Preparation of full-length deubiquitinating complex Ubp3/Bre5 and characterization of interaction with Cdc48

Su Wencheng, Lyu Cao, Shi Lili, Jing Xiaofei,Gai Yuanming, Zhang Jie, Tan Huanbo, Wang Pengju,Xia Lixin, Zou Peijian, and Qin Gang?

1) College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, P.R.China 2) National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences, Tianjin 300308, P.R.China 3) Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical Research,Tianjin 300193, P.R.China 4) Health Science Center, Shenzhen University, State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University,Shenzhen 518060, P.R.China

?

【生物工程 / Bioengineering】

Preparation of full-length deubiquitinating complex Ubp3/Bre5 and characterization of interaction with Cdc48

Su Wencheng1,2, Lyu Cao1,2, Shi Lili3, Jing Xiaofei1,2,Gai Yuanming2, Zhang Jie2, Tan Huanbo2, Wang Pengju2,Xia Lixin4, Zou Peijian2, and Qin Gang2?

1) College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, P.R.China 2) National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences, Tianjin 300308, P.R.China 3) Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical Research,Tianjin 300193, P.R.China 4) Health Science Center, Shenzhen University, State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University,Shenzhen 518060, P.R.China

Ubiquitination modification is a dynamic process essential for eukaryotic cell physiology. Ubp3, theSaccharomycescerevisiaehomologueofhumandeubiquitinaseUSP10,togetherwithitscofactorBre5,playsanactiveroleinnumerouscellularprocesses.AlthoughBre5isessentialforUbp3functioninvivo,unfortunately,duetodifficultyinpreparingcriticalquantitiesofintactfunctionalUbp3andUbp3/Bre5reconstitute,systemiccharacterizationonthiscomplexislacking.Hence,howexactlyBre5regulatesUbp3activitystillremainselusive.Tofillthisgap,wereportthesuccessfulexpressionandpurificationofrecombinantUbp3andBre5inEscherichiacoliinmonomericandcomplexform.Toourknowledge,thisisthefirstreportthesuccessfulpreparationoffull-lengthUbp3/Bre5proteincomplexinlargescale,whichallowsustoobtainfurtherunderstandingofmolecularbases.ThestoichiometricinteractionbetweenpurifiedUbp3andBre5confirmedproperfoldingoftheseproteins.ToassesstheproposeddirectinteractionsbetweenUbp3andBre5withtheubiquitinselectiveATPaseassociatedwithavarietyofcellularactivities(AAAATPase)Cdc48,seriesofpull-downassaysareperformed;resultsrevealthat,neitherUbp3norBre5aloneisabletobindCdc48.However,theUbp3/Bre5complexcouldbindCdc48efficiently,whichprovidsnovelinsightonUbp3/Bre5-Cdc48interactionmode.Insummary,ourresultslaythefoundationforfuturemechanisticevaluationbybothbiochemicalandstructuralmeans.

protein binding; deubiquitinase Ubp3; cofactor Bre5; ATPase Cdc48; deubiquitinating complex; GST-pulldown; direct interaction

The dynamic balance between ubiquitination and deubiquitination, the two reversal post-translational regulation processes, plays a vital role in eukaryotic cell physiology facilitated by highly specific catalytic machinery[1]. Deubiquitination is catalyzed by deubiquitinating proteases (DUBs), which are mainly categorized into five groups based on structural homology: ubiquitin-specific processing proteases (USPs/UBPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor domain-containing proteases (OTUs), Ataxin-3-like proteases and Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+)(JAmmol/L) proteases[2-3]. Among DUBs, UBPs represent the most abundant type. In theSaccharomycescerevisiaegenome,atleast16UBPencodingsequenceshavebeendiscovered.Noneofthemisessentialforcellviability,butcertainindividualmutantsexhibitpleiotropicabnormalities,implicatingimportantandwidespreadrolesincellularfunctions[4-5].

Ubp3,theSaccharomycescerevisiaehomologueofhumanUSP10,hasbeenshowntobeinvolvedinregulatingmultiplecellularprocesses,includingDNArepair[6-7],transcriptionregulation[8-9],signaltransduction[10-11],anterograde/retrogradetransport[12]andribophagy[13].OnepositiveregulatornamedBre5directlyinteractswithUbp3andisindispensableforUbp3function[12].InitialstructuralcharacterizationhasrevealedthatUbp3andBre5formasymmetricheterotetramerinwhichtheBre5NTF2-likedomaindimerinteractswithtwoN-terminalmotifsofUbp3withapparent1∶1stoichiometry[14-15].TheeffectofcofactorBre5onUbp3istoeitherfacilitatesubstratetargeting,ortomodulateitscatalyticactivityortoachieveboth;unfortunately,directprooffrombiochemicalandstructuralaspectsisstilldeficientduetodifficultyinpreparingthelargeUbp3andfull-lengthfunctionalUbp3/Bre5reconstitute.Recently,oneintriguingconnectionoftheubiquitin-selectivechaperonCdc48,anditscofactorUfd3toUbp3/Bre5mediatedribophagywasproposed.Ubp3andBre5wereshowntointeractwithCdc48andUfd3directly[16].However,themolecularbasisontheseinteractionsandfunctionalmechanismunderlyingthemremaintobepreciselyevaluated.

