Cance aha novdi XR ebaetreigadcl mobility cell and Renaudin targeting Xavier membrane CXCR5 in neddylation involved FANCA-modulated pathway a reveals analysis Proteomic ARTICLE RESEARCH 3546 2014 June 2 Accepted 2014; January 27 Received ` 2012). Rape, (Chen 1 localization and or subcellular or (Komander degradation function proteasomal their protein regulate for to target protein acts ligase lysine marks specific the ubiquitin a an Ubiquitylation to on with transfer ubiquitin interact the (K) then complete will will enzyme that which (E3) ubiquitin-conjugating E2), a ubiquitin-activating E1 to or the transferred (Ubc to then attached is first The It is process. It enzyme. kDa. molecular three-step a 8.5 with a of polypeptide mass in 76-amino-acid of Among a proteins addition is 1992). protein the target ubiquitin Martinage, on to based and chains is Han ubiquitin modifications ubiquitylation 2012; PTMs, are known al., post-translational several localization et through (Cai subcellular (PTMs) and/or regulated stability precisely activity, Protein INTRODUCTION motility, Cell NEDD8, Leukemia CXCR5, anemia, Fanconi cell WORDS: KEY the to motility. in FANCA cell involving migration receptor and pathway cell a neddylation uncovered the stimulates has work neddylation Our targeting motility. CXCR5 and modulate (d) in is and to involved CXCR5 membrane; CXCR5 appears (c) is mechanism; FANCC receptor unknown neddylation not Finally, an chemokine through but membrane. neddylation the of CXCR5 FANCA cell (a) majority (b) the that The neddylated; to manner. demonstrate localize FANCC-dependent we modified targets post-translationally or potential are FANCA- these that a proteins complex. in FANCcore other the of identify targets only We the be FANCD2 to with appear monoubiquitylation. treatment FANCI FANCD2 after and of cells inducer control an rescued of hydroxyurea, those FANCcore-complex- to from cells proteins deficient use compare we to monoubiquitylates Here, stress. spectrometry that replicative mass to ubiquitin-ligase response in E3 FANCI and an FANCD2 complex, as FANCcore nuclear functions the which in the participate marrow proteins of cancer. bone FANC to with predisposition Eight substrates a associated and genetic novel abnormalities is the developmental identify which failure, to leads anemia, to which Fanconi was of disorder inactivation study the this complex, FANCcore of aim The ABSTRACT Peetades NEMU08 eted ehrh nCance en Recherche de Paris. Centre 75013 U1068, Corvisart, INSERM address: Rue *Present 14 Cancer, le Contre asil,France. Marseille, uhrfrcrepnec ([email protected]) correspondence for Author Universite 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,34–54doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ´ ooi utv osy 40 ilji,France. Villejuif, 94805 Roussy, Gustave rologie ´ ai-u,940Osy France. Orsay, 91400 Paris-Sud, 1,2,3 enHge Guervilly Jean-Hugues , 2 NSUR80 ntttde Institut – 8200 UMR CNRS 3 qieLabellise Equipe ´ ooi,13009 rologie, 1,2,3, ´ ,Si Aoufouchi Said *, Ligue e Bsotntre h aepoen ttesm ie as sites same the ubiquitylation the is the regulate NEDD8 at might UBLs, and proteins ubiquitin identified to same relative several closest the the been Among the ubiquitin. target ubiquitin. of have to that often to analogous (ISG15) similar UBLs roles processes play product and undergo pathway ubiquitylation 15 proteins these gene and identified; (NEDD8) stimulated 8 precursor downregulated interferon neural (UBLs), developmentally (SUMO), proteins expressed modifier cell ubiquitin-like ubiquitin-like small other as such Furthermore, et (Clague rapid 2012). a of proteins the al., ubiquitylated family in plays of a involved deubiquitylation of ubiquitylation specific are and identification that that the (DUBs) enzymes by hypothesis deubiquitylating supported is The role 2009). regulatory Sun, and alr,gntcisaiiy rdsoiint ct myeloid acute to predisposition marrow a bone include instability, syndrome genetic this identified of failure, Fanconi 16 symptoms syndrome the (DDR) The response of anemia. damage 2012). DNA any D’Andrea, rare and in the Kim causes mutation 2013; al., biallelic et (Bogliolo A are Kashiyama recombination which 2013; homologous al., respectively), in et ERCC4, PALB2, involved or BRIP1, directly XPF BRCA2, more FANCO, and as cascade SXL4 known FANCN, signaling (also RAD51C, FANCJ, FANCQ a six and FANCD1, includes FANCP also of pathway proteins, This portion FANCE, pathway. downstream FANC upper the identified as FANCC, two to the referred its complex and constitute FANCB, FANCcore FANCM, targets and 2006). FANCA, FANCL al., FANCG, et FANCF, including