PSMC5, 19S Proteasomal ATPase, e laola Koineine Action ho Nucleus Accumbens (2015)

PLoS One. 2015 May 11; 10 (5): e0126710. Doi: 10.1371 / journal.pone.0126710. eCollection 2015.

Ahnishi YH1, Ahnishi YN2, Nakamura T3, Ohno M4, Kennethy PJ5, Yasuyuki O6, Nishi A7, Neve R8, Tsuzuki T4, Nestler EJ5.

inahaneloang

OsBFosB ke sengoloa se tsitsitseng sa sengoloa se bokellanang ho li-nucleus accumbens (NAc), e leng karolo ea mantlha ea potoloho ea moputso oa boko, ho arabela ho pepesehela khafetsa k'hok'heine kapa lithethefatsi tse ling tsa tlhekefetso. Le ha osBFosB e tsejoa e le heterodimerize le setho sa lelapa sa Jun ho theha sengoloa se sebetsang sa sengoloa, ha ho so be le phuputso e bulehileng ea balekane ba bang ba tlamang ba ΔFosB bokong. Mona, ka ts'ebeliso ea tomoso ea liteko tse nyalisitsoeng tse peli, re khetholla PSMC5-e tsejoang hape e le SUG1, subunit e nang le ATPase ea 19S proteasomal complex-joalo ka protheine e sebelisanang le protheine le ΔFosB. Re netefatsa litšebelisano tse joalo lipakeng tsa ΔFosB le PSMC5 e fumanehang ho NAc mme re bonts'a hore liprotheine ka bobeli li theha moaho le liprotheine tse ling tsa taolo ea chromatin tse amanang le ts'ebetso ea liphatsa tsa lefutso. Re tsoelapele ho bonts'a hore cocaine e sa foleng e eketsa maemo a nyutlelie, empa eseng cytoplasmic, a PSMC5 ho NAc le hore khatello e fetelletseng ea PSMC5 sebakeng sena sa boko e ts'ehetsa likarabo tsa locomotor ka cocaine. Hammoho, liphuputso tsena li hlalosa mokhoa o mocha o tlatsetsang liketsong tsa ΔFosB mme, ka lekhetlo la pele, e ama PSMC5 ho polasetiki ea molek'hule le boits'oaro e bakoang ke cocaine.

Tlhaloso: Ahnishi YH, Ohnishi YN, Nakamura T, Ohno M, Kennedy PJ, Yasuyuki O, et al. (2015) PSMC5, XPNUMXS Proteasomal ATPase, e Laola Ketso ea Cocaine ho LiNumus Accumbens. PloS ONE 19 (10): e5. Doi: 0126710 / journal.pone.10.1371

Mohlophisi oa thuto: James Edgar McCutcheon, Univesithing ea Leicester, UNITED KINGDOM

Re amohetse: December 10, 2014; E amohetse: April 7, 2015; E phatlalalitsoe: E se eka 11, 2015

Copyright: © 2015 Ohnishi et al. Ena ke sengolo se bulehileng sa phihlello se tsamaisitsoeng tlasa maemo a License ea Boikarabelo ba Creative Commons, e lumellang tšebeliso e sa thibeloang, phetiso, le phetiso ea mofuta o fe kapa o fe, ha fela ho ngolisitsoe mongoli oa mantlha le mohloli

Ho fumaneha ha Data: Lintlha tsohle tse amehang li ka har'a pampiri.

Lithuso: Mosebetsi ona o ne o tšehelitsoe ka lithuso tse tsoang ho National Institutes of Health, National Institute on Drug Abuse, le Ishibashi Foundation le Japan Society bakeng sa Kholiso ea Saense (linomoro tsa KAKENHI: 24591735, 26290064, 25116010). Bafani ba lichelete ba ne ba sena karolo morerong oa ho ithuta, ho bokella data le ho hlahlobisisa, qeto ea ho phatlalatsa, kapa ho hlophisa sengoloa.

Lithahasello tse tsitsitseng: Bangoli ba boletse hore ho na le lithahasello tsa tlhōlisano tse teng.

Selelekela

ΔFosB, sehlahisoa se qhekelletsoeng sa FosB gene, ke ea lelapa la Fos la lintlha tse tsoang, le tse kenyeletsang c-Fos, FosB ea bolelele bo felletseng, Fra-1, le Fra-2. ΔFosB, joalo ka liprotheine tse ling tsa Fos, heterodimerize e na le protheine ea lelapa la Jun — c-Jun, JunB, kapa JunD — ho etsa mofuta o sebetsang oa phepollo ea AP-1 (activator protein-1), e etsang kapa e hatellang tlhahiso ea mefuta e ikhethang ea sepheo [1,2].

ΔFosB e bontšitsoe e bapala karolo ea bohlokoa tšebelisong ea lithethefatsi [2]. E ikhethang har'a liprotheine tsa malapa tsa Fos, e bokellana ka li-nucleus accumbens (NAc) le likarolo tse ling tse amanang le moputso ka mor'a taolo e khutšoane ea lithethefatsi ka lebaka la boemo ba eona bo phahameng ba botsitso [3,4], e kopantsoeng le ho haella ha libaka tsa C-terminal tsa tšenyo ea C le phosphorylation ke liprotheine tse 'maloa tsa kinase [5-7]. Ho kenella ho joalo ha ΔFosB ho li-medic tsa NAc ho ile ha eketsa likarabo tsa boitšoaro ho lithethefatsi tsa tlhekefetso. Kahoo, ho fetella hoa ΔFosB sebakeng sena sa boko sa liphoofolo tse kholo, ekaba ka ts'ebeliso ea vaerase ea litšoelesa kapa litoeba tse sa sebetseng hantle, ho eketsa maikutlo a phoofolo mabapi le litlamorao tse matlafatsang le tse matlafatsang tsa koae le opoates hammoho le sepheo sa phoofolo sa ho itlhomme. koae [7-11]. Ka lehlakoreng le leng, ho hlonama ho matla ha bahanyetsi ba fosahetseng ba ΔFosB ho baka boitšoaro bo fapaneng ba boitšoaro [10-12].

