside igarukira mu buryo bwa fotosensitire irashobora kugenzurwa.Dushingiye kuri iki gitekerezo, twahisemo gukoresha ogisijeni imwe kugirango tugere ku kimenyetso cya poroteyine mu ngirabuzimafatizo.Twateje imbere uburyo bwa PDPL chemoproteomic kugirango dusohoze imirimo ine: (1) guhagarika ibisekuruza bya ogisijeni ikora imwe isa nuburyo bwa PL enzymatique;(2) gutanga ibimenyetso byakemuwe igihe cyo gutangiza urumuri;.
Photosensitizers irashobora kugabanywamo ibyiciro bibiri harimo fluorofore ntoya (urugero: roza bengal, methylene ubururu) 22 hamwe na proteine ntoya (urugero nka miniSOG, KillerRed) 23.Kugirango tugere ku gishushanyo mbonera, twateje imbere urwego rwa mbere rwa PDPL twongeramo poroteyine za fotosensitizer (PS) kuri POI24,25 (Ishusho 1a).Iyo urumuri rwumucyo wubururu, ogisijeni imwe rukumbi ihindura ibisigazwa bya nucleophilique amino acide, bikavamo polarite umpolung iba electrophilique kandi irashobora kongera kwitwara hamwe na amine probe nucleophile16,17.Iperereza ryakozwe hamwe nigitoki cya alkyne kugirango yemere gukanda chimie hanyuma akure hasi kubiranga LC / MS / MS.
Igishushanyo cyerekana ibimenyetso bya protein bigizwe na miniSOG.Iyo ihuye nurumuri rwubururu, selile zerekana miniSOG-POI zitanga ogisijeni imwe rukumbi, ihindura poroteyine zikorana ariko ntizihuza poroteyine.Ibicuruzwa biciriritse byo gufotora bifatwa na labels ya relay ya amine chimique ya amine kugirango ibe inyongeramusaruro.Itsinda rya alkynyl kuri chimie probe ryemerera gukanda chimie kugirango itungwe no gukururwa hakurikiraho kubara LC-MS / MS.b Imiterere yimiti ya amine probes 1-4.c Isesengura rya fluorescent gel isesengura mitochondrial yaho ya miniSOG-yunganiwe na proteomic marike ikoresheje probe 1-4 hamwe no kugereranya ugereranije na gel densitometrie.Ikigereranyo cyerekana ibimenyetso byerekana imiti yasuzumwe hifashishijwe ubushakashatsi bubi ukuyemo urumuri rwubururu cyangwa gukoresha selile HEK293T nta mvugo ya miniSOG.n = ibyitegererezo 2 byigenga.Buri kadomo kagereranya kopi yibinyabuzima.d Guhagararira abahagarariye no kugereranya PDPL ukoresheje probe optimiz 3 ihari cyangwa idahari ibice bya PDPL byerekanwe nka c.n = 3 ibyitegererezo byigenga.Buri kadomo kagereranya kopi yibinyabuzima.Hagati hamwe na whiskers byerekana uburyo na ± gutandukana bisanzwe.CBB: Coomassie Brilliant Ubururu.e Kwerekana amashusho ya ogisijeni imwe hamwe na Si-DMA itukura cyane.Umwanya munini: 10 µm.Ubushakashatsi bwa Gel hamwe nibigeragezo byasubiwemo byibuze inshuro ebyiri nibisubizo bisa.
Twabanje kugerageza ubushobozi bwamafoto akuze miniSOG26 na KillerRed23, yerekanwe neza muri HEK293T, kugirango duhuze propargylamine yanditseho proteome nkubushakashatsi bwimiti (Ishusho yinyongera 1a).Isesengura rya Gel fluorescence ryerekanye ko label ya proteome yose yagezweho hakoreshejwe miniSOG hamwe nubururu bwurumuri rwubururu, mugihe nta bicuruzwa byerekana ibimenyetso byagaragaye hamwe na KillerRed.Kugirango tunonosore ibipimo byerekana ibimenyetso, hanyuma twagerageje gushiraho imiti irimo aniline (1 na 3), propylamine (2), cyangwa benzylamine (4).Twabonye ko selile HEK293T ubwayo yari ifite ibimenyetso byerekana inyuma ugereranije nta mucyo w'ubururu, bishoboka ko biterwa na endogenous riboflavin photosensitizer, flavin mononucleotide (FMN) 27. Ubushakashatsi bwa Aniline bushingiye ku miti 1 na 3 bwatanze ibisobanuro birambuye, hamwe na HEK293T yerekana neza miniSOG muri mitochondria yerekana ubwiyongere bwikubye inshuro 8 ibimenyetso bya probe 3, mugihe probe 2 yakoreshejwe muburyo bwa RNA-label CAP-seq yerekana gusa ~ 2.5- ibimenyetso byiyongera, birashoboka bitewe nuburyo butandukanye bwo gukora hagati ya RNA na proteyine (Ishusho 1b, c). Ubushakashatsi bwa Aniline bushingiye ku miti 1 na 3 bwatanze ibisobanuro birambuye, hamwe na HEK293T yerekana neza miniSOG muri mitochondria yerekana ubwiyongere bwikubye inshuro 8 ibimenyetso bya probe 3, mugihe probe 2 yakoreshejwe muburyo bwa RNA-label CAP-seq yerekana gusa ~ 2.5- ibimenyetso byiyongera, birashoboka bitewe nuburyo butandukanye bwo gukora hagati ya RNA na proteyine (Ishusho 1b, c).Imiti ya Aniline ishingiye kuri 1 na 3 yerekanye umwihariko: HEK293T, igaragaza neza miniSOG muri mitochondriya, yerekana ubwiyongere bwikubye inshuro 8 ibimenyetso bya probe 3, mugihe probe 2, yakoreshejwe muburyo bwo kuranga CAP-seq RNA, gusa yerekana ~ inshuro 2,5 yikimenyetso cyiyongera, birashoboka bitewe nuburyo butandukanye bwo guhitamo hagati ya RNA na proteyine (Ishusho 1b, c).基于 苯胺 的 化学 探针 1 和 3 具有 更好 的 特异性 ， HEK293T 在 线粒体 中 稳定 表达 miniSOG ， 探针 3 的 信号 增加> 8 倍 ， 而 用于 RNA 标记 方法 CAP-seq 的 探针 2 仅 显示 ~ 2.5- 倍 信号 增加 ， 可能 是 由于 RNA 和 蛋白质 之间 的 不同 反应 偏好 （图 1b ， c）。基于 苯胺 的 化学 探针 1 和 3 具有 更 的 特异性 ， hek293t 在 粒 体 中 稳定 表达 minisog ， 探针 3 的 信号 增加 增加> 8 倍 而 于 na rna 标记 cap-eq 的 2 仅 ~ ~ ~ ~ 2.5 - 倍 信号 增加 ， 可能 是 由于 RNAUbushakashatsi bwa Aniline bushingiye kuri 1 na 3 bwari bufite umwihariko, HEK293T yerekanye miniSOG muri mitochondria, naho probe 3 yariyongereyeho inshuro 8 ibimenyetso, mugihe iperereza 2 ryuburyo bwa CAP-seq RNA ryerekana ko ryiyongereyeho ~ 2,5 gusa.mu kimenyetso, birashoboka bitewe nuburyo butandukanye bwo kwitwara hagati ya RNA na proteyine (Ishusho 1b, c).Byongeye kandi, ubushakashatsi bwa 3 isomers hamwe na hydrazine (probe 5, 6, 7) byageragejwe, byemeza ko iperereza rya 3 ryakozwe neza (Ishusho yinyongera. 1b, c).Mu buryo nk'ubwo, isesengura rya gel-fluorescence ryagaragaje ibindi bipimo ngenderwaho byubushakashatsi: uburebure bwa irrasiyoyasi (460 nm), ubushakashatsi bwa chimique (mM 1), nigihe cyo kurasa (20 min) (Ishusho yinyongera 2a - c).Kureka ikintu icyo ari cyo cyose cyangwa intambwe muri protocole ya PDPL byavuyemo ibimenyetso bikomeye bihinduka inyuma (Ishusho 1d).Ikigaragara ni uko ibimenyetso bya poroteyine byagabanutse cyane imbere ya sodium azide cyangwa trolox, izwiho kuzimya ogisijeni imwe.Kubaho kwa D2O, bizwiho guhagarika umwuka wa ogisijeni umwe, byongera ibimenyetso byerekana ibimenyetso.Kugira ngo hakorwe iperereza ku ruhare rw’andi moko ya ogisijeni ikora mu kuranga, mannitol na vitamine C byongeweho kugira ngo hashyizweho hydroxyl na superoxide radical scavengers, 18, 29, ariko ntibyabonetse kugabanya ibimenyetso.Kwiyongera kwa H2O2, ariko ntibimurikirwa, ntabwo byavuyemo kuranga (Ishusho yinyongera. 3a).Fluorescence yerekana amashusho ya ogisijeni yerekana amashusho ya Si-DMA yemeje ko hari ogisijeni imwe mu nsinga ya HEK293T-miniSOG, ariko ntabwo iri mu nsinga ya HEK293T.Byongeye kandi, mitoSOX Umutuku ntishobora kumenya umusaruro wa superoxide nyuma yo kumurikirwa (Igicapo 1e hamwe n’inyongera.Cytotoxicity ya PDPL yasuzumwe harimo imirasire yubururu bwubururu hamwe nubushakashatsi bwa chimique, kandi nta cytotoxicitike ihambaye yagaragaye (Ishusho yinyongera. 4a).