Priorattemptstopurifyrecombinantfull-lengthUbp3werenotverysuccessful[15].WefindthatthiswasmostlyduetotheintrinsicinstabilityofN-terminalregionofUbp3 (ourunpublisheddata).ToinitiatesystemiccharacterizationofthedeubiquitinatingcomplexUbp3/Bre5,wereportsuccessfulexpressionandpurificationofrecombinantUbp3andBre5inamonomericandcomplexform,throughtheEscherichiacoli(E.coli)expressionsystem.Utilizingthesepurifiedsamples,wecarefullyassesstheinteractionsbetweenCdc48andUbp3/Bre5invitro.Asfarasweknow,thisisthefirstreportonthesuccessfulpreparationofafull-lengthUbp3/Bre5complexinlargescale,whichallowsustoobtainfurtherunderstandingofmolecularbasisoftheUbp3/Bre5viabiochemicalandstructuralbiologymeans.

1 Materials and methods

1.1Materials

E.coliDH5α,E.coliBL21 (DE3),E.colitrx(DE3)andT4DNALigasewerepurchasedfromBeijingTransGenBiotech(Beijing,China).pGEX-4T-1,pET-28aandpET-32-Bre5 were obtained from our laboratory. Restriction enzymes were purchased from Fermentas Life Sciences (Vilnius, Lithuania). Es Taq DNA Polymerases were purchased from Beijing CoWin Bioscience Co., Ltd (Beijing, China). Extraction Kit, Plasmid Mini Kit and Cycle-pure Kit were purchased from OMEGA Bio-Tek (Norcross,GA). Primers were ordered from Shanghai Sangon Biotechnology (Shanghai, China). IPTG was purchased from Sigma (CA, USA). Recombinant glutathione S-transferase (GST), GST-Cdc48 and 6×His-Cdc48 were previously prepared as reported[17].

1.2 Construction of the expression plasmids

TheUbp3encodingsequencewasamplifiedfromSaccharomycescerevisiaegenome,withprimersdesignedwithrestrictionendonucleasecloningsitesEcoRIandXhoI(Table1).ThePCRreactionswerecarriedoutas:Step1 94 ℃,2min;Step2 94 ℃,30s, 55 ℃,30s, 72 ℃,2min;Step3 72 ℃, 10min,with25cyclesofstep2.TherecoveredPCRproductandvectorpGEX-4T-1weredigestedwithEcoRIandXhoIrestrictionenzymesandwereligatedviaT4ligase,thentransformedintoE.coliDH5αcells.PositiveclonesdesignatedaspGEX-4T-1-Ubp3wereselectedbycolonyPCRandwereverifiedbyDNAsequencing.

DNAfragmentsencodingBre5genewasamplifiedbyPCRfromapreviouslyconstructedpET-32-Bre5 plasmid with primers designed with restriction endonuclease cloning sitesNcoIandXhoI(Table1).PCRreactionswerecarriedoutas:Step1 95 ℃, 2min;Step2 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min30s;Step3 72 ℃, 10min,with25cyclesofstep2.TheamplifiedBre5fragmentwasclonedintoapET-28avectorbythesimilarprocessdescribedabove.Positivecloneswereselectedviadoubledigestion,andthesequencingverifiedplasmidwasdesignatedaspET-28a-Bre5.

Table 1 PCR primer sequences1)圖1 PCR擴增使用引物

1)Restriction sites are underlined.

1.3 Expression trials of recombinant Ubp3 and Bre5

pGEX-4T-1-Ubp3 and pET-28a-Bre5 plasmids were transformed intoE.colitrx(DE3)andE.coliBL21(DE3)cellsrespectively.AsinglecolonywasinoculatedintoaLBmediumsupplementedwithproperantibiotics(100μg/mLampicillinor50μg/mLkanamycin)andcultivatedovernightat37 ℃withvigorousshaking.Theovernightculturesweredilutedinfreshpre-warmedmedium(includingproperantibiotics)andgrownat37 ℃withvigorousshaking,untiltheOD600reached0.5-0.7.ProteinexpressionwasinducedbyaddingIPTG(finalconcentrationat0.2mmol/LforGST-Ubp3 induction and 0.1 mmol/L for 6×His-Bre5 induction), and cultures were collected after overnight growth at either 25 ℃ or 16 ℃. Samples were sonicated and fractionated, and whole cell lysate, supernatant, and pellet fractions were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Coomassie staining.

The co-expression experiment was essentially described above, except that both pGEX-4T-1-Ubp3 and pET-28a-Bre5 were co-transformed intoE.coliBL21(DE3)inthepresenceofantibiotics(100μg/mLampicillinplus50μg/mLkanamycin).