Rona le ba bang re kile ra netefatsa, ka ts'ebeliso ea li-gel shift assays, hore JunD le mohlomong liprotheine tse ling tsa lelapa la Jun ke balekane ba mantlha ba tlamang ba ΔFosB bokong ho vivo [13-15]. Leha ho le joalo, ho fihlela joale, ha ho so ka ho ba le tlhahlobo e bulehileng, e sa thekeseleng ea balekane ba ΔFosB ba tlamang bokong. Mona, re ne re batla ho tseba balekane ba tlamang ba ΔFosB ba mahlale a maiketsetso ka tšebeliso ea mofuta oa peo e kopanetsoeng ea mefuta e 'meli.16,17]. Lintlha tsa rona li senotse hore PSMC5, e tsejoang hape e le SUG1, ke molekane ea matla oa ΔFosB ka bobeli ka vitro le NAc ka vivo, moo e kenang le ΔFosB e le karolo ea ts'ebetso ea ts'ebetso ea phepelo ea koae, e boetse e na le protheine ea CBP (CREB e tlamang. ) le p300 — ka bobeli li-histone acetyltransferases (HATs) hammoho le BRG1 (protheine ea ntlafatso ea chromatin). Re tsoela pele ho bonts'a ho pepeseha ho sa feleng ha koae ea cocaine e fetola maemo a nyutlelie ea PSMC5, e leng subPit e nang le 19S proteinasomal tata, ho NAc le hore PSMC5 ka nako e 'ngoe e laola likarabelo tsa boitšoaro ho koae.

Litaba le Mekhoa

Tlhahlobo ea tomoso e habeli

Lisele tsa MaV203 yeast cell (Invitrogen Life Technologies) li ne li tsamaisoa ka kopanelo le pDBLeu e khanna likarolo tse fapaneng tsa protheine ea ΔFosB, mme laebrari ea boko ea mouse e ile ea patoa ho PPC86 (Invitrogen Life Technologies). Lisele tse fetotsoeng li ne li lengoe ho SC-medium e haellang leucine, tryptophan, le histidine, 'me e na le 10 mM ea 3-aminotriazole. Ho tlama pakeng tsa likarolo tsa FosB le molekane oa mokhethoa ho etsa mefuta e meraro ea moqolotsi (His3, Ura3, 'me LacZ), mme ho kenella ho etsa hore ba fetohileng ba khone ho phela tlasa maemo a cultured a sebelisitsoeng. Maqhubu a nepahetseng a ile a hlakoloa ka likotoana tsa pDBLeu-ΔFosB tse ncha ka ho khutlisetsoa ka seleng ea MaV203.

Mehala ea sele

Lisele tsa Mouse Neuro 2A neuroblastoma (ATCC) li ile tsa bolokoa maemong a tlase a bohlokoa a Eagle (EMEM) (ATCC), ea tlatsetsoa ka 10% ea serum (FBS) ho 37 ° C le 5% CO2. Lisele tsa Rat 1A ke mpho e tsoang ho Yusaku Nakabeppu (Fukuoka, Japan) [18] hape e bolokiloe ho Dulbecco's MEM (DMEM) (Life Technologies), e tlatselletsoang ka 10% FBS ho 37 ° C le 5% CO2. Phetoho ea lisele tse nang le DNA ea plasmid e ile ea phethoa ho sebelisoa Effectene (Qiagen) ho latela litaelo tsa moetsi.

PSMC5 le li-ΔFosB li bopa

Mefuta e 'maloa e feto-fetohang ea PSMC5, e' ngoe le e 'ngoe ea FLAG e emisitsoeng ho N-terminus ea bona, e ne e hlahisoa bakeng sa tšebeliso ea boits'ireletso kapa litekong tsa phetisetso ea bong. Tsena li kenyellelitsoe: PSMC5-K196M, PSMC5-Δcoiled-coil domain (PSMC5-ΔCC, e haelloang ke li-amino acid 27-68), PSMC5-NT (e nang le sekhechana sa N-terminal sa protheine, amino acid le CNXXNXNXNXXNXNXNXXNXXNXXX and XNXX -C (e na le sekhechana sa C-terminal sa protheine, 1 amino acid) (bona Setšoantšo sa 1). Re boetse re sebelisitse mefuta ea N-terminal ea MYC-tagged ea wildtype ΔFosB hammoho le ΔFosB e nang le phetoho tsamaisong ea eona ea leucine-zipper (phetoho ea amino acid 182 ho 205 e tsejoang e felisa heterodimerization le liprotheine tsa lelapa tsa Jun [6].

ponahalo e nyane
Mofuta oa 1. ΔFosB e tlamahanya le PSMC5 ka vitro.