Kugirango twige uburyo bwo kuranga no gutuma proteomic imenyekanisha poroteyine dukoresheje LC-MS / MS, dukeneye mbere na mbere kumenya aside amine yahinduwe hamwe na delta ya misa ya labels.Methionine, histidine, tryptophan na tyrosine byavuzwe ko byahinduwe na ogisijeni imwe rukumbi 14,15.Duhuza ibikorwa bya TOP-ABPP31 hamwe nubushakashatsi butabogamye butangwa na platform ya compte ya FragPipe ishingiye kuri MSFragger32.Nyuma yo guhindura ogisijeni ya ogisijeni hamwe na label ya chimique, kanda chimie yakozwe hakoreshejwe ikirango cyo kugabanya biotine kirimo umuhuza uhuza, hanyuma ukurikirwa na neutravidin urambuye hamwe na digestion.Peptide yahinduwe, iracyahambiriye kuri resin, yafotowe kugirango isesengurwe LC-MS / MS (Ishusho 2a hamwe namakuru yinyongera 1).Umubare munini wibihinduka byabaye muri proteome hamwe nikarita ya peptide irenga 50 (PSM) ihuza urutonde (Ishusho 2b).Igitangaje ni uko twabonye gusa ihinduka rya histidine, birashoboka ko biterwa na reaction nyinshi ya okiside ya okiside ya histidine yerekeza kuri aniline kurusha aside aside amine.Ukurikije uburyo bwashyizwe ahagaragara bwa okiside ya histidine ikoresheje ogisijeni imwe, 21,33 imiterere ya delta-mass ya +229 Da ihuye niyongerwaho rya probe 3 hamwe na 2-oxo-histidine nyuma ya okiside ebyiri, naho +247 Da nigicuruzwa cya hydrolysis ya +229 Da (Ishusho y'inyongera. 5).Isuzumabumenyi rya MS2 ryerekanwe kwizerwa ryinshi ryo kumenya ibyinshi muri y na b ion, harimo no kumenya ibice byahinduwe (y na b) (Ishusho 2c).Isesengura ry'imiterere y'uruhererekane rwaho rwa PDPL rwahinduwe na histidine ryerekanye icyerekezo giciriritse gikunda ibisigazwa bito bya hydrophobi ku myanya ± 1 (Ishusho yinyongera. 4b).Ugereranije, 1.4 histidine yamenyekanye kuri poroteyine, kandi ibibanza byibi bimenyetso byagenwe nubuso bworoshye bushobora kuboneka (SASA) hamwe nisesengura ryagereranijwe (RSA) (Isanamu yinyongera. 4c, d).
Ibikorwa bitabogamye byo kwiga guhitamo ibisigaye ukoresheje porogaramu yo kubara ya FragPipe ikoreshwa na MSFragger.Ihuza risobanutse rikoreshwa muri Kanda chimie kugirango yemere gufotora peptide yahinduwe kuva streptavidin resin.Ishakisha ryuguruye ryatangijwe kugirango hamenyekane byinshi byahinduwe, kimwe nibisigisigi bijyanye.b Shyira misa yo guhindura iboneka muri proteome.Ikarita ya Peptide PSM.c MS2 yerekana ibisobanuro byimbuga za histidine yahinduwe hamwe na probe 3. Nkurugero ruhagarariye, reaction ya covalent hamwe na probe 3 yongeyeho +229.0938 Da kuri aside amine yahinduwe.d Guhindura mutation ikoreshwa mugupima ibimenyetso bya PDPL.PRDX3 (H155A, H225A) na PRDX1 (H10A, H81A, H169A) byandujwe na plasima yo mu bwoko bwa gasozi kugirango bamenye ibendera.e Peptide ya syntetique yakozwe hamwe na miniSOG isukuye imbere ya probe 3 kandi ibicuruzwa bijyanye na Δm +247 na +229 byagaragaye muri LC-MS.f Muri vitro protein-to-protein imikoranire yagereranijwe na miniSOG-6xHis-tag na antibody anti-6xHis.Antibiotine (streptavidin-HRP) hamwe na anti-imbeba Western blot isesengura rya miniSOG-6xHis / anti-6xIbigo bya antibody yanditseho probe 3, bitewe nigihe cyo kumurika.Ibirango bya poroteyine kugiti cye bigaragarira muburemere bwa molekuline ihuye: LC antibody yumucyo, urunigi ruremereye rwa HC antibody.Ubu bushakashatsi bwagarutsweho bwigenga byibuze kabiri nibisubizo bisa.
Kugenzura ibinyabuzima byerekana urubuga rwanditseho, PRDX3 na PRDX1 byagaragajwe na mass spectrometrie byahinduwe biva kuri histidine bihinduka alanine kandi ugereranije nubwoko bwishyamba mubushakashatsi bwanduye.Ibisubizo bya PDPL byerekanaga ko ihinduka ryagabanutse cyane kuranga (Ishusho 2d).Hagati aho, urutonde rwa peptide rwerekanwe mubushakashatsi bweruye rwashizwemo kandi rusubizwa muri vitro hamwe na miniSOG isukuye imbere ya probe 3 n’umucyo w'ubururu, itanga ibicuruzwa bifite ihinduka ryinshi rya +247 na +229 Da igihe byamenyekanye na LC-MS (Ishusho . 2e).).Kugirango tumenye niba imikoranire ya poroteyine yegeranye ishobora kwandikwa muri vitro mu gusubiza ifoto ya miniSOG, twashizeho uburyo bwo kuba hafi y’ubukorikori dukoresheje poroteyine ya miniSOG-6xHis na antibody irwanya monoclonal muri vitro (Ishusho 2f).Muri ubu bushakashatsi, twategereje kuranga hafi ya antibody iremereye kandi yoroheje hamwe na miniSOG.Mubyukuri, anti-imbeba (kumenya iminyururu iremereye kandi yoroheje ya anti-6xHis yanditseho antibody) hamwe na streptavidin blots yiburengerazuba yerekanaga biotinylation ikomeye yumunyururu uremereye kandi woroshye.Ikigaragara ni uko twabonye miniSOG autobiotinylation bitewe na tagi ya 6xHis hamwe no guhuza imiyoboro yoroheje nu munyururu uremereye, bishobora kuba bifitanye isano n’ikinyuranyo cyasobanuwe mbere ya lysine na 2-oxo-histidine igisubizo cyegeranye.Mu gusoza, twanzuye ko PDPL ihindura histidine muburyo bushingiye.
Intego yacu itaha kwari ukuranga proteome zidasanzwe kugirango tugerageze umwihariko wa labels.Kubwibyo, twagaragaje miniSOG muri nucleus, matrise ya mitochondrial, cyangwa ER membrane yo hanze ya selile HEK293T (Ishusho 3a).Isesengura rya Gel fluorescence ryerekanye imirongo myinshi yanditseho ahantu hatatu hamwe nuburyo butandukanye bwo gushushanya (Ishusho 3b).Isesengura ryerekana amashusho ya Fluorescence ryerekanye umwihariko wa PDPL (Ishusho 3c).Ibikorwa bya PDPL byakurikiwe no gukanda reaction hamwe namabara ya rhodamine kugirango tumenye proteomes zo mu bwoko bwa microscopi ya fluorescence, kandi ibimenyetso bya PDPL byakoreshwaga hamwe na DAPI, abakurikirana mitochondrial, cyangwa ER bakurikirana, byemeza ko PDPL ari iyo kwizerwa cyane.Kubice bitatu bya organelle, kugereranya kuruhande rwa PDPL na TurboID ukoresheje avidin Western blot yerekanaga ko PDPL yanditseho cyane ugereranije nubugenzuzi bwabo.Mubihe bya PDPL, bande yanditseho bande nyinshi, byerekana proteine nyinshi zanditseho PDPL (Ishusho yinyongera. 6a-d).
Igishushanyo cyerekana miniSOG-yunganirwa na organelle yihariye ya proteome.miniSOG yibasira matrise ya mitochondrial ikoresheje guhuza kuri N-terminal 23 aminide acide ya muntu ya COX4 (mito-miniSOG), nucleus ikoresheje guhuza H2B (nucleus-miniSOG), na Sec61β ikoresheje uruhande rwa cytoplasmeque ya ER membrane (ER-miniSOG ).Ibyerekana birimo amashusho ya gel, gufata amashusho, hamwe na sprometrike.b Ishusho ya gel ihagarariye amashusho atatu ya organelle yihariye ya PDPL.CBB Coomassie Brilliant Ubururu.c Amashusho yerekana amashusho ya selile HEK293T agaragaza neza miniSOG hamwe nuduce dutandukanye twagaragaye na antibody yanditseho V5 (umutuku).Ibimenyetso bya subcellular bikoreshwa kuri mitochondria na ER (icyatsi).Ibikorwa bya PDPL birimo gutahura miniSOG (umuhondo) yanditseho proteomes subcellular proteomes ukoresheje Cy3-azide kanda chimie.Umwanya munini: 10 µm.d Ibirunga byibirunga bya PDPL byashyizweho na proteomes muri organelles zitandukanye bigereranywa numubare utabigenewe (n = 3 ubushakashatsi bwibinyabuzima bwigenga).T-ikizamini cyimirizo ibiri yumunyeshuri yakoreshejwe kubibanza byibirunga.Ubwoko bwa HEK293T bwakoreshejwe nkigenzura ribi. Poroteyine zahinduwe ku buryo bugaragara zigaragara mu mutuku (p <0.05 na> inshuro 2 ion ubukana butandukanye). Poroteyine zahinduwe ku buryo bugaragara zigaragara mu mutuku (p <0.05 na> inshuro 2 ion ubukana butandukanye). Значительно измененные белки выделены красным цветом (p <0,05 и> 2-кратная разница в интингности ионов). Poroteyine zahinduwe ku buryo bugaragara zigaragara mu mutuku (p <0.05 na> itandukaniro inshuro 2 mu bukana bwa ion).显 着 变化 的 蛋白质 以 <<<p <0.05 和> 2 倍 离子 强度 差异）。显 着 变化 的 蛋白质 以 红色 突出 <p <0.05 和> 2 Значительно измененные белки выделены красным цветом (p <0,05 и> 2-кратная разница в ионной силе). Poroteyine zahinduwe kuburyo bugaragara zigaragara mumutuku (p <0.05 na> itandukaniro ryikubye kabiri imbaraga za ionic).Poroteyine zifitanye isano ningirakamaro kuri HEK293T-miniSOG ariko ntabwo ari ngombwa kuri HEK293T zerekanwa mubyatsi.e Isesengura ryihariye ryamakuru ya proteomic duhereye kubigeragezo d.Umubare rusange wa poroteyine zifite imibare ihambaye muri buri organelle (utudomo dutukura nicyatsi) twerekanwe hejuru.Histogramu yerekana poroteyine ziri muri organelles zishingiye kuri MitoCarta 3.0, GO isesengura na A. Ting et al.abantu.Gutandukanya imibare ya mitochondria, nuclei, na ER.Ubu bushakashatsi bwagarutsweho bwigenga byibuze kabiri nibisubizo bisa.Amakuru mabi atangwa muburyo bwa dosiye yibanze.