Machida 2007; 2008; al., partners, et al., (Alpi to FANCI et monoubiquitin and collaboration a Alpi FANCD2 of in targets transfer which, the downstream catalyzes the FANCL, UBE2T, ligase E2 the RING with E3 and the Deshaies contains 2009; Sun, and with Chen interacts 2009; the 2009). protein includes al., Joazeiro, that target et proteins complex the (Acconcia multi-protein E3 while E3 a and E2 of protein, the component target (RING) proteins only another the E3 to and gene’ bind of E2 new that the both link interesting the consists directly subfamily of ‘really that RING-E3 members a E6-AP The the contain the domain. and The to group groups. domain, ‘homologous two other (HECT) the comprises contain terminus’ and family group carboxyl E3 and one ubiquitylation The of E3 DUBs. the members 90 and E2, of and E1s E1, two E3s circuitry involves few processes reversible the deubiquitylation Whereas date, and to 2004). identified been dynamic have al., their enzymes for NEDD8 et necessary neddylation deconjugating are which (Pan that in changes proteins, activity of conformational family the NEDD8 Cullin induces addition, its the In 2006). targets al., stimulates and also et internalization (Oved and recycling receptor the or for (EGFR) degradation required targets is receptor NEDD8 which factor ubiquitylation, example, growth For epidermal 2008). (Xirodimas, process h ACoecmlxi nE utpoencmlxthat complex multiprotein E3 an is complex FANCcore The 1,2 n iip Rosselli Filippo and 1,2,3, ` , FANC 0Es 600 E2s, 40 genes

Journal of Cell Science igeFN rti rteeitneo te currently other of existence DDR the a a or of of activities consequence protein alternative by a caused FANC be as also single metabolism might it tissue and of but clinical disturbance defect, and This general cell 1992). a from al., the result et could Rosselli al., heterogeneity 2001; et cellular al., (Briot and et responses 2012), Pang and al., 2008; production et cytokine (Pagano in free altered detoxification abnormalities 2011), and (Soulier, generation cancers neck radical and head and leukemia ARTICLE RESEARCH on ob infcnl ifrn nbt AC-adFNCdfcetclscmae ihtersetv orce controls. FANCA corrected of respective screens modifie the the post-translationally with to whose compared common proteins cells were indicate FANCC-deficient that spots and proteins Green FANCA- 287 exclusion. both the of in for region different obtained the significantly ratios FANCC-defic define be the and quantified lines FANCA- to indicate the the Blue found circles in of cells. (*) identified Green comparison FANCC-deficient proteins eventually orange. A ubiquitylated and that in potentially (E) proteins deficient of circled non-nuclear immunoprecipitation. overlap the are K-GG the indicate FANCI showing after of circles diagram and lines Gray Venn proficient:deficient FANCD2 A proteins. of (D) cells. nuclear-localized transmembrane. (ratio (C) the TM, proteins FANCA indicate main membrane. circles significant and the Red of (B) as proteins. FANCC network FANCI added for interacting and manually shown FANCD2 The is identifies (B,C) Software, counterparts immunoprecipitation. IP, Stringdatabase corrected h. their 8 and after cells complex. collected FANCC-deficient FANCCore and the FANCA- of of targets analysis Ubiquitylome 1. Fig. migration cell optimizing this in FANCA- of the and targeting membrane motility. mechanism the a and cell that in unknown the involved in to demonstrate is currently receptor neddylation neddylated a CXCR5 we that through is and Finally, manner CXCR5 membrane. dependent FANCcore receptor localize that nuclear cell targets proteins chemokine the FANCC the other or of FANCA the to targets potential of as direct majority identified were true the only Surprisingly, complex. the appear FANCI to be and aimed FANCD2 ubiquitin-ligase to complex. we E3 FANCcore the study, the of of this targets activity in novel characterize pathway, and FANC identify the of inactivation complex. FANCcore the of targets unidentified ofrhreuiaeteclua n lnclcneune of consequences clinical and cellular the elucidate further To A h nihetsrtg o bqiyae etdsi hw.Clswr rae ih5m yrxue H)and (HU) hydroxyurea mM 5 with treated were Cells shown. is peptides ubiquitylated for strategy enrichment The (A) 0(upeetr aeilTbe 1adS) eepce to expected We S2). and calculated S1 Tables a material had (supplementary least and 20 at cells of change corrected fold the FANCcore-proficient:FANCcore-deficient in identified unambiguously over-represented were as FANCI and FANCD2 in complex monoubiquitylation. FANCD2 of and inducer activity strong complex a is FANCcore which