 

A. Schematic ea ΔFosB, ΔFosB-LZM moo leucine zipper domain e fetotsoe ho felisa ΔFosB heterodimerization le liprotheine tsa Jun, le Δ2ΔFosB e se nang li-amino acid tsa pele tsa 78 tsa ΔFosB N-terminus. B. Schematic ea PSMC5, PSMC5-NT e nang le li-amino acid tsa pele tsa 151 tsa PSMC5, PSMC5-CT e haelloang ke li-amino acid tsa 235 tsa pele tsa PSMC5, 'me PSMC5-ΔCC e setseng e na le li-coUM28 -NUMX68. . Sebaka sa AAA se tsamaisana le motif, ATPases E amahanngoa le Lits'ebetso tse fapaneng tsa cellular, tse fumanehang ho li-ATPases tse ngata. C. 2.4 μg ea pcDNA3.1-ΔFosB (litselana 1-4) kapa ΔFosB-LZM (lane 5) e ile ea fetoloa ka kopanelo le 2.4 μg ea FLAG-tagged PSMC5 kapa mefuta e fapaneng ea ho tlosoa liseleng tsa Neuro2a. Matsatsi a mabeli kamora phetisetso, lisele li ile tsa lisoa le ho kenngoa ka ente ka anti anti-FLAG mme ea ntšoa Bophirimela ka anti-ΔFosB kapa anti-FLAG antibody. Hlokomela hore ΔFosB, empa eseng ΔFosB-LZM, e sebetsa hantle ho PSMC5 kapa PSMC5-NT, empa eseng PSMC5-CT kapa PSMC5-ΔCC. Lintlha tse bontšitsoeng setšoantšong sena li ne li hatisoa ka makhetlo a mararo ho e 'ngoe ea liteko tse tharo tse arohaneng.

doi: 10.1371 / journal.pone.0126710.g001

Animals

Limpho tsa banna tsa XXUMUMB tsa beke tse robeli ho isa ho 11BL / 57J (The Labor Labor Labor) li ile tsa sebelisoa bakeng sa liteko tsohle. Liphoofolo li ne li lula mokatong o lefifi oa 6-h ka khanya le lijo le metsi ad libitum mme ba ne ba lula habeli 1 bekeng pele ho liteko. Ho ile ha sebelisoa mekhoa e 'meli ea kalafo ea koae. Ho ithuta ka litlamorao tsa biochemical tsa cocaine, liphoofolo li ile tsa fuoa litekanyetso tsa letsatsi le letsatsi tsa 7 tsa cocaine (20 mg / kg) kapa saline, mme tsa bolaoa ke decapitation 24 hr kamora ho ente ea ho qetela. Ena ke protocol e tloaelehileng, e bonts'itsoeng ho hlahisa likarabo tse ngata tsa limolek'hule le tsa cellular ho lithethefatsi [7]. Ho ithuta tšusumetso ea PSMC5 ho li-nucleus bokellanang ka karabelo ea boitšoaro ho koae, re sebelisitse lethal dose ea lithethefatsi (7.5 mg / kg; bona sensomization ea ka tlase) ho latela monahano oa hore PSMC5 e ka eketsa kutloelo-pele ea phoofolo ho koae [8]. Liteko tsohle tsa liphoofolo li amohetsoe ke Komiti ea Tlhokomelo ea Liphoofolo le Ts'ebeliso ea Litsebeletso Thabeng ea Sinai.

Immunoprecipitation le Western blotting

Lisele tsa Neuro 2A li ile tsa fetisetsoa ka mefuta ea wildtype kapa e feto-fetohang ea PSMC5. Matsatsi a mabeli kamora phetisetso, lisele li ile tsa hlatsuoa ho PBS, e lister RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 10 mM sodium butyrate . Lysates li ile tsa aroloa mme tsa kenngoa ka antibodies e seng ea mofuta oa IgG (Sigma) kapa anti-FLAG (Sigma) ea 3 hr ho 4 ° C. Immunoprecipitation e entsoe ka lieta tsa Protein G (Attitrogen) joalo ka ha ho hlalositsoe [19]. Ka bokhutšoanyane, liprotheine tse sa sebetseng li ile tsa kenngoa ho SDS-PAGE mme tsa hlahlojoa ke Bophirima ba ts'ebeliso ba sebelisa anti-FosB / ΔFosB (Cell Signaling Technology) ba ipapisitse le protocol e phatlalalitsoeng [7]. Bakeng sa lits'oants'o tse kopanyang tsa protheine ea vivo, re sebelisitse likarolo tse hloekisitsoeng tsa nyutlelie ea litoeba tse tsoang ho punch-dissected NAc ea litoeba kamora kalafo e sa foleng ea koae (20 mg / kg IP letsatsi le letsatsi bakeng sa matsatsi a 7, ka litoeba tse sebelisitsoeng 24 hr ka mora ente ea ho qetela). Co-immunoprecipitation ho tsoa likaroloana tsa nyutlelie e ne e etsoa ka thepa ea Nuclear Complex Co-IP (Active Motif) kamora litaelo tsa moetsi. Li-antibodies tse latelang li sebelisitsoe: MYC kapa ß-Actin, Cell Signaling Technology (Danvers, MA), PSMC5 le histone H3, Abcam (Cambridge, MA), CBP, p300 le BRG1, Santa Cruz Biotechnology (Santa Cruz, CA), le FLAG M2, Sigma.

Immunohistochemistry

Immunohistochemistry e entsoe ho latela mekhoa e phatlalalitsoeng [20]. Lice li ne li sa sebetsoa li bile li entsoe ka mokhoa o ikhethileng ka 4% paraformaldehyde ho PBS. Brain li ne li reneketsoa ka 30% sucrose, ebe li hoamisoa le ho bolokoa ho -80 ° C ho fihlela e sebelisoa. Likarolo tsa Coronal (40 μm) li ne li khaoloa holim'a cryostat mme li sebetsoa bakeng sa immunohistochemistry. Likarolo tse phaphametseng tsa mahala li ne li kenelletse kahare ho buffer e thibelang e nang le 0.3% Triton le 3% serum e tloaelehileng ea esele. ΔFosB e fumanoe e sebelisa poli ea polyclonal antibody e phahamisitsoeng khahlanong le karolo ea N-terminal ea protheine (1 / 1000 Santa Cruz Biotechnology). PSMC5 e fumanoe e sebelisa rabi ea polyclonal antibody (1 / 100 Abcam, Cambridge, MA). Litšoantšo li nkuoe ka microscope e potelletseng (60x kukuza; Zeiss).