1.4PurificationofUbp3andBre5

TopurifyrecombinantGST-Ubp3inlargescale,cultivatedcellswithoptimalinductionwereharvestedbycentrifugation(4 500r/min, 20min, 4 ℃)andre-suspendedinicecoldbuffer(PBS,pH=7.4, 0.4mmol/LofPMSF, 1×proteaseinhibitor, 3mmol/LDTT)andlysedviaFrenchpress.Thelysatewasthencentrifugedat15 000r/minfor30minat4 ℃,withthesupernatantappliedtoGlutathioneSepharoseresin(GEHealthcarecat. #17-5132-03)pre-equilibratedwithPBSandincubatedwithrotatingfor4hat4 ℃.Thecolumnwaswashedwithequilibrationbuffer(PBScontaining3mmol/LDTT, 0.1%TritonX-100),andtheproteinwaselutedwithelutionbuffer(50mmol/LTris-HCl,pH=8.0, 200mmol/LNaCl, 10mmol/Lglutathione).TheelutionfractionswereanalyzedviaSDS-PAGEandCoomassiestaining.Selectedelutionfractionswerecombinedanddialyzedagainstice-colddialysisbuffer(50mmol/LTris-HCl,pH=7.5, 50mmol/LNaCl, 5%glycerol),thenappliedtotheSPSepharoseFF(GEHealthcarecat. #17-0929-01)pre-equilibratedwithanice-colddialysisbuffer.Afterbeingwashedwith10bedvolumesofthesamebuffer,thecolumnwaselutedwithanelutionbuffercontainingastepwiseincreaseinsaltconcentration(0.2, 0.3and0.4mol/LNaCl,respectively).TheelutionfractionswereanalyzedviaSDS-PAGEandCoomassiestaining.TheconcentrationofpurifiedrecombinantGST-Ubp3 protein was determined by using bovine serum albumin as a standard.

To purify recombinant 6×His-Bre5 on a in large scale, cells were harvested by centrifugation (4 500 r/min, 20 min, 4 ℃) and pellets were re-suspended in 100 mL of lysis buffer (50 mmol/L Tris-HCl, pH=7.5, 150 mmol/L NaCl and 20 mmol/L imidazole, 0.4 mmol/L of PMSF, 5 mmol/L β-mercaptoethanol). Cells were lysed via French Press. Lysates were clarified (15 000 r/min, 30 min, 4 ℃), and the supernatants were transferred to Ni Sepharose FF (GE Healthcare cat. #17-5318-03) pre-equilibrated with lysis buffer and rotated for 2 h at 4 ℃. The column was sequentially washed with a wash buffer (50 mmol/L Tris, pH=7.5, 150 mmol/L NaCl, 0.1% Triton X-100, 5 mmol/L β-mercaptoethanol) containing a stepwise increase of imidazole concentrations (20, 50 and 100 mmol/L). Then the 6×His-Bre5 protein was eluted with elution buffer (50 mmol/L Tris, pH=7.5, 150 mmol/L NaCl, 250 mmol/L imidazole, 5 mmol/L β-mercaptoethanol), and elution fractions were analyzed via SDS-PAGE and Coomassie staining. The concentration of purified recombinant 6×His-Bre5 protein was determined by using bovine serum albumin as a standard.

1.5 Preparation of Ubp3 /Bre5 complex

For the large-scale Ubp3/Bre5 complex purification, GST-Ubp3 was purified as described above, except that after GST-Ubp3 binding, sufficient amount of purified 6×His-Bre5 was applied to glutathione column and incubated for 1 h at 4 ℃.

1.6 LC-MS/MS analysis of recombinant Ubp3 and Bre5

For protein identification LC-MS/MS analysis was conducted using LTQ XL from Thermo Fisher (ESI-MS/MS). The instrument was operated with a spray voltage of 3.5 kV and an ion transfer tube temperature of 25 ℃. The information-dependent acquisition (IDA) mode of operation was employed in which a survey scan fromm/z400to1 800wasacquiredfollowedbycollision-induceddissociation(CID),andforMS/MS,usinganormalizedcollisionenergyof35%withanactivationqof0.25for30ms.IonselectionthresholdsforMSandMS/MSwere1 000and500counts,respectively.

TandemmassspectrawereextractedbytheXcaliburversion1.0.0.2.AllMS/MSsampleswereanalyzedusingSequest.IodoacetamidederivativeofCys,de-amidationofAsnandGln,oxidationofMetwerespecifiedinSequestasvariablemodifications.ProteomeDiscoverer1.2wasusedtovalidateMS/MSbasedpeptideandproteinidentifications.Peptideidentificationswereacceptediftheycouldbeestablishedatprobabilitygreaterthan95.0%asspecifiedbytheresultfilter,whichisXcorr> 1.9 if the charge is 1,Xcorr> 2.2 if the charge is 2,Xcorr> 3.75 if the charge is 3. Protein identifications were accepted if they were established at probability greater than 99.0% and contained at least 2 identified unique peptides.