hydroxyurea, inhibitor with replication treatment the counterparts after corrected the respectively) HSC-536corr, their and of compared (HSC-72corr those We with lymphoblasts complex- 1A). deficient FANCcore tryptic (Fig. (FANCC-deficient) the HSC-536 (FANCA- and extract HSC-72 after deficient) growing cellular exponentially residues from extracted lysine a proteins modified of the moiety digestion using on K-GG chains the by remains against ISG-15) modified directed that specifically and are are (NEDD8 that that antibodies ubiquitin-like the peptides or on based of ubiquitin approach enrichment developed heterogeneous recently its immunoaffinity a in used the involved we of be activity phenotype, might ligase that ubiquitin complex the of FANCcore substrates novel identify To by complex modified FANCcore post-translationally the are that proteins Identifying RESULTS h etdscnann h yietree yteFANCcore the by targeted lysine the containing peptides The ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal . .) sdtrie ythe by determined as 2.5), soit ihthe with associate etd was peptide d etcell ient 3547 -

Journal of Cell Science iia ota acltdfrFND n AC,which of FANCI, targets only ratio and the a be FANCD2 complex. might with FANCcore for FANCI the and proteins calculated FANCD2 other that that suggests identify to not analysis. FANCC similar did for and observed FANCA we the those both to Surprisingly, in similar and FANCI were corrected and that the FANCD2 cells between anemia changes Fanconi fold the the with in concentrations and high cells at corrected proteins other from peptides identify ARTICLE RESEARCH civ u ol hrfr,tepoemc eut rmthis to from specific results and 3548 proteomics sensitive the sufficiently Therefore, was goal. study our significant achieve this in no used pathway. FANC was the mass of that proficiency levels the there the monoubiquitylation with PCNA correlated or to MCM7 however, the According in difference results, S2C). Fig. spectrometry monoubiquitylated Using is material form. MCM7 MCM7 that (supplementary ubiquitylated demonstrate in the we technique, analysis detect this blot and enrich western to to order prior antibodies or (FK2) anti-MCM7 with anti-ubiquitin immunoprecipitation an perform to but needed S2A,B), we Fig. easily material was (supplementary PCNA immunoblot DNA-replication- by and corrected detected FANCD2 and the of anemia monoubiquitylation Fanconi The from the cells. between the peptides peptide in these difference of significant a no level was There and the MCM7. monoubiquitylated factor PCNA licensing Among replication-stress-induced of identified approach. peptide we well-known our proteins, of the modified sensitivity post-translationally we complex, the FANCcore identified the the examined of at targets next novel localized identify be Because to S1). failed to Fig. the we material appeared supplementary specifically 1B,C; (Fig. by and membranes nucleus outside localized the be ubiquitylation to of appeared proteins identified direct the of majority known already consistent complex. significant FANCcore their was highly a which with had ratio, first however, 1998). FANCI, the FANC-corrected:FANC-deficient al., and et of FANCD2 (Triepels part Only complex chain is respiratory were which mitochondrial of proteins subunit oxidoreductase, NDUFA8 three nuclear-encoded NADH:ubiquinone 1E; the These (Fig. and S3). analysis FANCI FANCcore-complex- FANCC FANCD2, Table the high material in identified a supplementary also were had three ( only 1D,E; that (Fig. ratio analysis analysis proficient:deficient in FANCA identified FANCA proteins the 76 the analysis, the in Of FANCC S3). Table our identified material in supplementary also identified proteins, proteins were the Table 287 unique Of material 1C). (supplementary 471 Fig. FANCI S2; identified and FANCD2 was we including that cells, change FANCI. fold and FANCA-corrected a FANCD2 for exhibited obtained lectin – values C-type 2011) the to prolectin) al., – similar or et (CLEC17A cells 1 (Koh isoform B one protein A member transmembrane dividing 17, Only family uncharacterized on domain 1B). Fig. the receptor S1; to glycan-binding found Table belonging we material significant polypeptide ( FANCI, a (supplementary and had ratio robust of FANCD2 that addition material to proteins a 415 the (supplementary addition few revealed moiety by In relatively ubiquitin-like S1). line modified a Table were cell or that FANCC-corrected ubiquitin proteins the individual to deficient hnw oprdteFNAdfcetclswt the with cells FANCA-deficient the compared we When FANCC- the comparing when identified were that peptides The