Batewe inkunga na gel hamwe nibisubizo byerekana amashusho, umubare utarimo ikirango wakoreshejwe mukugereranya proteome yamenyekanye muri buri organelle (Amakuru yinyongera 2).HEK293T idakingiwe yakoreshejwe nkigenzura ribi kugirango ikureho ibimenyetso byinyuma. Isesengura ryibibanza byibirunga ryerekanye poroteyine zikungahaye cyane (p <0.05 na> ubukana bwa ion inshuro 2) kimwe na poroteyine ya singleton iboneka gusa mumirongo yerekana miniSOG (Ishusho ya 3d utudomo nicyatsi kibisi). Isesengura ryibibanza byibirunga ryerekanye poroteyine zikungahaye cyane (p <0.05 na> ubukana bwa ion inshuro 2) kimwe na poroteyine ya singleton iboneka gusa mumirongo yerekana miniSOG (Ishusho ya 3d utudomo nicyatsi kibisi). Анжа графикика вулкана показал значительно обогщенные белки (p <0, 05 и> 2-кратная ин интингность ионов), а также одиночные белки, кот че присутствуют только в бакиях, рисе зы зы. Isesengura ry’ibibanza by’ibirunga ryerekanye poroteyine zikungahaye cyane (p <0.05 na> ubukana bwa ion inshuro 2) kimwe na poroteyine imwe igaragara gusa ku murongo ugaragaza miniSOG (Ishusho 3d, utudomo dutukura n’icyatsi).火山 图 分析 显示 出 显 着 富集 <<p <0.05 和> 2 倍 离子 强度） 以及 在于 miniSOG 表达 系 中 的 单一 蛋白质 图 d 3d 红色 和 绿色 点）。火山 图 分析 显示 出 显 <<<p <0.05 和> 2 倍 离子 强度） 仅 存 在于 minisog 表达 系 的 单一 蛋白质 d 3d 红色 绿色 点 。。。 d）））））） Анжа графика вулкана вявил значительно обогщенные белки (p <0, 05 и> 2x ионная сила), а также отдельные белки, присутствующие только врресрес чиной миииSS (красные и зеленые точки). Isesengura ry’ibirunga ryerekanye poroteyine zikungahaye cyane (p <0.05 na> 2x imbaraga za ionic) kimwe na poroteyine imwe gusa igaragara kumurongo wa imvugo ya miniSOG (utudomo nicyatsi kibisi ku gishushanyo 3d).Dufatanije naya makuru, twabonye poroteyine 1364, 461, na 911 zifite imibare ikomeye ya kirimbuzi, mitochondrial, na ER yo hanze ya membrane.Kugira ngo dusesengure neza PDPL ya organelle iherereye, twakoresheje MitoCarta 3.0, Gene Ontology (GO), na A. Ting n'abandi.amakuru set8 yakoreshejwe kuri mitochondriya, nucleus, na ER kugirango agerageze umwihariko wa organelle ya poroteyine zavumbuwe, bihuye neza na 73.4, 78.5, na 73.0% (Ishusho 3e).Umwihariko wa PDPL wemeza ko PDPL nigikoresho cyiza cyo kumenya proteome yihariye.Ikigaragara ni uko isesengura ryakozwe na poroteyine za mitochondial ryerekanye ko proteome yafashwe yagabanijwe cyane cyane muri matrise no mu gice cy’imbere (226 na 106), bingana na 91.7% (362) by’umubare rusange wa poroteyine za mitochondial.urwego rwo hejuru rwa PDPL rwemejwe byongeye (Ishusho yinyongera. 7a).Mu buryo nk'ubwo, isesengura rya kirimbuzi ryerekanye ko proteome yafashwe yagabanijwe cyane cyane muri nucleus, nucleoplasm, na nucleolus (Ishusho y'inyongera. 7b).Isesengura rya poroteyine ya kirimbuzi hamwe na peptide yerekana ibimenyetso bya kirimbuzi (3xNLS) byerekanaga neza ko byubatswe na H2B (Ishusho yinyongera. 7c - h).Kugirango umenye umwihariko wikimenyetso cya PDPL, laminine A ya kirimbuzi yatoranijwe nkumutego wa POI7 wihariye.PDPL yerekanye poroteyine 36 zikungahaye cyane, muri zo 12 za poroteyine (30.0% harimo na lamin A) zaranzwe neza na lamin A poroteyine zikorana zasobanuwe na String base, hamwe nijanisha ryinshi kuruta uburyo bwa BioID (poroteyine 122) 28 ya 28., 22.9 )Isesengura rya GO ryerekanye ko poroteyine zamenyekanye cyane cyane zari muri nucleoplasme (26), membrane nucleaire (10), membrane nucleaire (9), na pore de kirimbuzi (5).Hamwe na hamwe, izo poroteyine ziherereye mu kirere zifite 80% bya poroteyine zikungahaye, bikagaragaza umwihariko wa PDPL (Ishusho y'inyongera 8a - d).
Tumaze gushiraho ubushobozi bwa PDPL bwo gukora ibimenyetso byegeranye muri organelles, noneho twagerageje niba PDPL ishobora gukoreshwa mugusesengura abafatanyabikorwa ba POI.By'umwihariko, twashatse gusobanura PDPL isesengura rya poroteyine za cytosoliki, zifatwa nkintego zitoroshye kuruta bagenzi babo baho bitewe na kamere yabo ifite imbaraga nyinshi.Poroteyine ya Bromodomain na extraterminal (BET) BRD4 yakuruye ibitekerezo byacu kubera uruhare runini mu ndwara zitandukanye 35, 36.Urusobekerane rwakozwe na BRD4 ni coactivator ya transcriptional kandi intego nyamukuru yo kuvura.Mugutegeka imvugo ya c-myc na Wnt5a transcription, BRD4 itekereza ko ari ikintu cyingenzi kigena indwara ya acute myeloid leukemia (AML), myeloma nyinshi, lymphoma ya Burkitt, kanseri y'amara n'indwara zitera indwara 37,38.Byongeye kandi, virusi zimwe na zimwe zibasira BRD4 kugirango zandure virusi na selile, nka papillomavirus, VIH, na SARS-CoV-236,39.
Gushushanya ikarita ya BRD4 dukoresheje PDPL, twahujije miniSOG hamwe na N- cyangwa C-terminal isoform ya BRD4.Ibisubizo bya poroteyine byagaragaje urwego rwo hejuru rwuzuzanya hagati yuburyo bubiri (Ishusho yinyongera. 9a).Poroteyine ya kirimbuzi yagaragajwe na miniSOG-H2B ikubiyemo 77,6% bya poroteyine zikorana na BRD4 (Ishusho y'inyongera. 9b).Noneho, ibihe bitandukanye byo kumurika (2, 5, 10, 20 min) byakoreshejwe muguhindura marike (Ishusho 4a hamwe namakuru yinyongera 3).Twanzuye ko kuri Photoperiods ngufi, PDPL izashyiraho mbere na mbere abafatanyabikorwa bahuza, mugihe igihe kirekire kizaba kirimo poroteyine zagaragaye mugihe gito cyo gufotora kimwe nintego zitaziguye mubirango byanditse.Mubyukuri, twasanze guhuzagurika gukomeye hagati yigihe cyagenwe (84,6% kuminota 2 na 5; 87.7% kuminota 5 na 10; 98.7% kuminota 10 na 20) (Ishusho 4b hamwe ninyongera. 9c).Mu matsinda yose yubushakashatsi, ntitwasanze BRD4 yiyitirira gusa, ahubwo twasanze intego nyinshi zizwi nka MED1, CHD8, BICRA, NIPBL, SMC1A, na HMGB1 zasobanuwe mumibare yububiko.Imbaraga za ionic zizi ntego ziragereranywa nigihe cyo kwerekana (Igishusho 4c hamwe ninyongera. 9d).GO isesengura rya poroteyine zagaragaye mu itsinda ry’iminota 2 ryerekanye ko poroteyine zamenyekanye zashyizwe muri nucleus kandi zagize uruhare mu kuvugurura chromatin no mu mikorere ya RNA polymerase.Imikorere ya molekile ya poroteyine yari ikungahaye kuri chromatin ihuza cyangwa coactivation ya transcriptional, ihuza n'imikorere ya BRD4 (Ishusho 4d).Isesengura ryimikorere ya poroteyine yerekana isesengura ryerekanye urwego rwa mbere rwimikoranire itaziguye hagati ya BRD4 na HDAC umuryango uhuza ibigo nka SIN3A, NCOR2, BCOR, na SAP130 (Igicapo 4e nigishushanyo cya 9e), gihuza na BRD4 na HDAC ihuza amateka ya acetylated. ..Byongeye kandi, intego zihagarariye zagaragajwe na LC-MS / MS, zirimo Sin3A, NSUN2, Fus, na SFPQ, byemejwe n’iburengerazuba (Ishusho 4f).Vuba aha, isoform ngufi ya BRD4 byavuzwe ko ikora nuclei ifite ibintu bitandukanya amazi-yamazi (LLPS).RNA ihuza poroteyine Fus na SFPQ zihuza LLPS yimikorere itandukanye ya selile kandi byagaragaye hano nka proteine BRD4 itanditse.Imikoranire hagati ya BRD4 na SFPQ yemejwe n’ubushakashatsi bwakozwe na co-immunoprecipitation (co-IP) (Ishusho ya 4g), byerekana ubundi buryo bwo gutandukanya BRD4-yunganirwa n’amazi y’amazi akwiye gukorwaho iperereza.Ufatiye hamwe, ibisubizo byerekana ko PDPL ari urubuga rwiza rwo kumenya BRD4 izwi ikorana kimwe na poroteyine zitazwi.
Igishushanyo cyerekana miniSOG-yunganirwa na BRD4 yerekana hafi, igihe cyo kwerekana: 2, 5, 10, na 20 min.b Guteranya poroteyine zagaragaye mu bihe bitandukanye byo kumurika.Gutunganyiriza poroteyine byagaragaye muri HEK293T-miniSOG-BRD4 byari bifite imibare ihambaye ugereranije n'ubwoko bwa HEK293T.c Ion ubukana iyo ugereranije uhagarariye udashyizweho ikimenyetso uzwi na BRD4-ihuza poroteyine mugihe cyagenwe.n = 3 ibyitegererezo byigenga.Amakuru yatanzwe asobanura ± gutandukana bisanzwe.d Isesengura rya Gene ontologiya (GO) ya poroteyine zagaragaye mumatsinda yiminota 2.Amagambo icumi ya mbere ya GO arutonde.Ibibyimba bifite amabara ukurikije icyiciro cya GO, kandi ingano yubunini iragereranya numubare wa poroteyine ziboneka muri buri gihembwe.e Isesengura ryingingo za poroteyine zikorana na BRD4.Uruziga rw'umuhondo ni kole itaziguye kandi uruziga rw'imvi ni urwego rwa mbere rwa kole itaziguye.Imirongo itukura yerekana imikoranire yageragejwe naho imirongo yubururu igereranya imikoranire yahanuwe.f Intego za BRD4 zihuza intego zagaragaye muri LC-MS / MS zagenzuwe n’iburengerazuba.g Ubushakashatsi bwa immunoprecipitation bwemeza imikoranire hagati ya SFPQ na BRD4.Ubu bushakashatsi bwagarutsweho bwigenga byibuze kabiri nibisubizo bisa.Amakuru mabi atangwa muburyo bwa dosiye yibanze.