1.7 GST-pulldown experiments

For pull-down assays, GST or GST fusion proteins were first incubated with 50 μL of pre-equilibrated glutathione-Sepharose beads in buffer A (50 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L DTT, 0.1% triton X-100, pH=7.5) for 1 h at 4 ℃. The beads were washed once with 500 μL of buffer A to remove unbound material and then incubated with prey proteins for 1 h at 4 ℃. Beads were washed three times with 1 mL of buffer A, followed by three times of wash with buffer B (50 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L DTT, pH=7.5), then mixed with an SDS-PAGE loading buffer and analyzed on SDS-PAGE.

2 Results

2.1 Construction of the expression plasmids

Due to the exceptional ability of GST tag to greatly enhance the solubility and stability of fused proteins, GST tag has been widely used for facilitating recombinant protein preparation; therefore we introduce a GST domain fused at the N-term ofUbp3. TheUpb3 gene was amplified usingSaccharomycescerevisiaegenomeDNAasatemplate,asinglebandatabout2.8kbwasobtained,inaccordancewiththesizeofUpb3 coding region (Fig.1(a)); the encoding fragment was inserted into bacterial expression vector pGEX-4T-1, and a positive plasmid designated as pGEX-4T-1-Ubp3 was selected via colony PCR (Fig.1(b)) and verified via DNA sequencing. TheBre5genewasamplifiedsimilarly,withafragmentofabout1.5kbobtained(Fig.1(c)),theencodingfragmentwasinsertedintopET-28atointroducea6×HistagatN-termofBre5.Thepositiveplasmid,designatedpET-28a-Bre5, was selected by double restriction enzyme digestion (Fig.1(d)) and confirmed via DNA sequencing.

(a) PCR amplification of Ubp3 coding region from Saccharomyces cerevisiae genome. Lane 1, DNA marker; Lane 2, PCR product. (b) Verification of expression plasmids pGEX-4T-1-Ubp3 by colony PCR. Lane 1, DNA marker; Lane 2-3, PCR amplified fragments verifying two positive clones. (c) PCR amplification of Bre5 coding region from a previously constructed plasmid pET-32-Bre5. Lane 1, DNA marker; Lane 2, PCR product. (d) Verification of expression plasmids pET-28a-Bre5 by restriction enzyme digestion. Lane 1, DNA marker; Lane 2-3, two positive clones digested with Nco I and Xho I.Fig.1 Ubp3 and Bre5 coding fragments amplified by PCR and verification of recombinant expression plasmids圖1 PCR擴增Ubp3和Bre5編碼片段及質粒構建驗證

2.2 Expression and purification of Ubp3

Small scale expression trials of Ubp3 inE.colitrx(DE3)wereperformedatvariousinductionconditions.Comparedtonon-inducedcondition,onebandmigratingatabout130kDabecameapparentafterIsopropylβ-D-1-thiogalactopyranoside(IPTG)inductionwith0.2mmol/LIPTGat16 ℃,whichcorrespondstotheGST-Ubp3fusion,thusresultedinarelativelyhighersolubilityoftheinducedprotein(Fig.2(a));therefore,wechosethesameinductionconditionforlargescalepreparation.AsshowninFig.2(b),afterasingle-stepglutathionecolumnpurification,GST-Ubp3wasenrichedinelutionfractions.However,thesamefractionsalsocontainedseveralcontaminatingcomponentsofdiversemolecularweight.Thesizeofaprominentcontaminantwasabout26kDa(Fig.2(b),Lane6),similartoanintactGSTdomain,whichisnotsurprisingsinceGSTtruncatesarefrequentlyco-purifiedwithGSTfusionproteins,especiallywhenthefusedpartnercontainsdegradation-proneareas.OthermajorcontaminatingproteinsappearedtobethedegradationintermediatesofUbp3,sincethesebandsremainedratherunstable,almostdisappearedduringdialysis(comparedFig.2(b),Lane6withFig.2(c),Lane1).TofurtherimprovethepurityofcombinedGST-Ubp3poolandespeciallytoremoveGSTtruncates,wecontinuedwithion-exchangechromatography.BasedontheestimatedpIsforUbp3andGST(7.9forUbp3versus4.5forGST),SPsepharosewasselected,andtheefficacyofcontaminantremovalwasshowninFig.2(c).GSTtruncatesindialysisbufferremainedpoorlyboundtoSPresinclearlyhencelargelyexistedinflowthrough(Fig.2(c),Lane2);incontrast,mostGST-Ubp3adsorbedtoSPresinatthesamecondition,andwasabletobeefficientlyelutedwhenNaClconcentrationwasincreasedto0.2-0.3mol/L(Fig.2(c),Lane6-9).Recoveredfull-lengthGST-Ubp3exhibitedsignificantimprovementonpurity(>85%),itsidentitywasuniquelyverifiedbyLC-MS/MS(Fig.3).TotalyieldsofrecombinantGST-Ubp3arelistedinTable2.