eas fteefnig,w r ofdn htteapproach the that confident are we findings, these of Because the analyses, FANCC and FANCA the both for Surprisingly, . .)FNCpoiin:AC-eiin cell FANCC-proficient:FANCC-deficient 2.5) . .)ado significant a and/or 2.5) Z soeand/or -score Z -score, opnnsmgtatidpnetyo n nte nother in another one its locations. of cellular of in independently and some involved pathways act that only might and is monoubiquitylation components complex FANCI FANCcore and the FANCD2 that suggest study iia XR xrsinlvl,ecuigba ntemass the the have from in extracted counterparts proteins bias the Next, corrected excluding 2A). The (Fig. levels, their data hydroxyurea. spectrometry expression and with at CXCR5 cells treatment level similar expression following anemia CXCR5 and Fanconi the used state we determine steady overexpression, to CXCR5 a blotting difference FANCA-induced Fanconi-anemia-derived of western a the result the of in the or whether CXCR5 was cells of analysis determine spectrometry expression To mass modified the 1999). in al., observed and et invasion cancer Pevzner (Mu progression, diseases cancer pro-inflammatory a and behavior, plays auto-immune cell our which B in CXCR5, in of observed receptor role PTM observed differences chemokine the transmembrane the of the FANCA- characterization in our FANCA a To continued we for of mechanisms. study, role unknown targets a currently potential confirm through be activity CXCR5, might analysis, regulated SLAMF7 FANCA the and in B2M identified proteins its the in involved Among is FANCA and protein, neddylation neddylated a is CXCR5 rmorpoemc nlss n ehv hw htFANCA, results that the shown of have validating we member proteins, and new analysis, neddylated a proteomics of our as from group CXCR5 small identified the have we approaches, the 2G). with (Fig. the cells compared but anemia when of Fanconi FANCA-corrected lymphoblasts respective the level FANCC-corrected in expression the elevated not is the form that CXCR5 2F). neddylated demonstrates (Fig. cells immunoblot corrected and 2E). cells The anemia (Fig. Fanconi the cell cellular (PLA) from using per extracts immunoprecipitation NEDD8 spots a assay performed CXCR5–NEDD8 we anti-CXCR5 of Finally, ligation and significantly number inhibitor the NEDD8 anti-NEDD8 the proximity reduced to with Exposure 2D). cells used (Fig. without antibodies fixed Fig. S2B) we on neddylated material technology Fig. known (supplementary Second, level the material CXCR5 S2A). eliminate (supplementary the of to Cul4A modification 2012) able al., of is et treated form (Wei which cells from ML4924 2C), samples inhibitor (Fig. in NEDD8-specific present the longer with no is the albeit neddylated- band presumed confirm the CXCR5 specific, complementary that To demonstrated two we 2B). a anti-CXCR5 followed First, (Fig. approaches. we of CXCR5 an neddylation, CXCR5 of presence observed using form the and neddylated analysis revealed antibody minor, anti-CXCR5 which immunoblot an antibody, or lymphoblasts an anti-NEDD8 we HSC93 conducted an Consequently, either (EBV)-transformed FANC-proficient PTMs. using from mass virus a ISG15 extract also the Epstein-Barr specific protein and is the the before peptides NEDD8 immunoprecipitated by ubiquitin-modified of extract recognized the enrich signature is by to protein that created used the is antibody and that analysis of moiety spectrometry K-GG digestion The tryptic (supplementary S3A). CXCR5 of Fig. form conducted we material ubiquitylated a Unexpectedly, a identify was immunoprecipitation. as to antibody the failed analysis anti-PCNA for corrected an control immunoblot and positive antibody their an anti-CXCR5 an anti-ubiquitin and and using the FK2, using cells antibody immunoprecipitated FANCC-deficient were counterparts and FANCA- ncnlso,b sn obnto fdifferent of combination a using by conclusion, In ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal le ta. 2003; al., et ¨ller