Senomotor sensitization

Malinyane kaofela a ne a fuoa li-sindine tsa IP tsa letsoai letsatsi le leng le le leng bakeng sa matsatsi a 3 ho li beha tlas'a khatello ea liente. Letsatsing le hlahlamang, litoeba li ile tsa kenngoa IP ka saline kapa lethal dose ea cocaine (7.5 mg / kg; bona tlasa Liphoofolo tse kaholimo) 'me tsa beoa hang-hang ka mabokoseng a lipale tsa locomotor. Ts'ebetso ea litoeba tsa litoeba e tlalehiloe ho sebelisoa sisteme ea Photobeam e le likhahla tsa ambulatory beam for 30 min. Litlhare tsena li ne li phetoa letsatsi le letsatsi bakeng sa matsatsi a 3.

Phetiso ea lefutso e kopaneng le vaerase

Re sebelisitse mekhoa e phatlalalitsoeng haholo bakeng sa phetiso ea lefutso e tsamaeang le vaerase [7,8,11,19]. Ka bokhutšoanyane, li-plasmid tsa polelo bakeng sa PSMC5 kapa bakeng sa liphetoho tse 'maloa tsa eona (bona PSMC5 le ΔFosB construtures holimo) li ile tsa fallisoa ka bicistronic p1005 (+) HSV plasmid e bonts'ang GFP tlasa taolo ea morekisi oa CMV, le PSMC5 kapa li-mutants tsa eona. Morekisi oa IE4 / 5. Tlas'a ts'oaetso ea ketamine (100 mg / kg) / xylazine (10 mg / kg) anesthesia, litoeba li ne li behiloe ka har'a sesebelisoa sa stereotaxic e nyane ea liphoofolo, 'me bokaholimo ba craneal bo ile ba pepesetsoa. Ho ne ho sebelisoa li-sinding tsa mashome a mararo a metso e meraro bakeng sa ho kenya 0.5 μl ea vet ea HSV ho NAc maemong a 10 ° (AP + 1.6; ML + 1.5; DV -4.4) ka sekhahla sa 0.1 μl / min. Liphoofolo tse amohelang liente tsa HSV li ile tsa lumelloa ho fola matsatsi a 2 kamora ho buuoa pele ho liteko.

Lipalo-palo li

Li-ANOVA le liteko tsa moithuti li ile tsa sebelisoa, tsa lokisoa bakeng sa lipapiso tse ngata, 'me bohlokoa ba tsona bo behiloe ho p <0.05.

Results

PSMC5: Molekane ea tlatsang oa rialo oa ΔFosB

Re entse liteko tsa pele ho fumana sekhechana se loketseng sa ΔFosB se sebelisitseng lithebetso tse peli tse sa tsoaloang ntle le ho thula tsamaiso. Holo-ΔFosB e ile ea susumetsa ts'ebetso ea mofuta oa reporter ka bo eona, joalo ka karolo ea N-terminal 1-78 amino acid ea protheine. Leha ho le joalo, N-terminal e qobelletse ΔFosB (Setšoantšo sa 1A), e bitsoang Δ2ΔFosB, e haellang li-amino acid tsa 78 tsa pele tsa protheine, ha e na phello. Ka hona, re sebelisitse Δ2ΔFosB e le protheine ea Bait.

Ho shebella balekane ba ka re tlamang, re sebelisitse laebrari ea boko ea panya e patiloeng ho pPC86. Re fumane likhetho tsa 11 bakeng sa balekane ba tlamang. Le ha liprotheine tsena li ne li kenyelletsa balekane ba tsejoang ba ΔFosB ba heterodimerization, c-Jun le JunD (Lethathamo 1), moemeli ea hlaheletseng ka ho fetesisa e ne e le PSMC5. Le ha ho makatsa, ena e ne e le tharollo e khahlisang, hobane PSMC5 e bonts'itsoe tlalehong e le 'ngoe lilemong tse fetileng ho tlama ho c-Fos in vitro [21]. Leha ho le joalo, ha ho na litlaleho tsa pele tsa ho nka karolo ha PSMC5 ts'ebetsong ea koae. Leha ho le joalo, ka lebaka la matla a lets'oao la PSMC5 molemong oa mofuta oa bobeli oa mofuta oa tsoekere, re ikemiselitse ho tsoela pele ho ithuta litšebelisano tsa ΔFosB-PSMC5.

ponahalo e nyane
Tafole 1. Liphetho tsa ho hlahlojoa ha Yeast Two-Hybrid le Δ2ΔFosB.

doi: 10.1371 / journal.pone.0126710.t001

Taba ea mantlha, ho netefatsa tšebelisano pakeng tsa ΔFosB le PSMC5, re ile ra etsa liteko tsa vitro co-immunoprecipitation. Re fumane hore CRAG-tagged PSMC5 (Setšoantšo sa 1B), e fetisetsoang lisele tsa Neuro 2A, e theotsoe ka tlase ΔFosB (Setšoantšo sa 1C). Taba ea bobeli, ho supa sebaka ho PSMC5 e ikarabellang ho ΔFosB, re hlahisitse PSMC5 mutants tse 'maloa tsa FLAGCNUMX (Setšoantšo sa 1B) mme a pheta tlhahlobo ea kopanetsoeng ea kopanetsoeng. ΔFosB e theotsoe fatše ka katleho le li-amino acid tsa N-terminal 151 tsa PSMC5 (PSMC5-NT), empa eseng ka C-terminal 172 amino acid sekhechana sa protheine (PSMC5-CT) (Setšoantšo sa 1C). PSMC5 e haelloa ke "coil domain" (PSMC5-ΔCC) e ne e boetse e sa sebetse ho ΔFosB. Liphumano tsena li fana ka maikutlo a hore PSMC5 e kopanya ΔFosB ka se-coil-coil domain ea eona (amino acid acid 27-68). Ntle le moo, FLMC5 e kentsoeng ke CRAG ha ea ka ea fana ka phetoho ea ofFosB e nang le domain e fetotsoe ea leucine zipper (domainFosB-LZM) (Setšoantšo sa 1C), e bonts'a hore ΔFosB e tlama PSMC5 ho tsoa ka domain name kapa, mohlomong, hore ΔFosB heterodimerization ea hlokahala bakeng sa PSMC5 binding.