(a)Small scale expression trials of GST-Ubp3 induced at 16 ℃ (Lane 1-4) and 25 ℃ (Lane 5-8) respectively. Lane 1 and 5, total proteins of uninduced cells; Lane 2 and 6, total proteins of induced cells; Lane 3 and 7, soluble fraction of induced cells; Lane 4 and 8, insoluble fraction of induced cells. (b) GST-Ubp3 purification through glutathione column chromatography. Lane 1, soluble fraction of induced cells; Lane 2, flowthrough; Lane 3-4, wash; Lane 5-8, elution fraction. (c) Further purification of GST-Ubp3 through SP cation-exchange chromatography. Lane 1, dialyzed GST-Ubp3 pool from glutathione column chromatography; Lane 2, flowthrough; Lane 3, wash; Lane 4-6, 0.2 mol/L NaCl elution fraction; Lane 7-9, 0.3 mol/L NaCl elution fraction; Lane 10, 0.4 mol/L NaCl elution fraction.Fig.2 SDS-PAGE analysis on expression and purification of Ubp3 in E.coli圖2 蛋白電泳分析 Ubp3在大腸桿菌內的表達及純化

Amino acid sequence corresponding to Ubp3 was shown, with identified unique peptides highlighted in gray.Fig.3 (Color online) Identity verification of purified Ubp3 via tandem MS/MS圖3 MS/MS鑒定純化的Ubp3蛋白

proteinnamepurificationstagesV/mLm(targetprotein)/mgyield/%celllysate10060100glutathioneaffini-tychromatography161627GST-Ubp3Spcation-ex-changechromatography2010176×His-Bre5celllysate100150100nickleaffinitychromatography246040

2.3 Expression and purification of Bre5

In experiments parallel to Ubp3, expression trials of Bre5 inE.coliBL21(DE3)werealsoperformed.Uponinduction,oneproteinwithamolecularweightofabout70kDaappears(Fig.4(a)),whichislargerthantheexpectedsizeof6×His-Bre5 (about58kDa);thisismostlikelyduetounusualmobilityofBre5inSDS-PAGE,sincetheidentityofpurifiedproteinwasconfidentlyverifiedasBre5byLC-MS/MS(Fig.5).Targetproteininducedwith0.1mmol/LIPTGat16 ℃exhibitedrelativelybettersolubility(Fig.4(a)),sameinductionconditionwasalsoappliedtolargescalepurification. 6×His-Bre5waspreparedaccordingtostandardone-stepnickelaffinitychromatographyprocedure,asshowninFig.4(b).Theimidazoleelutionfractionswerepooledanddialyzed,totalyieldsofrecombinant6×His-Bre5aresummarizedinTable2.

(a) Small scale expression trials of 6×His-Bre5 induced at 16 ℃ (Lane 1-4) and 25 ℃ (Lane 5-8) respectively. Lane 1 and 5, total proteins of uninduced cells; Lane 2 and 6, total proteins of induced cells; Lane 3 and 7, soluble fraction of induced cells; Lane 4 and 8, insoluble fraction of induced cells. (b) 6×His-Bre5 purification through nickel affinity chromatography. Lane 1, soluble fraction of induced cells; Lane 2, flowthrough; Lane 3, wash; Lane 4, 50 mmol/L imidazole elution fraction; Lane 5-6, 80 mmol/L imidazole elution fraction; Lane 7-9, 100 mmol/L imidazole elution fraction; Lane 10-12, 250 mmol/L imidazole elution fraction; Lane 13, 500 mmol/L imidazole elution fraction.Fig.4 SDS-PAGE analysis on expression and purification of Bre5 in E.coli圖4 蛋白電泳分析Bre5蛋白在大腸桿菌內的表達及純化

Amino acid sequence corresponding to Bre5 was shown, with identified unique peptides highlighted in gray.Fig.5 (Color online) Identity verification of purified Bre5 via tandem MS/MS圖5 MS/MS鑒定純化的Bre5蛋白

2.4FunctionaltestofrecombinantUbp3andBre5

HavingsuccessfullyobtainedsolubleUbp3andBre5inhighpurity,wesoughttoconfirmwhethertheyareproperlyfoldedornot.Previously,ithasbeenwellestablishedthatUbp3andBre5physicallyinteractwitheachotherinvivoandinvitro[12, 14-15];thus,weperformedaGST-pulldowntodirectlyexaminetheinteraction.AsshowninFig.6,incontrasttoGST(Lane4),GST-Ubp3displaysastoichiometricinteractionwithBre5 (Lane5),consistentwithpreviousreports[14-15].Basedonthesedata,weconcludthatourpreparedrecombinantUbp3andBre5arefunctional.