Usibye kumenya intego za POI zitanditswe, turakeka ko PDPL izaba ikwiriye kumenya insimburangingo ya enzymes, bisaba kuranga poroteyine zihuza mu buryo butaziguye mu bigo binini kugira ngo zisobanure insimburangingo.Parkin (yashizweho na PARK2) ni ligase ya E3 kandi ihinduka ryimiterere muri parkin rizwiho gutera indwara ya autosomal recessive juvenile Parkinson (AR-JP) 42.Byongeye kandi, parkin yasobanuwe nkibyingenzi kuri mitofagy (mitochondrial autophagy) no kuvanaho ubwoko bwa ogisijeni ikora.Nubwo, nubwo hamenyekanye insimburangingo nyinshi za parike, uruhare rwa parike muri iyi ndwara ntirurasobanuka neza.Kugirango utangaze insimburangingo zidafite imiterere, PDPL yageragejwe hongerwaho miniSOG kuri N- cyangwa C-terminus ya parkin.Ingirabuzimafatizo zavuwe na carboneyl cyanide proton itwara m-chlorophenylhydrazone (CCCP) kugirango ikore parkin ikoresheje inzira ya PINK1-Parkin.Ugereranije n'ibisubizo byacu bya BRD4 PDPL, guhuza parike ya N-terminus byagaragaje urutonde runini rwa poroteyine, nubwo byari bikubiyemo igice kinini cya C-terminus (177 kuri 210) (Ishusho 5a, b na Data wongeyeho 4).ibisubizo bihuye na raporo zerekana ko tagi ya N-terminal ishobora gukora cyane Parkin44.Igitangaje ni uko hari poroteyine 18 zuzuzanya gusa mu makuru yacu hamwe n’ibisubizo byatangajwe na AP-MS kuri Parkin43, bishoboka ko biterwa no gutandukanya imirongo ya selile nakazi ka proteomics.Usibye poroteyine enye zizwi (ARDM1, HSPA8, PSMD14, na PSMC3) zagaragajwe nuburyo bubiri (Ishusho 5c) 43.Kugirango turusheho kwemeza ibisubizo bya LC-MS / MS, ubuvuzi bwa PDPL hamwe n’iburengerazuba bwakoreshejwe bwakoreshejwe kugirango ugereranye ibisubizo by’ababyeyi ba HEK293T hamwe n’umurongo uhamye wa N-terminal.Intego zitamenyekanye mbere CDK2, DUT, CTBP1, na PSMC4 zapimwe hamwe na binder izwi, DNAJB1 (Ishusho 5d).
Ikirunga cya poroteyine zikorana na parike muri selile HEK293T hamwe na miniSOG yerekanwe neza yahujwe na N- cyangwa C-terminus ya parkin (n = 3 ubushakashatsi bwibinyabuzima bwigenga).T-ikizamini cyimirizo ibiri yumunyeshuri yakoreshejwe kubibanza byibirunga.HEK293T yakoreshejwe nkigenzura ribi. Poroteyine zahinduwe ku buryo bugaragara zigaragara mu mutuku (p <0.05 na> inshuro 2 ion ubukana butandukanye). Poroteyine zahinduwe ku buryo bugaragara zigaragara mu mutuku (p <0.05 na> inshuro 2 ion ubukana butandukanye). Значительно измененные белки выделены красным цветом (p <0,05 и> 2-кратная разница в интингности ионов). Poroteyine zahinduwe ku buryo bugaragara zigaragara mu mutuku (p <0.05 na> itandukaniro inshuro 2 mu bukana bwa ion).显 着 变化 的 蛋白质 以 <<<p <0.05 和> 2 倍 离子 强度 差异）。显 着 变化 的 蛋白质 以 红色 突出 <p <0.05 和> 2 Значительно измененные белки выделены красным цветом (p <0,05 и> 2-кратная разница в ионной силе). Poroteyine zahinduwe kuburyo bugaragara zigaragara mumutuku (p <0.05 na> itandukaniro ryikubye kabiri imbaraga za ionic).Poroteyine zifitanye isano ningirakamaro kuri HEK293T-miniSOG ariko ntabwo ari ngombwa kuri HEK293T zerekanwa mubyatsi.b Igishushanyo cya Venn cyerekana poroteyine zuzuzanya hagati ya N-terminal na C-terminal.Ikirangantego cya N-kirashobora gukora cyane parike kandi bikavamo poroteyine zimenyekana.c Igishushanyo cya Venn cyerekana poroteyine zuzuzanya hagati ya PDPL na AP-MS.Imikoranire izwi irashyizwe ku rutonde, harimo 4 kuri 18 zuzuye za poroteyine na 11 kuri 159 za poroteyine zagaragaye cyane muri PDPL.d Intego zihagarariwe zagaragajwe na LC-MS / MS zagenzuwe na Western blotting.e Ssu72 na SNW1 byagaragaye ko ari parike ya parike itanditswe.Izi poroteyine zashyizweho na FLAG zandujwe muri HEK293T na HEK293T-Parkin-miniSOG zikurikirwa no kuvura CCCP mu bihe bitandukanye.Gutesha agaciro byagaragaye cyane kumurongo wa Parkin ukabije.f Ukoresheje proteasome inhibitor MG132, hemejwe ko inzira yo gutesha agaciro Ssu72 na SNW1 ihuzwa na proteasome-ubiquitination.Ubu bushakashatsi bwagarutsweho bwigenga byibuze kabiri nibisubizo bisa.Amakuru mabi atangwa muburyo bwa dosiye yibanze.
Ikigaragara ni uko poroteyine zagaragajwe na PDPL zigomba kuba zirimo poroteyine zihuza parike na insimburangingo.Kugirango tumenye insimburangingo ya parkin itanditswe, twahisemo poroteyine ndwi zamenyekanye (PUF60, PSPC1, UCHL3, PPP1R8, CACYBP, Ssu72 na SNW1) hanyuma twanduza plasmide kugirango tumenye izo gen zisanzwe HEK293T hanyuma dusobanure neza HEK293T ya miniSOG-Parkin.Urwego rwa poroteyine za Ssu72 na SNW1 rwaragabanutse cyane mumurongo uhamye wa miniSOG-Parkin (Ishusho 5e).Kuvura hamwe na CCCP kumasaha 12 byaviriyemo kwangirika cyane kwa substrate zombi.Kugira ngo hakorwe iperereza niba iyangirika rya Ssu72 na SNW1 rigengwa na proteasome-ubiquitination, proteasome inhibitor MG132 yongewemo kugirango ibuze ibikorwa bya proteasome, kandi mubyukuri twasanze inzira yo gutesha agaciro yabujijwe (Ishusho 5f).Intego zinyongera zitari substrate zemejwe nkimikoranire ya Parkin ikoresheje blotting yuburengerazuba (Ishusho yinyongera 10), yerekanaga ibisubizo bihuye na LC-MS / MS.Mu gusoza, guhuza ibikorwa bya PDPL hamwe no kugenzura poroteyine yo kugenzura ituma habaho kumenya E3 ligase substrate itanditswe.
Twateje imbere urubuga rusanzwe rwerekana ibimenyetso bigufasha kumenya mumwanya nigihe cyo guhuza POIs.Ihuriro rishingiye kuri poroteyine ya miniSOG ya miniSOG, igera kuri 12 kDa gusa, munsi ya kimwe cya kabiri cyubunini bwa enzyme ya APEX2 ikuze (27 kDa) na kimwe cya gatatu kingana na TurboID (35 kDa).Ingano ntoya igomba kwaguka cyane murwego rwo kwiga poroteyine nto.Ubundi bushakashatsi bwiyongera kumafoto yinyongera, yaba proteine zifite genetike cyangwa molekile ntoya, birakenewe kugirango umusaruro wa kwanti wa ogisijeni umwe wongere kandi wongere ibyiyumvo byubu buryo.Kuri verisiyo yubu ya miniSOG, ibyemezo byigihe gito birashobora kugerwaho ukoresheje kumurika ubururu kugirango ukore ibimenyetso byegeranye.Byongeye kandi, igihe kinini cyo kumurika cyasohoye "igicu" kinini cya ogisijeni imwe rukumbi, bivamo guhindura ibisigisigi bya kure bya histidine ya kure, kwiyongera kwa radiyo, hamwe nubushobozi bwo guhuza neza imiterere ya PDPL.Twagerageje kandi ubushakashatsi burindwi bwa chimique kugirango twongere ibimenyetso-byerekana inyuma kandi dushakisha uburyo bwa molekile iri inyuma yubu buryo.Akazi ka TOP-ABPP gahujwe no gushakisha kutabogamye byemeje ko impinduka zabaye gusa muri histidine kandi ko nta bidukikije bihoraho byagaragaye kugirango hongerwe amateka ya histidine, usibye guhitamo gukabije kuri histidine mukarere ka loop.