PSMC5-ΔFosB e tlama ho NAc kamora taolo ea koae e sa foleng

Ho ipapisitsoe le liphumano tsena tse fumanehang ka vitro, re ithutile hore na maemo a PSMC5 ho NAc a fetotsoe ho latela taolo ea koae e sa foleng. Re fumane ka subcellular dikarolo le Western blotting hore cocaine e sa foleng e eketsa maemo a nyutlelie ea PSMC5 sebakeng sena sa boko ntle le phetoho maemong a cytoplasmic (Setšoantšo sa 2A). Tšusumetso ena ha ea ka ea bonoa kamora tekanyetso e le 'ngoe ea cocaine (ya data e sa bontsitsoeng). Ka mor'a moo re ile ra hlahloba ho fumana tikoloho ea PSMC5 le ΔFosB ho NAc ka li-microscopy tsa confocal. Re ile ra sekaseka litoeba 24 hr kamora lethal dose la ho qetela la koae, e leng nako eo ka eona ΔFosB e leng eona feela e ka bonoang FosB sehlahisoa sa gene (bona Nestler 2008). Re fumane liente tse matla tsa PSMC5 ho NAc, ho kenyelletsa le lets'oao le matla la nyutlelie. ~ 85% ea ΔFosB + nuclei e kopantsoeng le PSMC5 (Setšoantšo sa 2B). Ntle le moo, re entse liteko tsa Co-immunoprecipitation ho li-extc tsa NAc mme ra fumana hore, kamora kalafo e sa foleng ea koae, ΔFosB e ile ea theoloa fatše ka mokhoa o motle ke anti-PSMC5 antibody (Setšoantšo sa 2C). Ka lehlakoreng le leng, tlhahlobo ea NA drugve NAc ea lithethefatsi (kamora ho entoa khafetsa ka saline) e senotse hore ΔFosB ha e hlahe (data e sa bonts'itsoeng). Lintlha tsena li lumellana le seo re se fumaneng moetlong oa sele le ho netefatsa hore ΔFosB le PSMC5 li ikopanya ho NAc ho vivo.

ponahalo e nyane
Mofuta oa 2. PSMC5 regulation ho mouse NAc.

 

Ho hlakola ha Bophirimela likaroloana tsa nyutlelie le cytosolic tsa NAc ea litoeba tse tšoaroang letsatsi le letsatsi ka letsoai kapa k'hok'heine (20 mg / kg) matsatsi a 7, le liphoofolo li hlahlobiloe 24 hr kamora ente ea ho qetela. Cocaine e eketsa nyutlelie empa eseng cytosolic ea PSMC5. Histone H3 le ß-actin, tse neng li sa angoa ke k'hok'heine, li sebelisitsoe e le taolo ea ho kenya thepa. Lintlha li bolela ± SEM (n = 8-10 / sehlopha, * p <0.05). Co-localization ea endo native PSMC5 (green) le ΔFosB (putsoa) ho NAc ea litoeba tse alafshoang khafetsa ka cocaine joalo ka li-lysates tsa nyutlelie tsa AC kamora kalafo e sa foleng ea cocaine e ile ea fuoa immunoprecipitation ka anti-PSMC5 antibody kapa mouse IgG joalo ka taolo , 'me joale Bophirimela ba hlakoloa ka lesole la anti-FosB / ΔFosB. Palo e bonts'a litšebelisano tsa PSMC5-ΔFosB ho NAc ho vivo. Boitsebiso ho B le C bo ile ba phetoa ka makhetlo a mararo ho e 'ngoe le e' ngoe ea liteko tse arohaneng.

doi: 10.1371 / journal.pone.0126710.g002

PSMC5 e ntlafatsa polelo ea ΔFosB ka vitro

Kaha PSMC5 ke setho se tsebahalang sa mofuta oa protheine, re ile ra leka hore na e laola maemo a ΔFosB a sebelisa lisele tsa Rat 1A. PSMC5 overexpression e ne e sena phello maemong a basil a ΔFosB, empa e bakile ntlafatso e nyane empa e le ea bohlokoa ea ho kenyelletsoa ha uponFosB holim 'a lisele tse khothatsang tsa lisele (Setšoantšo sa 3A). Mokhoa o tšoanang o ile oa bonoa bakeng sa FosB ea bolelele bo felletseng empa litlamorao ha lia ka tsa fihlela bohlokoa ba lipalo. Ka lehlakoreng le leng, khatello ea polelo ea tlhaho ea PSMC5 liseleng tsa Rat 1A, e fihlelletsoeng ka ts'ebeliso ea li-siRNA tse lebileng PSMC5, ha ea ka ea ama maemo a basal ΔFosB empa a thibela ka matla ΔFosB ho kenella ka serum e susumetsang (Setšoantšo sa 3B). Litlamorao tse tšoanang li ile tsa bonoa bakeng sa FosB ea bolelele bo felletseng. Lintlha tsena li fana ka maikutlo a hore PSMC5 ha e khothaletse ho senyeha hoa protheine ea ΔFosB, joalo ka ha ho ka lebelloa joalo ka karoloana ea mantlha ea protheine, empa ho e-na le moo e hlokoa ho bokelloa ka botlalo FosB Lihlahisoa tsa gene ka vitro, mohlomong ka ho tiisa protheine.

ponahalo e nyane
Mofuta oa 3. PSMC5 regulation of FosB / ΔFosB expression in Rat 1A cell.