Lane 1, GST; Lane 2, GST-Ubp3; Lane 3, 6×His-Bre5; Lane 4, pulldown sample using GST as bait and 6×His-Bre5 as prey; Lane 5, pulldown sample using GST-Ubp3 as bait and 6×His-Bre5 as prey.Fig.6 GST-pulldown assay between recombinant Ubp3 and Bre5圖6 GST-pulldown 檢測Ubp3與Bre5結合

2.5Large-scalepreparationofUbp3/Bre5complex

ThesuccessfulpurificationoffunctionalUbp3andBre5individuallypromptedustotrypreparingUbp3/Bre5complexdirectly,whichisessentialforfurtherfunctionalandstructuralcharacterization.Initially,wetookoureffortonco-expressingpGEX-4T-1-Ubp3 and pET-28a-Bre5 inE.coli.UnfortunatelytheinductionofUbp3andBre5isnotatcomparablelevel(Fig.7(a)),GST-Ubp3inductionisnearlyundetectable),preventingproductivecomplexassemblyinvivo.Tosolvethisproblem,wedevelopeda‘hybrid’procedureasshowninFig.7(b),GST-Ubp3waspurifiedaccordingtotheestablishedtwo-stepproceduredescribedabove,exceptthatafterGST-Ubp3bindingtoglutathionecolumn,sufficientamountofrecombinant6×His-Bre5wasaddedtotriggertheon-columncomplexassembly.Byfollowingthisstrategy,theglutathioneelutionfractionsdisplayednearlystoichiometricdistributions

ofUbp3andBre5,indicatingsuccessfulcomplexformationonglutathionecolumn(Fig.7(c)).Moreover,successiveSPcation-exchangechromatographysignificantlyenhancedthepurityofUbp3/Bre5complexbyefficientlyremovingGSTtruncatesandothercontaminants,aprocedurecomparabletoindividualUbp3purification(Fig.7(d));interestingly,thepreformedUbp3/Bre5complexwelltoleratedhighsaltelutioncondition(0.4-0.5mol/LNaCl),presentingexceptionalstability.

(a) Small-scale co-expression trials of GST-Ubp3 and 6×His-Bre5 induced at 16 ℃ (Lane 1-4) and 25 ℃ (Lane 5-8) respectively. Lane 1 and 5, total proteins of un-induced cells; Lane 2 and 6, total proteins of induced cells; Lane 3 and 7, soluble fraction of induced cells; Lane 4 and 8, insoluble fraction of induced cells. (b) Procedure for two-round GST-Ubp3/6×His-Bre5 complex preparation. (c) The first round of GST-Ubp3/6×His-Bre5 complex preparation through glutathione column chromatography. Lane 1, soluble fraction of induced cells; Lane 2, flowthrough after 6×His-Bre5 incubation with glutathione column ; Lane 3-7, wash; Lane 8-11, elution fraction. (d) The second round of GST-Ubp3/6×His-Bre5 complex purification through SP cation-exchange chromatography. Lane 1, dialyzed GST-Ubp3/6×His-Bre5 pool from glutathione column chromatography; Lane 2, flowthrough; Lane 3-4, wash; Lane 5, 0.2 mol/L NaCl elution fraction; Lane 6-7, 0.3 mol/L NaCl elution fraction; Lane 8-10, 0.4 mol/L NaCl elution fraction.Fig.7 Preparation of recombinant Ubp3/Bre5 complex圖7 重組Ubp3/Bre5復合體的制備

2.6 Assessment of interactions between Ubp3 and Bre5 with Cdc48

Recently, the Ubp3/Bre5 complex has been linked to the ATPase associated with a variety of cellular activities (AAA ATPase) Cdc48. Ossareh-Nazari et al[16]proposed a direct interaction between Ubp3 and Bre5 with Cdc48 respectively, hence providing further evidence that Cdc48 has close crosstalk with deubiquitinating pathways. Taking advantages of Ubp3, Bre5 and Ubp3/Bre5 preparations, we directly examined the proposed interactions by performing a series of GST-pulldown experiments. To our surprise, we could hardly observe any interaction between GST-Ubp3 and Cdc48 (Fig.8(a), Lane 5), consistently. GST-Cdc48 also failed to pulldown Bre5 (Fig.8(b), Lane 3); these results are obviously contradictory to the former finding from Ossareh-Nazari et al[16]. Intriguingly, however, when the preformed GST-Ubp3/Bre5 complex was used as pulldown bait, a significant binding of Cdc48 was observed (Fig.8(c), Lane 3), indicating the assembled Ubp3/Bre5 complex is indeed able to physically interact with Cdc48. At this moment, we could not explain the discrepancy, but our results strongly suggest that more careful experiments need to be performed to uncover the real Ubp3/Bre5-Cdc48 interaction mode.

(a) GST-Ubp3 fails to interact with 6×His-Cdc48. Lane 1, 6×His-Bre5; Lane 2, 6×His-Cdc48; Lane 3, pulldown sample using GST as bait and 6×His-Cdc48 as prey; Lane 4, pulldown sample using GST-Ubp3 as bait and 6×His-Bre5 as prey; Lane 5, pulldown sample using GST-Ubp3 as bait and 6×His-Cdc48 as prey. (b) GST-Cdc48 fails to interact with 6×His-Bre5. Lane 1, 6×His-Bre5; Lane 2, pulldown sample using GST as bait and 6×His-Bre5 as prey; Lane 3, pulldown sample using GST-Cdc48 as bait and 6×His-Bre5 as prey. (c) GST-Ubp3 /6×His-Bre5 complex interacts with 6×His-Cdc48. Lane 1, 6×His-Cdc48; Lane 2, pulldown sample using GST as bait and 6×His-Cdc48 as prey; Lane 3, pulldown sample using GST-Ubp3/6×His-Bre5 complex as bait and 6×His-Cdc48 as prey.Fig.8 GST-pulldown assays between Ubp3 and Bre5 with Cdc48圖8 GST-pulldown檢測Ubp3 /Bre5 與Cdc48的結合