PDPL yakoreshejwe kandi mu kuranga poroteyine zo mu bwoko bwa proteome zifite umwihariko wa poroteyine no gukwirakwiza byibuze ugereranije n’ubundi buryo bwo gushyira hafi hamwe n’uburyo bwihariye bwa chimique ya organelle.Ibimenyetso byegeranye nabyo byakoreshejwe neza kuranga ubuso, lysosomal, hamwe na proteomes zifitanye isano na 46,47.Twizera ko PDPL izahuzwa nizi ngirabuzimafatizo.Twongeyeho, twamaganye PDPL tumenye intego zo guhuza poroteyine za cytosoliki zigoye kuruta poroteyine zifitanye isano na membrane bitewe n'imiterere yazo kandi zigira uruhare mu mikoranire yigihe gito.PDPL yakoreshejwe kuri poroteyine ebyiri, coactivator transcriptional BRD4 hamwe na ligase E3 Parkin ifitanye isano n'indwara.Izi poroteyine zombi ntizatoranijwe gusa ku bikorwa by’ibanze by’ibinyabuzima, ahubwo zatoranijwe n’ubuvuzi bwazo ndetse n’ubushobozi bwo kuvura.Kuri izi POIs ebyiri, hazwiho guhuza abafatanyabikorwa kimwe nintego zitanditswe.Ikigaragara ni uko icyiciro cyo gutandukana kijyanye na poroteyine SFPQ yemejwe na co-IP, ishobora kwerekana uburyo bushya BRD4 (isoform ngufi) igenga LLPS.Muri icyo gihe, twizera ko kumenyekanisha insimburangingo ya Parkin ari ibintu bisabwa kumenya ibimenyetso bifatika bitaziguye.Twabonye ibice bibiri bya parkin bitamenyekana kandi twemeza ko byangiritse kumuhanda ubiquitination-proteasome.Vuba aha, hashyizweho ingamba zishingiye ku mutego wo gufata hydrolase substrate mu kuyifata hamwe na enzymes.Nubwo ubu ari uburyo bukomeye cyane, ntibukwiriye gusesengura insimburangingo zigira uruhare mu gushinga ibigo binini kandi bisaba ko habaho isano ya covalent hagati ya enzyme na substrate.Turateganya ko PDPL ishobora kwagurwa kugirango yige izindi poroteyine hamwe nimiryango ya enzyme, nka deubiquitinase nimiryango ya metalloprotease.
Uburyo bushya bwa miniSOG, bwitwa SOPP3, bwakozwe hifashishijwe umusaruro mwiza wa ogisijeni umwe.Twagereranije miniSOG na SOPP3 dusanga imikorere yerekana ibimenyetso, nubwo igipimo cyerekana-urusaku nticyahindutse (Ishusho yinyongera 11).Twakekereje ko gutezimbere SOPP3 (urugero, binyuze mu bwihindurize bwerekanwe) byaganisha kuri poroteyine zikora neza zikenera igihe gito cyumucyo bityo bigatuma inzira zingirabuzimafatizo zifatwa.Ikigaragara ni uko verisiyo ya PDPL igarukira gusa kuri selile kuko isaba urumuri rwubururu kandi ntishobora kwinjira mubice byimbitse.Iyi mikorere ibuza kuyikoresha mubushakashatsi bwikitegererezo cyinyamaswa.Ariko, guhuza optogenetike na PDPL bishobora gutanga amahirwe yo gukora ubushakashatsi ku nyamaswa, cyane cyane mu bwonko.Mubyongeyeho, abandi ba injeniyeri ya infragre fotosensitifers nabo bakuraho iyi mbogamizi.Ubu ubushakashatsi burakomeje muri kano karere.
Umurongo w'akagari HEK293T wabonetse muri ATCC (CRL-3216).Umurongo w'akagari wagerageje kwandura mycoplasma kandi watsimbataye muri DMEM (Thermo, # C11995500BT) hiyongeraho 10% ya serumu y'inda y'inda (FBS, Vistech, # SE100-B) na 1% penisiline / streptomycine (Hyclone, # SV30010).gukura.
3-Aminophenylene (sample 3) na (4-Ethynylphenyl) methanamine (sample 4) yaguzwe na Bidepharm.Propylamine (probe 2) yaguzwe muri Energy-chimique.N- (2-Aminophenyl) pent-4-kwizeraide (probe 1) yashizwemo ukurikije uburyo bwatangajwe.
Imbonerahamwe yinyongera yerekana urutonde rwimiterere ikoreshwa muri ubu bushakashatsi.Urutonde rwa miniSOG na KillerRed rwakuwe mu mpano plasmid yatanzwe na P. Zou (Kaminuza ya Peking).Matrixondrial matrix yibanda kumurongo yakomotse kuri 23 N-terminal amino acide ya COX4 hanyuma ikoronizwa mubice byerekanwe hakoreshejwe inteko ya Gibson (Beyotime, # D7010S).Kugirango tumenye membrane na nucleus ya endoplasmic reticulum, ADN ya SEC61B (NM_006808.3) (NEB, # M0491L) yongerewe imbaraga na PCR ivuye mu isomero rya cDNA ry’utugari twa HEK293T, na ADN ya H2B (yatanzwe na D. Lin, Laboratoire ya Shenzhen Bay) na clon, nkuko byavuzwe haruguru.Keretse niba byerekanwe ukundi, izindi genes za poroteyine zikoreshwa muguhindura no kubaka imirongo ihamye ya selile PCR yongerewe mubitabo bya HEK293T selile cDNA.G3S (GGGS) na G4S (GGGGS) byakoreshejwe nk'umuhuza hagati ya poroteyine ya bait na miniSOG.Ikirangantego cya V5 (GKPIPNPLLGLDST) cyongewe kuriyi nyubako.Kugirango bigaragare mu nyamaswa z’inyamabere no gushyiraho umurongo uhamye wa selile, miniSOG fusion yubaka yashyizwe mubice bya pLX304.Kugirango bagaragaze bagiteri, miniSOG yashyizwe muri vector ya pET21a yanditseho 6xHis kuri C-terminus.
Ingirabuzimafatizo za HEK293T zatewe kuri selile 2.0 x 105 kuri buri riba mu masahani atandatu y’iriba hanyuma zanduzwa nyuma y’amasaha 24 hamwe na plasmide ya recombinant lentiviral (2.4 μg pLX304) hamwe na plasima yo gupakira virusi (1.5 μg psPAX2 na 1.2 μg pMD2.G) ukoresheje Lipo8000 (Beyotime , # C0533), hafi 80%.Nyuma yo kwandura ijoro ryose, uburyo bwarahinduwe kandi bwongerwaho andi masaha 24.Ikusanyamakuru rya virusi ryakozwe nyuma yamasaha 24, 48 na 72.Mbere yo kwanduza imirongo ya selile yagenewe, uburyo bwa virusi bwayungurujwe hifashishijwe akayunguruzo ka 0.8 mm (Merck, # millex-GP) na polybrene (Solarbio, # H8761) hiyongereyeho 8 μg / ml.Nyuma yamasaha 24, selile zemerewe gukira muguhindura uburyo.Ingirabuzimafatizo zatoranijwe hakoreshejwe 5 μg / ml blasticidin (Solarbio, # 3513-03-9) kubice bitatu byambere nkuguhitamo gukomeye.Noneho ukoreshe 20 μg / ml nkurwego rukomeye kubice bitatu bikurikira.
Ingirabuzimafatizo zatewe mu byumba 12 byiza (Ibidi, # 81201) ku bucucike bwa selile zigera ku 20.000 kuri buri riba.Kugirango urusheho gukomera kwa selile HEK293T, ongeramo 50 µg / ml fibronectine (Corning, # 356008) ivanze muri saline ya fosifate (PBS, Sangon, # B640435) kuri 37 ° C.Ibyumba byateguwe mbere yisaha 1 hanyuma bikurwaho na PBS.Nyuma ya 24 h, selile zogejwe rimwe hamwe na PBS, zishyiramo 1 mM probe 3 mumuti mushya wa Hanks uringaniye (HBSS, Gibco, # 14025092) kuri 1 h kuri 37 ° C, hanyuma ushyirwamo LED yubururu (460 nm) ).) byarakaye kuminota 10 mubushyuhe bwicyumba.Nyuma yibyo, selile zogejwe kabiri hamwe na PBS hanyuma zishyirwaho na 4% ya fordehide muri PBS (Sangon, # E672002) muminota 15 mubushyuhe bwicyumba.Kurenza formaldehyde yakuwe muri selile zihamye ukaraba inshuro eshatu hamwe na PBS.Ingirabuzimafatizo zashizwemo 0.5% Triton X-100 (Sangon, # A600198) muri PBS hanyuma yozwa inshuro 3 hamwe na PBS.Noneho kura chambre hanyuma wongere kuri buri sample 25 µl yo gukanda reaction ivanze irimo 50 µM Cy3-azide (Aladdin, # C196720), 2 mM CuSO4 (Sangon, # A603008), 1 mM BTTAA (Confluore, # BDJ-4) na 0.5 mg / ml sodium ascorbate (Aladdin, no S105024) hanyuma ikabikwa muminota 30 mubushyuhe bwicyumba.Nyuma yo gufata ibyemezo, selile zogejwe inshuro esheshatu hamwe na PBS irimo 0,05% Hagati ya 20 (Sangon, # A600560) (PBST) hanyuma ihagarikwa na 5% BSA (Abcone, # B24726) muri PBST muminota 30 mubushyuhe bwicyumba.
Kurinda immunocaline ya colocalisation, selile zashyizwemo antibodi yibanze ukurikije ibihe byerekanwe: imbeba anti-V5 tag mAb (1: 500, CST, # 80076), urukwavu rurwanya Hsp60 mAb (1: 1000), ABclonal, # A0564), urukwavu polyclone anti-calnexin antibody (1: 500, Abcam, # ab22595) cyangwa urukwavu rurwanya lamin A / C antibody ya monoclonal (1: 500; CST, # 2032) kuri 4 ° C ijoro ryose.Nyuma yo koza inshuro 3 hamwe na PBST, selile zashyizwemo antibodiyumu ya kabiri: ihene irwanya urukwavu Alexa Fluor 488 (Thermo, # A11034) yavanze 1: 1000, ihene irwanya imbeba Alexa Fluor 594 (CST, # 8889) yavanze 1: 1000.dilution Kuvomera ubushyuhe bwicyumba muminota 30.Ingirabuzimafatizo zogejwe inshuro 3 hamwe na PBST hanyuma zirwanya DAPI (Thermo, # D1306) muri PBS muminota 10 mubushyuhe bwicyumba.Nyuma yo gukaraba 3 hamwe na PBS, selile zafunzwe muri 50% glycerol (Sangon, # A600232) muri PBS kugirango ishusho.Amashusho ya Immunofluorescent yabonetse hakoreshejwe ZEISS LSM 900 Airyscan2 microscope ya conocal hamwe na software ya ZNE 3.5.