Journal of Cell Science EERHARTICLE RESEARCH edlto srqie o h pia agtn fCC5t the to CXCR5 that of and targeting optimal manner the FANCA-dependent for required a is in neddylation neddylated is EBV- cell CXCR5 analyzed the 3A,B). the (Fig. to lines latter among cell lymphoblastoid the receptor transformed level of highest membrane-associated to the targeting showed NEDD8 of cells the of FANCA-corrected in addition the membrane, the involved that is hypothesis lines. CXCR5 cell the several in with membrane plasma amount Consistent the the to quantified localized here. we CXCR5 identified analysis, of neddylation cytometry whether CXCR5 flow EGFR the determine by for to in Therefore, case wanted the involved we also be was 2006), this to al., et shown (Oved been trafficking has CXCR5 neddylation cell motility Because in the cell to for CXCR5 role and targeting membrane for a responsible is plays Neddylation complex, FANCcore the neddylation. not mean the show but data G, and E (G) For shown. counterparts. level expression also corrected CXCR5 is their the experiments and showing independent cells results Student’s FANCC-deficient blot three using Ne Western cells, from by CXCR5, (F) FANCA-deficient band calculated MLN4924. against in immunoprecipitated inhibitor antibodies Nedd8 NEDD8 the with the against of results 10 of antibody dots) quantification presence bar: the (red or Scale a PLA with absence ce (blue). with (D) in immunoprecipitation HSC-93 DAPI immunoprecipitation control. both, after in or Ct, with after (Ub) NEDD8 MLN4924. h stained band CXCR5, ubiquitin inhibitor 8 neddylated is against or NEDD8 for CXCR5 nucleus (Abs) CXCR5 the the mM The to NEDD8, showing (5 shown. exposed against blot are (HU) cells antibody Western both hydroxyurea or with (C) with cells (IP) IgG. untreated treatment immunoprecipitation rabbit in without after Rb, antibody or level IgG with expression wt). (corr), CXCR5 wild-type, counterparts the (FANC-pathway-proficient; corrected showing their blot and Western manner. cells (B) FANCA-dependent (HSC536) a FANCC-deficient in cells, neddylated (HSC72) is CXCR5 2. Fig. ocnimadepn norpeiu eut hwn that showing results previous our on expand and confirm To t -test. A etr ltrslssoigterltv xrsinlvlo XR nFANCA-deficient in CXCR5 of level expression relative the showing results blot Western (A) m .()Qatfcto ftePAdt nclsaaye yteadto fantibodies of addition the by analyzed cells in dots PLA the of Quantification (E) m. elln E51 hc a sltdfo ypai oe is node, lymphatic a from isolated was melanoma which metastatic membrane MEL501, the line in the cell MiTF factor to transcription oncogenic targeted less was 3D,E). CXCR5, the (Fig. as level wild-type Furthermore, same the at CXCR5. the expressed was of wild-type which membranes mutant, the overexpressing CXCR5-K339R on level cells protein there CXCR5 the cells, the in untransfected increase the mutant an with was (supplementary a Compared neddylated or S3C). be Fig. CXCR5 cells material cannot of U2OS that form transfected form wild-type we CXCR5-K339R full-length Next, a the surface. either cell supports with targeting the optimal to finding the of CXCR5 for of important This level is neddylation 3C). the that (Fig. hypothesis reduced a manner in cells treatment dose-dependent FANCA-corrected the MLN4924 MLN4924. in CXCR5 inhibitor the the membrane-associated NEDD8 treated expected, we the As First, with approaches. cells different FANCA-corrected used we membrane, ial,i a enrpre htteoeepeso fthe of overexpression the that reported been has it Finally, ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal 6 .. infcnewas significance s.d.; Relative NEDD8 d or dd8 ). 3549 lls

Journal of Cell Science EERHARTICLE RESEARCH 3550 3. Fig. e etpg o legend. for page next See ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal

Journal of Cell Science oe faNd8ihbtr(edi.Tewsenbo eut o Cullin4A for results blot western The different (Nedd8i). two inhibitor with Nedd8 of cells a amount the of the of doses are for treatment line (left) after cell results CXCR5 HSC93) analysis membrane-associated (wt; cytometry wild-type Flow one one (C) and HSC99), shown. (HSC72corr) and line EGF088 in cell HSC72, CXCR5 (FA; corrected membrane-associated lines of cell of amount FANCA-deficient results the The three for (B) shown. analysis the are cytometry in lines flow cell CXCR5 (corr) membrane-associated corrected of and amount FANCA-deficient the membrane for its analysis regulates manner.cytometry 339 FANCA-dependent lysine a on in CXCR5 association of Neddylation 3. Fig. ARTICLE RESEARCH tr ftewudasyi hw.UO el eetasetdwt idtp W)o 39 XR;atr4 ,teclswr cace ihafn pipett fine a with scratched were cells the 40 h, bar: 48 Scale after recovery. 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(kr) mutant each K339R taken the measurements or 12 CXCR5 wild-type least the with transfected cells for edlto hog urnl nnw ehns n eut ntemmrn soito fCC5ta losfrhg elmotility. cell high for allows that CXCR5 of association membrane the in results and mechanism unknown currently a through neddylation Student’s using calculated was t ts;ns,nnsgiiat C ipelna oe ihihigorrslsi hw.FNAoeepeso nue CXCR5 induces overexpression FANCA shown. is results our highlighting model linear simple A (C) non-significant. n.s., -test; A h eut fflow of results The (A) m .