 

Lisele tsa Rat 1A li fetisitsoe ka 4 μg ea PSMC5 kapa DNA ea taolo. Tlhaloso e feteletseng ea PSMC5 e ne e se na phello maemong a basal expression a FosB kapa ΔFosB protein joalo ka ha ho laetsoe ke Western blotting, empa e hlahisitse keketseho e nyane empa e le kholo ho kenngoe ha osBFosB ka ho hlohlelletsa serum (F (2,21) = 9.75, p = 0.001). Lisele tsa Rat 1A li fetisitsoe ka 5 pmol ea e 'ngoe ea li-siRNA tse peli kapa RNA e laoloang (control). Li-siRNA ka bobeli li fokotsehile hantle liprotheine tsa PSMC5 ha li bapisoa le maemo a taolo (siRNA # 1, 23 ± 5% ea taolo; siRNA # 2, 18 ± 6%; p <0.05; n = 4). Ho kokota ha PSMC5 ho ne ho se na tšusumetso maemong a basal a FosB kapa ΔFosB empa e entse hore ho kenngoe ha FosB le ΔFosB ka ho hlohlelletsa serum (FosB: F (2,6) = 20.99, p = 0.002; osBFosB: F (2,6) = 22.83 , p = 0.002).

doi: 10.1371 / journal.pone.0126710.g003

Foromo ea ΔFosB le PSMC5 e na le CBP, p300, le BRG1 ho NAc

Ho utloisisa ka nepo mekhoa e hatisang eo PSMC5 e ka susumetsang ts'ebetso ea ΔFosB, re fuputse lilekane tse ling tse tlamang bakeng sa liprotheine tse peli tsa NAc tlasa maemo a sa phekoleng a koae. Ho na le tlaleho e le 'ngoe eo PSMC5 e tlamellang ho CBP-a HAT-mme e eketsa histone H3 acetylation ho projeke ea proximal ea MHC-II liseleng tsa HeLa [22]. Ho feta moo, litoeba tse haelloang ke CBP li bonts'a maikutlo a fokotsehileng a boitšoaro ba "cocaine" hammoho le "histone acetylation" e fokolitsoeng FosB morekisi [23]. Kahoo re ile ra leka hore na PSMC5 e kanna ea tlama le ΔFosB e le karolo ea li-complexes tse nang le CBP mohlomong le li-activator tse ling tse hatisang.

Re qalile re bonts'a hore ΔFosB e thetsitse CBP le p300 ka katleho, e amanang le HAT, liseleng tsa Neuro2A (Setšoantšo sa 4A). Ka lehlakoreng le leng, mofuta oa "leucine zipper mutant" oa ΔFosB, joalo ka ha ho lebelletsoe, ha oa ka oa bontša ketsahalo ena. Ka mokhoa o ts'oanang, PSMC5 e hutsitse hantle CBP le p300 (Setšoantšo sa 4B). Ho khahlisang, phello ena e boetse e bonoa bakeng sa PSMC5-ΔCC, e sa kang ea theola ΔFosB, e bonts'a hore PSMC5 e sebelisana le CBP le p300 ka likarolo tse ling tsa protheine mme ka boikemisetso ba eona e tlama ho ΔFosB.

ponahalo e nyane
Mofuta oa 4. ΔFosB le PSMC5 e sebelisana le CBP, p300, le BRG1 ho vitro le vivo.

 

Lisele tsa Neuro2A li ile tsa fetisetsoa ka 2.4 μg ea MYC-tagged ΔFosB kapa MYC-tagged ΔFosB-LZM. Lintho tse qotsitsoeng ka seleng li ne li sa thibeloe ka anti-CBP kapa anti-p300 antibody, le li-precipitates li ne li koetsoe ka bophirima ka anti anti e tšoanang kapa ka anti-MYC antibody. Ka bobeli CBP le p300 li sebelisana le ΔFosB 'me litšebelisano tse joalo li hloka zipper e hlakileng ea leucine. Lisele tsa Ne NeX2A li ile tsa fetisoa ka 2.4 μg ea FLAGC tagged PSMC5 kapa FLAG-tagged PSMC5-ΔCC. Lintho tse qotsitsoeng ka seleng li ne li sa thibeloe ka anti-CBP kapa anti-p300 antibody, le li-precipitates li ne li koetsoe ka bophirima ka antibody e tšoanang kapa ka anti anti-FLAG. Ka bobeli CBP le p300 li sebelisana le PSMC5 'me litšebelisano tse joalo ha li hloke domain name ea CC. C. Li-lysates tsa nyutlelie ea mouse NAc kamora kalafo e sa foleng ea koae e ile ea kenngoa immunoprecipitation ka anti-CBP kapa anti-p300 antibody. Ho thibeloa hoa morao-rao ka bophirimela ha litla-morao tse tlisoang ke anti-FosB / osFosB li bonts'itse tšebelisano lipakeng tsa ΔFosB le CBP / p300. D. Aliquots ea lysate tse tšoanang tsa nyutlelie e ile ea kenngoa chankaneng ea anti-BRG1 kapa anti-PSMC5 antibody, ea lateloa ke ho thibeloa ha litheko ka bophirimela ka anti-FosB / ΔFosB kapa anti-BRG1 antibody. Liphetho li bonts'oa tšebelisano lipakeng tsa ΔFosB le BRG1, le BRG1 le PSMC5. E. Setšoantšo sa Schematic sa phetisetso ea phetisetso ea khatiso e entsoeng ke ΔFosB: li-heterodimers tsa JunD tse sebelisanang le CBP / p300, BRG1, le PSMC5.