3 Discussions and conclusions

The pleiotropic defects ofubp3mutantindicateitswidespreadcellularfunctions.DespitetheinvolvementofdeubiquitinatingactivityofUbp3/Bre5inmultiplecellularprocessesandpartialresolutionofUbp3/Bre5interactionmode,themolecularbasisonhowBre5regulatesUbp3activityisstilllargelyunknown.TopreciselydissectthepotentialroleofBre5instepofsubstraterecognition,catalyticactivationorcrosstalkwithotherinteractingpartners,asophisticatedinvitroreconstitutionsystemishighlydemanding.ObviouslythesuccessfulpreparationoffunctionalUbp3/Bre5complexinhighhomogeneityisaprerequisite.Inthiswork,wehaveestablishedanexpressionandpurificationsystemtofulfillthisrequirement.Withthedevelopmentofasimpleandefficientpurificationstrategywecouldobtainrecombinantfull-lengthUbp3,Bre5andalsoUbp3/Bre5complexinlargescale,whichtoourknowledgehasnotbeenreportedbefore.

Cdc48,ahighlyconservedcomponentinAAAATPasefamily,wasnoticedandstudiedrecently,becauseofitsdistinctubiquitin-selectiveproperty:thehomohexamerofCdc48canactasageneralplatformformulti-purposedecisionmakingofmolecularevents,dependingonitsabilitytointeractwithplentyofcofactors,amongwhichbothubiquitinligasesandDUBsareincluded[18-19].ThediscoveryofUbp3-Cdc48interactionfurtherenrichesthetoolboxofCdc48andextendsitsactioninribophagypathway.Toconfirmthisimportantnotionandobtaindeeperinsight,wesetupaseriesofinteractionassaystoassesstheproposedone-to-oneinteractionsbetweenUbp3,Bre5andCdc48.Importantly,inoursystemwecouldnotreproducethediscoveredphysicalinteractionbetweenUbp3andBre5withCdc48,however,wecoulddetectastableinteractionbetweenUbp3/Bre5complexandCdc48,implicatingsynergisticactionofUbp3andBre5uponCdc48binding.ThisperspectivecouldpotentiallyexplaintheobligatoryroleofBre5inUbp3functioning.Undoubtedly,revisionofthecurrentworkingmodelawaitsmorecarefulexperiments.

WebelievethatthehighpurityreconstitutesofrecombinantUbp3/Bre5willcontinuouslybringdeeperinsightonmolecularpropertiesofthiscomplexinfuture.Importantly,inanestablishedinvitroenzymaticactivityassay,theUbp3/Bre5complexcanexhibittypicaldeubiquitinatingenzymeactivity(manuscriptinpreparation),whichopensupapathforsystemiccatalyticmechanismcharacterizationofUbp3.

[1] Wilkinson K D. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome[J]. Seminars in Cell & Developmental Biology, 2000, 11(3):141-148.

[2] Nijman S M,Luna-Vargas M P,Velds A,et al.A genomic and functional inventory of deubiquitinating enzymes[J]. Cell, 2005, 123(5):773-786.

[3] Reyes-Turcu F E, Ventii K H, Wilkinson K D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes[J]. Annual Review of Biochemistry, 2009,78:363-397.

[4] Amerik A Y, Li S J, Hochstrasser M. Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae[J]. The Journal of Biological Chemistry, 2000, 381(9/10): 981 -992.

[5] Poulsen J W, Madsen C T, Young C, et al. Comprehensive profiling of proteome changes upon sequential deletion of deubiquitylating enzymes[J]. Journal of Proteomics, 2012, 75(13):3886-3897.

[6] Bilsland E, Hult M, Bell S D, et al. The Bre5/Ubp3 ubiquitin protease complex from budding yeast contributes to the cellular response to DNA damage[J]. DNA Repair (Amst), 2007, 6(10):1471-1484.

[7] Mao P, Smerdon M J. Yeast deubiquitinase Ubp3 interacts with the 26 S proteasome to facilitate Rad4 degradation[J]. Journal of Biological Chemistry, 2010, 285(48): 37542-37550.

[8] Chew B S, Siew W L, Xiao B, et al. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3[J]. Biochemical Journal, 2010, 431(3):391-399.

[9] Kvint K, Uhler J P, Taschner M J, et al. Reversal of RNA polymerase II ubiquitylation by the ubiquitin protease Ubp3[J]. Molecular Cell, 2008, 30(4):498-506.

[10] Li Y, Wang Y. Ras protein/cAMP-dependent protein kinase signaling is negatively regulated by a deubiquitinating enzyme, Ubp3, in yeast[J]. Journal of Biological Chemistry, 2013, 288(16):11358-11365.