()Gahsoigtepretg frcvr o nrnfce e)clsand cells (ev) untransfected for recovery of percentage the showing Graph (B) m. th n 75 and infcnl eue hnteK3RCC5mtn was defect. motility in mutant a was defect with a CXCR5 associated closure that is indicates neddylation K339R wound finding CXCR5 This the culture, 4A,B). (Fig. monolayer when overexpressed U2OS reduced CXCR5- scratch the significantly the a creating or in cells After neddylated. protein U2OS wound be cannot CXCR5 of that motility wild-type mutant K339R the a compare in either to patches expressing used assay we Peyer’s mobility, wound-healing cell in in a role a and plays neddylation spleen CXCR5 whether (Fo the CXCL13 ligand, its in to to response centers migration and germinal motility plasma cell the B the to in CXCR5 involved targeting is for CXCR5 important membrane. is PTM this FANCA that by regulated and mechanism a through NEDD8 of by addition modified the post-translationally is CXCR5 that demonstrated have of 3H). downregulation (Fig. siRNA-mediated FANCA the or by MiTF as MLN4924 the well inhibitor as neddylation 3G), the (Fig. with cells MEL501 treating fteFNcr ope nrsos orpiainsrs.By stress. replication to response in activity complex ligase FANCcore ubiquitin the the in of of substrates we altered novel phenotype, are identify heterogeneous to its that wanted in pathways involved and molecular anemia the Fanconi elucidate further To DISCUSSION n AC-eiin el oprdwt hi respective their with compared counterparts. cells FANCA- corrected the FANCC-deficient in under-represented identified being were and Surprisingly, as that confidence proteins cells. high only extracts the with FANCC-corrected the were FANCI and in and FANCD2 FANCA- overrepresented the the being of targets from as specific identified complex, are we which FANCcore digestion, peptides, tryptic FANCI after and FANCD2 moiety K-GG a peptides immunoprecipitated containing of analysis spectrometry mass using A ersnaieiaeo el fe h ellyrwssrthda the at scratched was layer cell the after cells of image representative A (A) th hs sn eea ifrn prahsadcl oes we models, cell and approaches different several using Thus, ecnie;tewikr hwtemnmmadmxmm significance maximum; and minimum the show whiskers the percentiles; ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal rtre l,19) odetermine To 1996). al., et ¨rster e,wt at with mes, 3551 tip e

Journal of Cell Science EERHARTICLE RESEARCH 3552 h 2Mcekon Yn ta. 02,wihaetwo are which 2012), al., of et Inhibition (Yang of involved. activation checkpoint also the G2/M and is stability the pathway CHK1 both FANC increases 2008), neddylation the Xirodimas, and 2002; which al., et findings in (Jones FANC and replication the remodeling and chromatin repair in by the DNA that involved observations. are published only mechanism proteins several by target between not also NEDD8 a but study supported of present interplay our is from existence precise pathways The neddylation 2012). the al., al., et regulates et (Hardtke clinical diseases Yu and pro-inflammatory 2005; progression, cell and cellular cancer that invasion B responses, cancer anemia in role immune involved Fanconi differentiation, is the terminal signaling the determine CXCR5-dependent in phenotypes. and plays neddylation FANCA CXCR5 how determine CXCR5 process, enzymes this 4C). regulates the in (Fig. role involved is identify precise directly function, are motility neddylation the CXCR5 that elucidate in and plays to CXCR5 migration neddylation needed that are by cell studies (d) in-depth optimized (c) Further receptor and or the mechanism, membrane of stimulated cell targeting unknown the the in currently to involved is a CXCR5 neddylation modulates CXCR5 FANCA through (b) protein, neddylation neddylated a is be CXCR5 to remains PTM studies. protein future in in determined involved al., regulate pathways in indirectly et FANCC or independent directly (Medhurst isolated subcomplexes and subcomplex these Whether FANCL, FANCE–FANCF been 2006). and a has with FANCB cytoplasmic interacts in FANCG, FANCA found with example, be of association several can For nucleus, proteins the subcomplexes. complex in FANCcore assembled though is the Even phenotype. complex anemia FANCcore play Fanconi the the could in function role this important an and transmembrane FANCA proteins, of that membrane-associated PTMs hypothesis the and was regulate membrane- the independently that supports FANCC i.e. and observation class This cells only class, FANCC-deficient 1). channel, and (Fig. the FANCA- protein receptor, the surprisingly in enriched the surface significantly