doi: 10.1371 / journal.pone.0126710.g004

Ho netefatsa hore litšebelisano tsena li etsahala le ho vivo, re ile ra tsamaisa koae ka mokhoa o sa tsitsitseng ho susumetsa maemo a ΔFosB le li-PSMC5 tsa nyutlelie, ebe re kenya li-extcacts tsa NAc ka antibodies ea anti-CBP kapa anti-p300. E lumellana le datha ea rona ea semelo sa sele, immunoprecipitation of CBP kapa ea p300 e theotsoe ka tlase ΔFosB (Setšoantšo sa 4C). Re ile ra leka hore na BRG1, semelo sa mantlha sa SWI-SNF chromatin remodeling tataiso, se ka boela sa tlama ho ΔFosB le PSMC5, ho latela tsebo ea rona ea pejana ea hore BRG1 e hloa mefuta e meng ea ΔFosB e kopaneng le ts'ebetso ea eona ho NAc kamora kalafo e sa foleng ea cocaine [24]. Re fumane hore immunoprecipitation ea BRG1 e theotsoe ΔFosB ho li-extracts tsa NAc, le hore ts'ireletso ea PSMC5 ka mokhoa o ts'oanang e kopantsoe le melao ea BRG1 (Setšoantšo sa 4D). Ha li kopantsoe hammoho, liphetho tsena li fana ka maikutlo a hore BFosB-PSMC5 form complexes in NAc eo hape e kenyeletsang CBP / p300 le BRG1 (4E ea setšoantšo).

PSMC5 overexpression e eketsa likarabo tsa locomotor ho koae

Tlamo e tummeng ea PSMC5 e nang le ΔFosB ho NAc e re susumelelitse ho etsa lipatlisiso hore na ho eketsa maemo a PSMC5 sebakeng sena sa boko ho laola likarabo tsa boitšoaro ho koae. Re hlahisitse vepta ea Herpes Simplex Virus (HSV) e atisang ho penta PSMC5 kapa e 'ngoe ea liphetoho tsa eona' me ra netefatsa lierekisi ho NAc ho vivo (Setšoantšo sa 5A). Polelo e kopanngoang le bongata ea li-predominates tsa PSMC5 ka har'a seleng ea sele (Setšoantšo sa 5B). MCC ea mofuta o mong o feteletseng oa mofuta oa hlaha oa PSMC5 ha ea ka ea bonts'a likarabo tse fetotsoeng litekanyetsong tsa cocaine tsa pele, empa e bonts'itse ts'ebetso e atolositsoeng ea karabelo ha e arabela litekanyetso tse phetoang tsa koae ha li bapisoa le litoeba tsa taolo ea GFP. Ho fapana le moo, litoeba tse ngata li hatisa mofuta o feto-fetohang oa PSMC5 e sa nang le sebaka sa eona sa coil-coil (PSMC5-ΔCC) ha ea hlahisa phello ena (Setšoantšo sa 5B). Ho khahlisang ke hore overexpression ea PSMC5-K196M, e haelloang ke ts'ebetso ea ATPase ea protheine ea naheng, le eona e ka 'na ea e-ba le likarabo tse ngata tsa koae.

ponahalo e nyane
Mofuta oa 5. PSMC5 overexpression ho NAc e eketsa likarabo tsa locomotor ho koae.

 

A. Moemeli oa HSV-mediated expression transgene ho NAc e bohareng. AC, ho qala ka pele. Likarolo tsa NAc tsa mantlha le likhetla li tsejoa ho palo eo. Bophahamo bo phahameng ba boemeli (60x) ba lits'oants'o tsa immunohistochemical tsa PSMC5 ho li-neuron tsa NAc kamora ente ea HSV-PSMC5 e bonts'ang hore protheine ke matla a nyutlelie joalo ka ha a tšoailoe ke DAPI. Litoeba li fumane liente tsa HSV tse kopaneng ho NAc e lateloa ke liente tsa IP tsa letsatsi le letsatsi tsa tekanyetso ea cocaine (7.5 mg / kg). Likarabo tsa locomotor li bonts'itsoe ho arabela ea pele le ea ho qetela ea litekanyetso tse 3 tsa letsatsi le letsatsi tsa moriana. Ho hatelloa haholo ha PSMC5 kapa PSMC5-K196M ho eketsa likarabo tsa motlakase ho cocaine e phetoang, phello e sa bonoeng le PSMC5-ΔCC. Ho ne ho se na tšusumetso e kholo ho li-transgen likarabong tsa motlakase ho litekanyo tsa pele tsa cocaine. ANOVA F (3,125) = 4.163, * p <0.05 ke teko ea Dunnett ea posthoc.

doi: 10.1371 / journal.pone.0126710.g005

Puisano

Liphetho tsa boithuto ba hona joale li senola mochine o mocha oo ΔFosB e arolelanang litlamorao ho bokong le mochine o mocha o kenyang ts'ebetso ea koae. Ka ts'ebeliso ea mokhoa o sa tsubeng, oa mofuta oa bobeli oa mofuta oa tsoekere, re khethile PSMC5 e le molekane ea tlamang oa ΔFosB. Re netefalitse phumano ena ka bobeli ka lisele tse cultured in vitro le NAc ho vivo ka ho bonts'a matla ea PSMC5-ΔFosB e tlamang. Habohlokoa, maemo a nyutlelie a PSMC5 a kenngoa ho NAc ke tsamaiso ea koae e sa foleng. Re bontšitse hape hore PSMC5-ΔFosB binding e etsahala ka har'a concert le liprotheine tse ling tse 'maloa tsa transactional activator, e leng CBP le p300 (li-HAT tse peli) le BRG1 (senotlolo sa mantlha sa mefuta-futa ea ntlafatso ea SWI-SNF chromatin). Hammoho, liphumano tsa rona li ts'ehetsa maikutlo a hore PSMC5 ke karolo ea ts'ebetso ea ts'ebetso ea mongolo e nkuoang bonyane mefuta e itseng ea ΔFosB e kentsoeng nakong ea tsamaiso ea cocaine e sa foleng.4E ea setšoantšo). Ho lumellana le khopolo ena ke ho fumana ho eketsehileng hore ho feta ha PSMC5 ho NAc, joalo ka vereFosB ka bo eona, e khothaletsa karabelo ea boitšoaro ho koae. E ka ba ho khahlisang lithutong tsa nako e tlang ho latela tsena liphatlalatsong tsa vivo tse nang le tšebelisano-hloko ea litšebelisano tsa PSMC5-ΔFosB-HAT-BRG1 ka tšebeliso ea lipapatso tsa mo-vitro.