[11] Wang Y, Zhu M, Ayalew M, et al. Down-regulation of Pkc1-mediated signaling by the deubiquitinating enzyme Ubp3[J]. Journal of Biological Chemistry, 2008, 283(4):1954-1961.

[12] Cohen M, Stutz F, Belgareh N, et al. Ubp3 requires a cofactor, Bre5, to specifically de-ubiquitinate the COPII protein, Sec23[J]. Nature Cell Biology, 2003, 5(7):661-667.

[13] Kraft C, Deplazes A, Sohrmann M, et al. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease[J]. Nature Cell Biology, 2008, 10(5):602-610.

[14] Li K, Ossareh-Nazari B, Liu X, et al. Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme[J]. Journal of Molecular Biology, 2007,372(1):194-204.

[15] Li K, Zhao K, Ossareh-Nazari B, et al. Structural basis for interaction between the Ubp3 deubiquitinating enzyme and its Bre5 cofactor[J]. Journal of Biological Chemistry, 2005, 280(32):29176-29185.

[16] Ossareh-Nazari B, Bonizec M, Cohen M, et al. Cdc48 and Ufd3, new partners of the ubiquitin protease Ubp3, are required for ribophagy[J]. EMBO reports, 2010, 11(7): 548-554.

[17] Rumpf S, Jentsch S. Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone[J]. Molecular Cell, 2006, 21(2):261-269.

[18] Hanzelmann P, Buchberger A, Schindelin H. Hierarchical binding of cofactors to the AAA ATPase p97[J]. Structure, 2011, 19(6):833-843.

[19] Stolz A, Hilt W, Buchberger A, et al. Cdc48: a power machine in protein degradation[J]. Trends in Biochemical Sciences, 2011, 36(10):515-523.

【中文責編：晨 兮；英文責編：艾 琳】

去泛素酶復合體Ubp3/Bre5的制備及與Cdc48作用

蘇文成1, 2，呂 操1,2，時麗麗3，景曉飛1,2，蓋園明2，張 潔2，譚煥波2，王鵬舉2， 夏立新4，鄒培建2，秦 剛2

1)天津工業(yè)技術大學生物技術學院, 天津 300457; 2)中國科學院天津工業(yè)生物技術研究所,國家工業(yè)酶重點實驗室,天津300308; 3)天津藥物研究院天津分子設計與藥物發(fā)現重點實驗室, 天津300193;4)深圳大學醫(yī)學部,呼吸疾病國家重點實驗室深圳大學變態(tài)反應分室, 深圳 518060

泛素化是一種存在于真核細胞中與生理功能密切相關的蛋白修飾，泛素化與去泛素化處于動態(tài)調節(jié)過程中. Ubp3是與人USP10同源的酵母去泛素化酶，結合輔引子Bre5在細胞內發(fā)揮廣泛作用.為研究該復合體的工作機制，制備重組蛋白復合體,在大腸桿菌中成功表達并純化重組Ubp3與Bre5單體及Ubp3/Bre5復合體，首次成功大規(guī)模制備重組Ubp3/Bre5復合體.通過一系列pulldown實驗，檢驗Ubp3/Bre5與AAA家族中泛素選擇性ATP酶Cdc48的相互作用模式，結果發(fā)現，Ubp3及Bre5無法單獨與Cdc48結合，但Ubp3/Bre5復合體可以有效與Cdc48相互作用.提出了Ubp3/Bre5-Cdc48相互作用的新模式，制備了高質量重組Ubp3/Bre5復合體.該研究為通過生化及結構生物學進行分子機制探索奠定了基礎．

結合蛋白質；Ubp3去泛素化酶；結合輔因子Bre5；ATP酶Cdc48；去泛素化復合體；與谷光苷肽巰基轉移酶沉淀試驗；直接相互作用

天津市科技支撐計劃資助項目(11ZCZDSY08100); 中國科學院百人計劃資助項目(KSCW2-YW-BR-4) ;國家自然科學基金資助項目(81273275)

蘇文成(1987—)，女(漢族)，內蒙古自治區(qū)呼和浩特市人，天津工業(yè)技術大學碩士，E-mail:woshisuwenc@yahoo.com

/References：

：Su Wencheng, Lyu Cao, Shi Lili, et al.Preparation of full-length deubiquitinating complex Ubp3/Bre5 and characterization of interaction with Cdc48[J]. Journal of Shenzhen University Science and Engineering, 2015, 32(1): 58-67.

Q 513 Document code：A

10.3724/SP.J.1249.2015.01058

Received：2014-01-17；Revised：2014-12-24；Accepted：2014-12-26

Foundation：The Program of Tianjin Municipal Science & Technology Project (11ZCZDSY08100); The Program of “One Hundred Talented People” of the Chinese Academy of Sciences (KSCW2-YW-BR-4); National Natural Science Foundation of China (81273275)

? Corresponding author：Associate professor Qin Gang, E-mail: qing@genequantum.com

引 文：蘇文成，呂 操，時麗麗，等. 去泛素酶復合體Ubp3/Bre5的制備及與Cdc48作用[J]. 深圳大學學報理工版，2015，32(1)：58-67.(英文版)