was cells, proteins, cell anemia associated and Fanconi the transporter in reduced siprati eladscnaylmhi ra behavior organ lymphoid secondary and that cell that CXCR5 B and shown in CXCR4 between important have crosstalk bone is studies functional a the Previous is to there syndrome. with contribute anemia associated could abnormalities Fanconi developmental defects and these failure C-X-C that marrow the that hypothesis of downstream the and occur supports pathway receptors which signaling the 2012), in al., defects of et migration (Barroca LSK the and cells in (MEFs) the fibroblasts changes embryonic in mouse of FANCG-knockout results defect inhibition signaling CXCL12 cellular Consequently, Fanconi-anemia-like 2012). a in in al., phenotype. results defect pathway et neddylation neddylation chromatin (Kee to involved potential relocalization are foci and the neddylation monoubiquitylation a and FANCD2 support NEDD8 in is FANC- Finally, further in anemia. there the Fanconi findings increased and that 2004), is These al., hypothesis factors et cells. transcription Xirodimas 2006; pathway-deficient these al., of et Gao activity negatively NF- anemia 2007; neddylation of al., activity Fanconi et Additionally, transcriptional 2011). with the regulates al., associated et features (Guervilly cellular important arc n olausrcnl eotdta CXCR4– a that reported recently colleagues and Barroca receptor chemokine the (a) that demonstrate we study, this In significantly were that PTM K-GG a with proteins the Among k n 5 (Abida p53 and B 2 osatnu ntttd Ge de Institut Constantinou; AGCCTCGTCAGGAGCCGC-3 GCGGCTCCTGACGAGGCTGGGCTGTAC-3 novdi bqiyain edlto n te PTMs other pathways and to signaling point processes. neddylation the developmental starting and syndrome. ubiquitylation, physiology between a cell anemia regulates as in crosstalk Fanconi serve involved the how might of findings discover receptor provide view our that membrane integrated mechanisms Moreover, potential and more new to a pathway allude CXCR5 and neddylation in signaling the changes the proteins, the study. on that this dependent speculate in the observed to is we of tempting defect that is neddylation cause this it FANCG-knockout of 2012), the the al., cause identify et and in (Barroca to 2006), defects cells al., failed signaling should et colleagues FANCG CXCL12–CXCR4 (Medhurst and and subcomplex FANCA Barroca cytoplasmic Because a 2013). form al., et (Schmidt 0 /lpncli,10u/lsrpoyi n . /lpuromycin g/ml 0.5 pyruvate, and sodium streptomycin mM DMEM 1 ug/ml or 100 serum, RPMI in penicillin, bovine grown U/ml fetal well type), were 100 Cells 10% as lines. (wild with deficient) cell supplemented (FANCA Mel501 HSC-93 and HSC-99 U2OS and HSC-536corr, as deficient) corrected and (FANCA respective EGF088 their HSC-72corr deficient), (FANCC counterparts HSC-536 (FANCA HSC-72 and lines deficient) cell lymphoblastoid EBV-immortalized treatments used and We conditions culture lines, Cell METHODS AND MATERIALS inln ehooy,ga nimue(eu,dne anti-rabbit donkey anti-actin (Cell (Tebu), anti-NEDD8 goat anti-mouse rabbit goat (Abcam), Cruz), Technology), (Santa anti-vinculin Signaling anti-PCNA BD mouse mouse and (Abcam), anti- (Abcam), mouse mouse (Genetex antibody), anti-CXCR5 CXCR5 (Abcam), Alexa-Fluor-488-conjugated the rabbit anti-FANCD2 for Biomol), Pharmingen rabbit mouse (FK2; (Abcam), Cruz), anti-MCM7 anti-ubiquitin rabbit (Santa antibodies: anti-FANCD2 following the used We Antibodies later. h protocol, 24 conducted manufacturer’s were the experiments to the transfected according and were (Fermentas) K339R Turbofect and DNASU) using (from CXCR5 wild-type France), 5 primers: following Site- the II QuikChange and the (Agilent) using using Kit cells (K339R) Mutagenesis into mutated later. Directed transfected was h 48 K339 was conducted purchased were CXCR5 (SMARTpool), nM) experiments the (20 were and MiTF (Polyplus), siRNA (SMARTpool) INTERFERin against FANCA Dharmacon. and from siRNA (5 (SMARTpool) Eurogentec. FANCC siRNA from purchased control The score confidence Transfection a analysis with interactions UbiScan StringDatabase protein for silico the in Technology using . The Signaling were determined antibody. Cell pellets branch were to ubiquitin cell the ice analysis, using dry (LC-MS/MS) on chromatography spectrometry sent liquid and mass immunoprecipitation tandem peptide K-GG the For analysis Proteomics 37 at Invitrogen) from (all 0m tc ouini MO eepromda h oe and doses the at a performed were as DMSO, prepared NEDD8 in the the indicated. solution Pharmaceutical), times with by stock Treatments Millennium performed mM 10 facility. were (MLN4924, core analysis inhibitor Ubiquitin proteomic Signaling and Cell spectrometry mass yrxue,fahfoe n trdat stored and frozen flash hydroxyurea, m 0.7. el eecletdb etiuaina fe xouet mM 5 to exposure after h 8 at centrifugation by collected were Cells ncnlso,orfnig eelacneto ewe FANC between connection a reveal findings our conclusion, In fteFNAFA-xrsigvco agf rmAngelos from gift (a vector FANCA–FLAG-expressing the of g ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal ˚ n ne %CO 5% under and C 9 -CGUCGACGGAAUACUUCGA-3 ´ ne ´ 9 iu uan NS Montpellier, CNRS, - Humaine tique o h vrxrsinexperiment, overexpression the For . 