Kumashusho ya ogisijeni ya fluorescent yerekana amashusho, selile zogejwe kabiri hamwe na buffer ya Hanks HEPES mbere yo kongeramo 100 nM Si-DMA muri buffer ya Hanks HEPES (DOJINDO, # MT05).Nyuma yo guhura n’umucyo, selile zashyizwe muri incubator ya CO2 kuri 37 ° C muminota 45.Ingirabuzimafatizo zogejwe kabiri hamwe na buffer ya HEPES ya Hanks hanyuma irwanya Hoechst muri buffer ya HEPES ya Hanks muminota 10 mubushyuhe bwicyumba hanyuma igaragara amashusho ikoresheje microscope ya ZEISS LSM 900., # M36008) muri buffer ya HBSS irimo calcium na magnesium.Nyuma yo guhura n’umucyo cyangwa doxorubicine (MCE, # HY-15142A), selile zashyizwe muri incubator ya CO2 kuri 37 ° C. muminota 10, zogejwe kabiri na buffer ya HBSS, hanyuma zishyirwa hamwe na Hoechst muri buffer ya HBSS mubushyuhe bwicyumba.iminota.Doxorubicin yakoreshejwe nkigenzura ryiza aho selile zavuwe hamwe na 20 μM doxorubicine muri HBSS irimo 1% BSA kuminota 30.Amashusho ya Immunofluorescent yabonetse hakoreshejwe microscope ya Zeiss LSM 900.
HEK293T ingirabuzimafatizo zigaragaza mito-miniSOG zatewe mu bucucike bwa 30% mu byokurya bya cm 15.Nyuma yamasaha 48, igihe ~ 80% byahuriraga, selile zogejwe rimwe hamwe na PBS, zishyirwa hamwe na 1 mM Probe 3 muri buffer nshya ya HBSS mumasaha 1 kuri 37 ° C hanyuma ikamurikirwa na LED yubururu muminota 10 mucyumba ubushyuhe..Nyuma yaho, selile zogejwe kabiri hamwe na PBS, zirakurwaho hanyuma zongera guhagarikwa muri buffer ya PBS ikonje irimo urubura rwa protease idafite EDTA (MCE, # HY-K0011).Ingirabuzimafatizo zatewe na sonicating tip kumunota 1 (isegonda 1 kumasegonda 1 kuri 35% amplitude).Uruvangitirane rwavuyemo rwashyizwe kuri 15.871 xg mu minota 10 kuri 4 ° C kugira ngo rukureho imyanda, maze intumbero ndengakamere ihinduka kuri mg / mL 4 ikoresheje ibikoresho bya poroteyine BCA (Beyotime, # P0009).Huza ml 1 ya lysate yavuzwe haruguru hamwe na 0.1 mM ya fotodegradable biotin azide (Confluore, # BBBD-14), 1 mM TCEP (Sangon, # A600974), 0.1 mM TBTA ligand (Aladdin, # T162437), na 1 mM CuSO4 incubator hamwe hepfo kuzunguruka hejuru yisaha 1 kubushyuhe bwicyumba.Nyuma yo gufata ibyokurya, ongeramo imvange kubisubizo byabanje kuvangwa (MeOH: CHCl3: H2O = 4 ml: 1 ml: 3 ml) mumashanyarazi ya ml 10.Ingero zaravanze kandi zashyizwe kuri 4500 g muminota 10 mubushyuhe bwicyumba.Ibisubizo byo hepfo no hejuru byajugunywe, imvura yogejwe kabiri hamwe na ml 1 ya methanol hanyuma yinjizwamo 15871 × g muminota 5 kuri 4 ° C.Ongeramo ml 1 ya 8 M urea (Aladdin, no U111902) muri mM 25 ya amonium bicarbonate (ABC, Aladdin, no.Ingero zasubiwemo hamwe na 10 mM dithiothreitol (Sangon, # A100281 muri 25 mM ABC) mu minota 40 kuri 55 ° C hakurikiraho kongeramo 15 mM nshya iodoacetamide (Sangon, # A600539) ku bushyuhe bwicyumba mu mwijima.Alkylation mu minota 30..Hiyongereyeho 5 mM dithiothreitol kugirango hongerwe reaction.Tegura hafi 100 µl NeutrAvidin agarose isaro (Thermo, # 29202) kuri buri cyitegererezo ukaraba inshuro 3 hamwe na ml 1 PBS.Umuti wa proteome wavuzwe haruguru wavanze na ml 5 PBS hanyuma ushyirwa mumasaro ya NeutrAvidin agarose yogejwe mbere yamasaha 4 mubushyuhe bwicyumba.Amasaro yogejwe inshuro 3 hamwe na ml 5 PBS irimo 0.2% SDS (Sangon, # A600485), inshuro 3 hamwe na ml 5 PBS irimo 1M urea, inshuro 3 hamwe na ml 5 ddH2O.Amasaro yahise asarurwa na centrifugation hanyuma ihagarikwa muri 200 μl ya 25 mM ABC irimo 1 M urea, 1 mM CaCl 2 (Macklin, # C805228) na 20 ng / μl trypsin (Promega, # V5280).Gerageza ijoro ryose kuri 37 ° C hamwe no kuzunguruka.Igisubizo cyahagaritswe no kongeramo aside (Thermo, # A117-50) kugeza pH igeze kuri 2-3.Amasaro yogejwe inshuro 3 hamwe na ml 1 ya PBS irimo 0.2% SDS, inshuro 3 hamwe na ml 1 ya PBS irimo 1 M urea, hanyuma inshuro 3 hamwe na ml 1 y'amazi yatoboye.Peptide yahinduwe yarekuwe na lysis yoroheje (365 nm) muminota 90 ukoresheje 200 μl ya 70% MeOH.Nyuma ya centrifugation, ndengakamere yakusanyijwe.Amasaro noneho yogejwe rimwe hamwe 100 μl ya 70% ya MeOH hanyuma ndengakamere zirahuzwa.Ingero zumye mu cyuma cyihuta cya Speedvac hanyuma zibikwa kuri -20 ° C kugeza zisesenguye.
Kugirango umenye no kugereranya peptide imwe ya ogisijeni yahinduwe peptide, ingero zongeye gushonga muri 0.1% acide formic na 1 μg za peptide zasesenguwe hakoreshejwe Orbitrap Fusion Lumos Tribrid mass spectrometer ifite ibikoresho bya nano ESI biva muri Tune na Xcalibur biva muri software y'abacuruzi 4.3.Ingero zatandukanijwe kuri cm 75 µm × 15 cm zipakiye imbere capillary inkingi hamwe na 3 µm C18 ibikoresho (ReproSil-pur, # r13.b9.) Hanyuma bihuzwa na EASY-nLC 1200 UHPLC (Thermo).Peptide yatandukanijwe n'umurongo wa minisiteri 95 ya chromatografi kuva kuri 8% solvent B kugeza 50% solvent B (A = 0.1% acide formique mumazi, B = 0.1% acide formic muri 80% acetonitrile), hanyuma yiyongera kumurongo kugeza kuri 98% B min mu minota 6 ku gipimo cya 300 nl / min.Orbitrap Fusion Lumos ikusanya amakuru muburyo butandukanye hagati ya MS scan yuzuye na MS2 scan bitewe namakuru.Umuvuduko w'amashanyarazi washyizwe kuri 2,1 kV n'ubushyuhe bwa capillary ya ion yari 320 ° C.MS spectra (350-2000 m / z) yakusanyirijwe hamwe ikemurwa 120.000, AGC 4 × 105, nigihe cyo kwinjiza ms 150.Ibintu 10 bikunze kugwiza inshuro nyinshi muri buri scan yuzuye yacitsemo ibice akoresheje HCD ifite ingufu zisanzwe zo kugongana zingana na 30%, idirishya ryo kwigunga rya quadrupole ya 1,6 m / z, hamwe no gukemura 30.000.Intego ya AGC kuri tandem mass spectrometry ukoresheje 5 × 104 nigihe kinini cyo kwinjiza 150 ms.Imbaraga zidasanzwe zashyizwe kumasegonda 30. Iion idashyizweho cyangwa abafite amafaranga ya 1+ na> 7+ banze kuri MS / MS. Iion idashyizweho cyangwa abafite amafaranga ya 1+ na> 7+ banze kuri MS / MS. Неназначенные ионы или ионы с зарядом 1+ и> 7+ были отклонены для МС / МС. Iion cyangwa ion zidashyizweho umukono wa 1+ na> 7+ zanze kuri MS / MS.未 指定 的 离子 或 电荷 为 1+ 和> 7+ 的 离子 被 拒绝 用于 MS / MS。未 指定 的 离子 或 电荷 为 1+ 和> 7+ 的 离子 被 拒绝 用于 MS / MS。 Нууказанные ионы или ионы с зарядами 1+ и> 7+ были отклонены для МС / МС. Ions cyangwa ion zidasobanutse zifite ibirego bya 1+ na> 7+ byanze kubera MS / MS.
Amakuru yibanze atunganywa hifashishijwe urubuga rwo kubara rwa FragPipe rushingiye kuri MSFragger.Kubogama kwa misa hamwe na aside amine ihuye byagenwe hakoreshejwe algorithm yo gushakisha ifunguye hamwe na kwihanganira imbaga ya -150 kugeza 500 Da.Peptide yahinduwe noneho yamenyekanye hifashishijwe ibyahinduwe na histidine hamwe ninyungu rusange za +229.0964 na +247.1069 Da muri PD (Proteome Discoverer 2.5, Thermo).
Ingirabuzimafatizo zerekana neza gene miniSOG yahujwe yashyizwe mu byokurya bya cm 6.Iyo ugeze ku masangano ~ 80%, selile zogejwe rimwe hamwe na HBSS (Gibco, # 14025092), hanyuma zishyirwa hamwe nubushakashatsi bwimiti muri HBSS kumasaha 1 kuri 37 ° C hanyuma rumurikirwa nubururu bwubururu.10W LED muminota 20 mubushyuhe bwicyumba.Kugirango umenye ubwoko bwubwoko bwa ogisijeni ikora muri PDPL, vitamine C ya mM 0,5 (MCE, # HY-B0166), 5 mM Trolox (MCE, # HY-101445), D2O (Sigma, # 7789-20-0), 100 mM mannitol (Ingufu za Shimi, # 69-65-8), 100 μM H2O2, 10 mM NaN3 zongerewe muri selile nkinyongera.Nyuma yo koza hamwe na PBS ikonje, selile zarasibwe, zegeranijwe muri tariyeri 1.5 ya centrifuge, hanyuma zishyirwa hamwe niminota 1 muri 200 μl ya PBS hamwe na 1x protease inhibitor idafite EDTA (1 s na 1 s idafite, amplitude 35%).Uruvange rwavuyemo rwashyizwe kuri 15.871 × g mu minota 10 kuri 4 ° C hanyuma intumbero ndengakamere ihinduka kuri mg / mL 1 ikoresheje ibikoresho bya poroteyine BCA.Hafi ya 50 µl ya lysate yavuzwe haruguru yashizwemo na azide 0.1 mM rhodamine azide (Aladdin, no.Nyuma yo gukanda reaction, imvura yaguye hamwe na acetone yakozwe hongerwaho 250 μl ya acetone yabanje gukonjeshwa kuri sample, gushiramo kuri -20 ° C muminota 20 na centrifuging kuri 6010 × g kuminota 10 kuri 4 ° C.Kusanya pellet hanyuma ubiteke muri 50 µl ya buffer ya 1x Laemmli kuminota 10 kuri 95 ° C.Ingero zasesenguwe kuri geles ndende ya SDS-PAGE hanyuma igaragara hifashishijwe sisitemu yo kwerekana amashusho ya Bio-rad ChemiDoc MP Touch hamwe na software ya Image Lab Touch.