Ho kenya letsoho ha PSMC5 ts'ebetsong ea koae ke pale e ncha. E tsebisoe pele e le setho sa lelapa le leholo la ATPases le bopileng protheine, PSMC5 e bonts'itsoe ho theosa le lilemo ho sebetsana le lintlha tse 'maloa tsa mongolo, ho kenyelletsa c-Fos, p53, li-receptor tsa lihormone tsa nyutlelie, le likarolo tsa mochine o moholo oa ho ngola oa basal [25], leha ho le joalo, ho entsoe lithuto tse fokolang tse sebetsang ho theosa le lilemo [26]. Ketso ea bona e hlophisitsoeng hantle ke ho nts'etsapele ts'ebetso ea lintlha tsa mongolo oa MYC liseleng tse ferekaneng [27]. Moelelo oa PSMC5 lits'ebetsong tsa mongolo o khothalelitse karolo e ka etsahalang bakeng sa ts'ebetso ea ubiquitination-proteinasomal taolong ea phetisetso ea liphatsa tsa lefutso, empa ho kenya letsoho ha PSMC5 molomong o joalo ho lula ho sa tsejoe ho fihlela joale [28,29].

Hanyane hanyane ho tsejoa ka ts'ebetso ea PSMC5 bokong. Phuputso ea pejana e bonts'a polelo e atileng ea PSMC5 mRNA ho pholletsa le bokong [30], empa ts'ebetso ea eona e sebetsang e lutse e sa laoloe. Seo re se fumaneng hona joale se etsa hore lipatlisiso tse eketsehileng tsa protheine ena e khahlisang li utloisise karolo ea tsona ea ho tsamaisa phetisetso ea liphatsa tsa lefutso le kamano ea tsona ho ts'ebetso ea li-ubiquitination-proteinasomal bokong. Tlamo ea PSMC5 ho ΔFosB e kopantsoe le sebaka sa coil-coil sa coMC5. Ho feta moo, bokhoni ba PSMC5 ba ho khothaletsa likarabo tsa koae, ha bo ntse bo hloka sebaka sa coil-coil, ha bo hloke tšebetso ea ATPase e kenelletseng liprotheine. Liphetho tsena li hlahisa monyetla oa hore, bonyane ka har'a sistimi ea rona, ts'ebetso ea mantlha ea PSMC5 e kanna ea kopanngoa ka ho tlama ho eona ho ΔFosB le liprotheine tse ling tsa taolo tse ngotsoeng le eseng ka ts'ebetso ea eona e amanang le proteinasom ka ngoe. Ho hlokahala mosebetsi o eketsehileng ho leka sena ka kotloloho le menyetla e meng. Motsoako o fetisang karabelo ea kankere ea methapo ea bongata ea PSMC5 o eketsehile mabapi le koae ka ho sebelisana le ΔFosB hoa utloahala, leha ho sebelisoa ts'ebeliso ea mofuta oa 3 oa kalafo ea koae, hobane hoa tsebahala hore maemo a ananelang osFosB a bokellana bokong ka nako ena.3].

Se fumanoeng hajoale se tiisa ts'ebeliso ea ts'ebeliso ea mekhoa e sa lekanyetsoang le e sa bonoang ea ho ithuta ka motheo oa molek'hule ea taolo ea boko. Tlhokomelo ea rona ea pele ho PSMC5 e ne e thehiloe feela boikhethong ba eona bo tummeng ba ho osFosB ka hara mofuta o le habeli oa mofuta o kopaneng oa hybrid, leha ho le joalo e bonahala e le karolo ea bohlokoa ea liphetoho tse ngotsoeng tse nkuoang hape ho NAc ke tsamaiso ea khafetsa ea koae. Kutloisiso e ntlafalitsoeng ea mekhoa e qaqileng eo maemo a nyutlelie ea PSMC5 a qobelloang ke koae, 'me ka nako eo, PSMC5 ebe e kenya letsoho ho li-activation tse hlahisang koae tseo e leng mohopolo lipatlisisong tsa hajoale. Khabareng, tlhahlobo ea rona ea boea e habeli habeli e senotse balekane ba 'maloa ba tlamang ba ΔFosB (Lethathamo 1) eo hape e netefatsang tlhahlobo e tobileng mefuteng ea koae. Ka kopanelo, mosebetsi ona o kenya letsoho kutlwisisong e ntseng e eketseha ea methapo e rarahaneng ea limolek'hule tseo mosebetsi oa cocaine alter NAc o sebetsang ho tsona.

lumela hore baa fokola

Mosebetsi ona o tšehelitsoe ka lithuso tse tsoang ho National Institute on Drug Abuse, le Ishibashi Foundation le Mokhatlo oa Japane bakeng sa Kholiso ea Saense (lipalo tsa KAKENHI: 24591735, 26290064, 25116010).

Menehelo ea Mongoli

O ile a amohela le ho hlophisa liteko: YHO YNO EJN. O entse liteko: YHO YNO PJK RN. Hlahloba data: YHO YNO EJN. Li-reagents tse kentsoeng / lisebelisoa / lisebelisoa tsa tlhahlobo: TN MO OY AN RN TT. Ngola pampiri: YHO EJN.

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