Kugaragaza no kweza mini recombinant miniSOG-6x Poroteyine ye yakozwe nkuko byasobanuwe mbere.Muri make, E. coli BL21 (DE3) selile (TransGen, # CD701-02) yahinduwe hamwe na pET21a-miniSOG-6xHis kandi imvugo ya protein yatewe na 0.5 mM IPTG (Sangon, # A600168).Nyuma ya lysis ya selile, proteyine zahanaguwe hifashishijwe amasaro ya Ni-NTA agarose (MCE, no 70666), zandikirwa kuri PBS, zibikwa kuri 80 ° C.
Kuri antibody ishingiye kuri vitro label yegeranye, vanga 100 μM isukuye miniSOG, 1 mM probe 3, na 1 μg anti-label imbeba monoclonal antibody (TransGen, # HT501-01) muri PBS kugeza kuri reaction ya 50 μl..Uruvangitirane rwa reaction rwashyizwemo urumuri rwa LED rwubururu kuri 0, 2, 5, 10, na 20 min mubushyuhe bwicyumba.Uruvange rwashyizwemo 0.1 mM biotine-PEG3-azide (Aladdin, # B122225), 1 mM TCEP, 0.1 mM TBTA ligand, na 1 mM CuSO4 kuri 1 h ku bushyuhe bwicyumba hejuru yicyuma kizamuka hejuru.Nyuma yo gufata ibyemezo, ongeramo buffer ya 4x Laemmli muvanga hanyuma ubiteke kuri 95 ° C muminota 10.Ingero zasesenguwe kuri geles ya SDS-PAGE hanyuma zisesengurwa no guhuza iburengerazuba hamwe na streptavidin-HRP (1: 1000, Solarbio, # SE068).
Peptide ya histidine irimo peptide ya syntetique hamwe na C-terminal amidation (LHDALDAK-CONH2) yakoreshejwe mu gusesengura peptide yegeranye ishingiye kuri vitro label.Muri ubu bushakashatsi, 100 μM yasukuye miniSOG, 10 mM probe 3 na 2 μg / ml peptide ya syntetique peptide yavanze muri PBS mubisubizo byuzuye bya 50 μl.Uruvangitirane rwa reaction rwashyizwemo urumuri rwa LED rwubururu kumasaha 1 mubushyuhe bwicyumba.Microliter imwe yicyitegererezo yasesenguwe hakoreshejwe sisitemu ya LC-MS (Amazi, SYNAPT XS Ions Mobility Igihe-cy-Indege ya misa ya ecran hamwe na software ya MassLynx isesengura).
HEK293T ingirabuzimafatizo zigaragaza neza gene ya miniSOG fusion yabibwe mumasahani ya cm 10 kumirongo ifite aho itandukaniye (Mito, ER, Nucleus) hamwe nibiryo bya cm 15 kumirongo ya Parkin-miniSOG na BRD4-miniSOG.Iyo ugeze ku masangano ~ 90%, selile zogejwe rimwe hamwe na HBSS, hanyuma zishyirwa hamwe na probe 3 muri HBSS kumasaha 1 kuri 37 ° C hanyuma zimurikirwa na LED 10 yubururu ku bushyuhe bwicyumba.Kubirango bidahuye na Parkin, 10 µM proton carboneyl cyanide itwara m-chlorophenylhydrazone CCCP (Solarbio, # C6700) hamwe na probe 3 muri HBSS yongeyeho isaha 1 kuri 37 ° C.Lysis selile, kanda chimie, kugabanya na alkylation intambwe nimwe nkuko byasobanuwe haruguru, usibye ko mg 2 za lysate zongewemo kandi biotine PEG3 azide yakoreshejwe mugukanda reaction aho gukoresha biotine azide ifotora.Nyuma yo gukungahaza, amasaro yogejwe inshuro 3 hamwe na ml 5 ya PBS irimo 0.2% SDS, inshuro 3 hamwe na ml 5 ya PBS irimo 1 M urea, inshuro 3 hamwe na ml 5 za PBS.Nyuma yaho, 2 µg trypsin yongewe kuri 300 µl 25 mM ABC irimo 1 M urea kugirango ikure proteine ijoro ryose kuri 37 ° C.Igisubizo cyahagaritswe kongeramo aside formike kugeza pH ya 2-3 igeze.Nyuma ya trypsinisation ku masaro, igisubizo cya peptide cyarasibwe hifashishijwe inkingi ya SOLAµ HRP (Thermo, # 60209-001) hanyuma yumishwa mu cyuma cyihuta cya Speedvac.Peptide yongeye gushonga muri 0.1% acide formic na 500 ng ya peptide yasesenguwe hakoreshejwe Orbitrap Fusion Lumos Tribrid mass spectrometer ifite ibikoresho bya nano-ESI byasobanuwe haruguru.Peptide yatandukanijwe kubucuruzi bwa RP-HPLC (75 mkm x 2 cm) (Thermo, no 164946) hamwe nisesengura rya RP-HPLC (75 μ m x 25 cm) (Thermo, no 164941), byombi byuzuye 2 mm.gradient kuva 8% kugeza 35% ACN muminota 60, hanyuma umurongo wiyongereye kugera kuri 98% B muminota 6 kumuvuduko wa 300 Nl / min.MS spectra (350-1500 m / z) yakusanyirijwe hamwe ikemurwa 60.000, AGC 4 × 105, nigihe ntarengwa cyo kwinjiza ms 50.Iyoni zatoranijwe zatandukanijwe bikurikiranye na HCD mukuzunguruka 3 s hamwe ningufu zisanzwe zo kugongana zingana na 30%, idirishya rya quadrupole yo kwigunga ya 1,6 m / z, hamwe nicyemezo cya 15000. Intego ya 5 × 104 tandem mass spectrometer AGC nigihe kinini cyo gutera inshinge. ya ms 22 yakoreshejwe.Gukuraho imbaraga byashyizwe kumasegonda 45. Iion idashyizweho cyangwa abafite amafaranga ya 1+ na> 7+ banze kuri MS / MS. Iion idashyizweho cyangwa abafite amafaranga ya 1+ na> 7+ banze kuri MS / MS. Неназначенные ионы или ионы с зарядом 1+ и> 7+ были отклонены для МС / МС. Iion cyangwa ion zidashyizweho umukono wa 1+ na> 7+ zanze kuri MS / MS.未 指定 的 离子 或 电荷 为 1+ 和> 7+ 的 离子 被 拒绝 用于 MS / MS。未 指定 的 离子 或 电荷 为 1+ 和> 7+ 的 离子 被 拒绝 用于 MS / MS。 Нууказанные ионы или ионы с зарядами 1+ и> 7+ были отклонены для МС / МС. Ions cyangwa ion zidasobanutse zifite ibirego bya 1+ na> 7+ byanze kubera MS / MS.
Icyitegererezo cyo gutegura intambwe iganisha ku gutunganya amashapure ya NeutrAvidin yari ameze nko mu isesengura rya LC-MS / MS ryasobanuwe haruguru.Hafi ya 50 μg ya lysate yakoreshejwe nk'igitekerezo cyo kugenzura imizigo na mg 2 za lysate yakoreshejwe mugukanda reaction.Nyuma yo gukungahaza no gukaraba hamwe na neutravidin, poroteyine zahambiriwe zongerwaho hongerwaho 50 μl ya buffer ya Laemmli ku masaro ya agarose hanyuma ikateka kuri 95 ° C. mu minota 5.Igenzura ryinjiza imitwaro hamwe namasaro akungahaye kumasaro yasesenguwe na SDS-PAGE hanyuma yimurirwa muri membrane ya PVDF (Millipore, # ISEQ00010) hakoreshejwe uburyo busanzwe bwiburengerazuba.Ibibyimba byafunzwe n'amata ya skim 5% (Sangon, # A600669) muri TBS arimo 0.1% hagati ya 20 (TBST) kandi yashizwemo hamwe na antibodi yibanze na kabiri.Antibodies yibanze yavomwe 1: 1000 mumata 5% ya skim muri TBST hanyuma yinjizwa ijoro ryose kuri 4 ° C.Antibodiyumu ya kabiri yakoreshejwe ku kigereranyo cya 1: 5000 kandi yashyizwemo isaha 1 ku bushyuhe bwicyumba.Ibibumbano byerekanwe na chemiluminescence ukoresheje sisitemu yo gufata amashusho ya Chemidoc MP.Scan zose zidakuweho za blots na geles mubishushanyo bitangwa nkamakuru yibanze.
Antibodiyumu zibanze zikoreshwa muri ubu bushakashatsi zirimo urukwavu rurwanya urukwavu rurwanya SFPQ (CST, no. AP), urukwavu rurwanya mSin3A antibody ya polyclone (Abcam, # ab3479), antibody anti-tag monoclonal antibody (TransGen, # HT201-02), imbeba anti-act-actin monoclonal antibody (TransGen, # HC201-01), urukwavu rurwanya -CDK2 antibody ya monoclonal (ABclonal, # A0094), antibody ya monoclonal antibody kuri CTBP1 (ABclonal, # A11600), antibody yinkwavu ya polyclone kuri DUT (ABclonal, # A2901), urukwavu rwitwa polyclone antibody kuri PSMC4 (ABclonal, # A2505), urukwavu rurwanya- Antibody ya ADNJB1 (ABclonal, # A5504).Izi antibodies zakoreshejwe mugihe cya 1: 1000 mumata ya skim 5% muri TBST.Antibodiyumu ya kabiri yakoreshejwe muri ubu bushakashatsi harimo kurwanya urukwavu IgG (TransGen, # HS101-01), kurwanya imbeba IgG (TransGen, # HS201-01) kuri 1: 5000.
Kugirango ukore iperereza niba BRD4 ikorana na SFPQ, selile zihamye za HEK293T na BRD4-miniSOG zirenze urugero HEK293T zashyizwe mubisahani bya cm 10.Ingirabuzimafatizo zogejwe na PBS ikonje hanyuma ziterwa muri ml 1 ya Pierce IP lysis buffer (Thermo Fisher, # 87787) hamwe na protease inhibitor ya EDTA idafite iminota 30 kuri 4 ° C.Nyuma yibyo, lysates yakusanyirijwe muri mili 1.5 ya centrifuge hanyuma igashyirwa kuri 15.871 xg muminota 10 kuri 4 ° C.Supernatant yasaruwe kandi yinjizwamo 5 µg ya anti-V5 yanditseho antibody ya monoclonal antibody (CST, # 80076) ijoro ryose kuri 4 ° C.Koza hafi 50 µl za poroteyine A / G amasaro ya magneti (MCE, # HY-K0202) kabiri hamwe na PBS irimo 0.5% Hagati ya 20.Noneho lysates ya selile yashizwemo amasaro ya magneti mumasaha 4 kuri 4 ° C hamwe no kuzunguruka kuva hasi kugeza hejuru.Noneho amasaro yogejwe inshuro enye hamwe na ml 1 ya buffer ya PBST hanyuma utekwa kuri 95 ° C muminota 5.Ingero zasesenguwe kuri geles ya SDS-PAGE hanyuma yimurirwa muri membrane ya PVDF hakoreshejwe uburyo busanzwe bwa blot.Ibibyimba byafunzwe mu mata ya skim 5% muri TBST hanyuma bikurikiranwa hamwe na antibodi yibanze na kabiri.Antibody Primary Rabbit anti-SFPQ antibody ya monoclonal (CST, # 71992) yakoreshejwe ku kigereranyo cya 1: 1000 mumata 5% ya skim muri TBST hanyuma ikorwa ijoro ryose kuri 4 ° C.Kurwanya urukwavu IgG byakoreshejwe ku kigereranyo cya 1: 5000 kandi byashyizwe mu isaha 1 ku bushyuhe bwicyumba.Ibibumbano byerekanwe na chemiluminescence ukoresheje sisitemu yo gufata amashusho ya Chemidoc MP.
Inzego zose zikoreshwa mu isesengura rya Solvent Accessible Surface Area (SASA) zabonetse muri Banki ya Protein Data Bank (PDB) 52 cyangwa Ububiko bwa AlphaFold Protein Database53.SASA Yuzuye yabazwe kuri buri gisigara ukoresheje gahunda ya FreeSASA.Gusa amakuru yuzuye kandi adasobanutse ya SASA ya label ya histidine nabaturanyi bayo yakoreshejwe kugirango babone SASA igereranijwe kuri buri nyubako.Kugereranya ibishobora kugerwaho (RSA) kuri buri histidine yabazwe mukugabanya agaciro ka SASA ntarengwa hamwe nubuso bushoboka bushoboka busigaye bushoboka kuboneka.Histidine yose noneho yashyizwe mubikorwa byihishe niba hagati ya RSA yari munsi ya 20%, ubundi yashyizwe ahagaragara 56.
Amadosiye mabi yabonetse muburyo bwa DDA yashakishijwe hakoreshejwe Proteome Discoverer (v2.5) cyangwa MSfragger (Fragpipe v15.0) mububiko bukwiye bwa SwissProt bwagenzuwe na protein ikubiyemo ibintu byanduye.Peptide isaba trypsin yuzuye hamwe na site ebyiri zabuze za clavage, methylation ya carbamoyl nkimpinduka ihamye hamwe na okiside ya methionine nkimpinduka ikomeye.Kwihanganira uburemere bwibice byashyizwe kuri 10 ppm na 0.02 Da (MS2 Orbitrap). Indwara zanduye zavanyweho, kandi poroteyine zirayungurura kugirango ubone igipimo cyo kuvumbura ibinyoma cya <1%. Indwara zanduye zavanyweho, kandi poroteyine zirayungurura kugirango ubone igipimo cyo kuvumbura ibinyoma cya <1%. Попадания загрязняющих веществ были удалены, а белки отфиль званя, ч чы получить коэффентент ложного обнаружения <1%. Indwara zanduye zavanyweho kandi poroteyine zirayungurura kugirango itange igipimo cyibinyoma cya <1%.去除 污染物 命中 ， 过滤 蛋白质 以 获得 <1% 的 错误 发现 率。 <1% 的 错误 发现 率。 Попадания загрязняющих веществ были удалены, а белки отфиль званя для достижения оровня ложных обнаружений <1%. Indwara zanduye zavanyweho kandi poroteyine zirayungurura kugirango igere ku gipimo cyiza cya <1%.Kubisesengura ryinshi udakoresheje ibirango, ibisanzwe bya poroteyine biva mubintu bitatu byasubiwemo.Isesengura rya poroteyine yo mu bwoko bwa poroteyine ryakozwe hifashishijwe isesengura rya Gene Ontology (GO) ryakozwe na DAVID Bioinformatics Resources, MitoCarta 3.0 hamwe na data base yakozwe kandi itangazwa nitsinda rya Alice Ting.Ikarita y'ibirunga yabonetse muri Perseus (v1.6.15.0). Impinduka za poroteyine nyinshi zageragejwe ku kamaro k’ibarurishamibare hifashishijwe t-test ebyiri, kandi hit za protein zagaragaye hamwe nimpinduka nyinshi> 2 (keretse bivuzwe ukundi) nagaciro k <0.05. Impinduka za poroteyine nyinshi zageragejwe ku kamaro k’ibarurishamibare hifashishijwe t-test ebyiri, kandi hit za protein zagaragaye hamwe nimpinduka nyinshi> 2 (keretse bivuzwe ukundi) nagaciro k <0.05. Изменения кратности содержания белка были проверены на статистическую значимость с использованием дву Мононнего t-критерия, и совпадения белков были идентифицирова с изменением содержя> 2 Guhindura intungamubiri za poroteyine byageragejwe ku kamaro k’ibarurishamibare ukoresheje t-test ebyiri, kandi guhuza poroteyine byagaragaye hamwe no guhindura ibirimo> 2 (keretse bivuzwe ukundi) nagaciro ka <0.05.使用 双边 t 检验 测试 蛋白质 丰 度 倍数 变化 统计 显 2 2 2 2 2 2 2 2 2使用 双边 t 检验 测试 蛋白质 倍数 变化 的 统计 着 性 2 2 变化 2 2 2 2 2 2 2 2 Статистическую значимость кратных изменений содержания белка проверяли с использованием дву мононнего t-критерия, а совпадения белков определяли для изменений содержя> 2 (если не ука зо пое). Ubusobanuro bwibarurishamibare bwibintu byahinduwe mubintu bya poroteyine byageragejwe hakoreshejwe t-test ebyiri, kandi guhuza poroteyine byagenwe kugirango bihindurwe> 2 (keretse byerekanwe ukundi) na p-gaciro <0.05.Isesengura ryimikorere ya poroteyine ryakozwe hifashishijwe isesengura rya GO hamwe na base de base.
Ibinyabuzima bitatu byigana byakozwe hamwe nibisubizo bisa.Isesengura mibare ryakozwe na GraphPad Prism (software ya GraphPad) kandi ibibanza byibirunga byakozwe na Perseus (v1.6.15.0).Kugereranya ayo matsinda yombi, p-indangagaciro zagenwe hakoreshejwe t-test yumurizo ibiri.Gusa poroteyine ya singleton yamenyekanye byibuze kabiri mumatsinda yubushakashatsi yashyizwe mubibanza byibirunga, kandi indangagaciro zabuze mu itsinda ryigenzura zasimbuwe na Perseus zivuye mubisanzwe kugirango p-agaciro gashobore kubarwa.Utubari twibeshya twerekana uburyo ± gutandukana bisanzwe.Mu isesengura rya proteomic yo gusesengura imibare, ubwinshi bwa poroteyine zagaragaye byibuze byibuze bibiri byigana.Uburyo bwibarurishamibare ntabwo bwakoreshejwe mbere yo kumenya ingano yicyitegererezo.Ubushakashatsi ntabwo ari impanuka.Abashakashatsi ntibahumye amaso imirimo mugihe cyo kugerageza no gusuzuma ibisubizo.
Kubindi bisobanuro kubijyanye nigishushanyo mbonera, reba Raporo yubushakashatsi bwibidukikije ifitanye isano niyi ngingo.
Amakuru rusange ya spekrometrike yabonetse muri ubu bushakashatsi yashyikirijwe ProteomeXchange Consortium ibinyujije mu bubiko bw'abafatanyabikorwa ba iProX57 munsi ya dataset ID PXD034811 (Dataset ya PDPL-MS).Amakuru mabi atangwa muburyo bwa dosiye yibanze.Iyi ngingo itanga amakuru yumwimerere.
Gingras, AC, Abe, KT & Raught, B. Kumenya abaturanyi: gukoresha biotinylation biterwa na hafi kugirango urangire poroteyine hamwe namakarita. Gingras, AC, Abe, KT & Raught, B. Kumenya abaturanyi: gukoresha biotinylation biterwa na hafi kugirango urangire poroteyine hamwe namakarita.Gingras, AS, Abe, KT na Raut, B. Kumenyera ibidukikije: gukoresha biotinylation yegeranye no kuranga poroteyine hamwe namakarita. Gingras, AC, Abe, KT & Raught, B. 了解 邻里 ： 使用 邻近 依赖性 生物 化 化 表征 表征 复合 物 并 绘制 细胞 器 图。 Gingras, AC, Abe, KT & Raught, B. Sobanukirwa abaturanyi: koresha umuturanyi ushingiye kubuzima bwibinyabuzima.Gingras, AS, Abe, KT na Raut, B. Gusobanukirwa hafi: kuranga poroteyine no gushushanya ingirangingo ukoresheje biotinylation yegeranye.Ibiriho.Igitekerezo cyanjye.Imiti.ibinyabuzima 48, 44–54 (2019).
Geri, JB n'abandi.Gushushanya microen ibidukikije wohereza ingufu za Dexter muri selile selile.Ubumenyi 367, 1091–1097 (2020).
Hertling, EL n'abandi.Imiyoboro ibiri-ya proteome yerekana selile yihariye yo guhindura imikorere yabantu.Ingirabuzimafatizo 184, 3022–3040.e3028 (2021).
Igihe cyo kohereza: Nzeri-15-2022