2 80 9 2 ˚ . n 5 and .Poenextraction, Protein C. 9 -GTACAGCCC- 9 was ) 9 -

Journal of Cell Science eaae ySSPG.Alwsenbo uniiain were quantifications software. blot ImageJ the western and measures All densitometry SDS-PAGE. using performed by separated eecniee ssignificant. as considered were ahsi ENbfe,teimn opee eeeue by eluted were 35 complexes in 1M immune min 5 the five buffer, for After conditions. NETN boiling same the in under antibody washes control isotype specific ZisAi bevrZ) mgswr atrduiga Orca-ER an using captured were Images Z1). (Hammamatsu). Camera Observer Axio (Zeiss ecpotao n eaue ybiig h rtis(25 proteins The boiling. 4 by protein and with denatured the (BioRad), and inhibitors combined assay temperature, mercaptoethanol Bradford room were phosphatase the at using samples and determined incubation was min protease concentration 20 After with (Roche)]. supplemented (Novus), h olce el eedsutdi yi ufr[0m Tris-HCl mM [50 MgCl mM buffer 1 lysis NaCl, in mM disrupted 40 were 7.9, pH cells collected The Dako. mouse from analysis blot obtained antibodies Western were isotype IgG The rabbit and (Tebu). IgG2a,b anti-goat and IgG1 donkey and (Tebu) ARTICLE RESEARCH eaegaeu oalmmeso h eteNtoa el Recherche la de National discussions. Centre stimulating the for of UMR8200 members Unit all Scientifique to grateful are We Acknowledgements two-tailed a using using by determined analyzed was Significance were analysis images microscope Statistical inverted The Zeiss at camera. a cell taken MRm with were the software. h) AxioCam pictures AxioVision 8 Next, and and an cells overnight. tip, 5 and The fine (0, medium a h. six- time-points 48 serum-free using in different for well scratched a plasmid per was in cells CXCR5 monolayer 200,000 with incubated of transfected density were and a plates at seeded well were cells U2OS assay Wound-healing 37 at min 30 for probe were 2% PLA cells with in PBS the incubated in then BSA, diluted and 2% antibody h primary 2 containing of for min. PBS solution BSA 5 for a in for in Sigma) (Sigma) blocked from containing incubated (both PBS paraformaldehyde being X-100 in Triton permeabilized 4% After 0.5% then and and with Tween-20 temperature 0.05% room fixed at min were 10 cells Briefly, assay ligation Proximity centrifugation before PBS cold in 150 once at washed and harvested were Cells analysis cytometry Flow and EDTA) a mM using 1 NaCl, 4 precleared at and were mM h (150 extracts 1 NP-40 The for buffer DNA. 0.5–1% antibody the non-specific NETN 8.0, fragment pH using to sonicated Tris-HCl extracted mM were 50 proteins The immunoprecipitation Protein eaue ybiigfraohr5mni h rsneo 1 of presence the in min 5 another for boiling by denatured omgei rti ed Mlioe htwr otdwt a with coated overnight were incubated that were beads (Millipore) and 4 extracts beads at the and G antibody, Protein specific magnetic to auatrrspooo.Fnly h ldswr one in mounted and 63 (Sigma) were the of DAPI following magnification slides with a performed supplemented the at (Dako) were analyzed Finally, medium amplification fluorescent protocol. and manufacturer’s ligation Next, oiesrmabmn(S)wt nAlexa-Fluor-488-conjugated an with (BSA) albumin serum bovine XR nioydltd110adicbtdfr1ha room at h 1 PBS, a (Accuri). using cold software analyzed for and CFlow with BSA with 3% washes incubated cytometer containing PBS C6 two in and incubated After were cells conditions. the 1:100 dark under diluted temperature antibody CXCR5 ˚ .Cnrlimnpeiiain eecnutduiganon- a using conducted were immunoprecipitations Control C. b -mercaptoethanol. g o i.Tepleswr eupne nPScnann 3% containing PBS in resuspended were pellets The min. 5 for m lof2 ˚ 6 .Tecerdetat eeadded were extracts cleared The C. 6 2 .%SSad1 Benzonase 1% and SDS 0.1% , 6 aml ufrcontaining buffer Laemmli sn loecnemicroscopy fluorescence using aml ufradwere and buffer Laemmli t -test. P -values m )were g) , m 0.05 lof ˚ C. b - a,K .adMriae A. Martinage, and K. K. Han, o,G,Lw . o,D,Yo . g .K,Wn,V . aeed,V,Lam, V., Vagenende, V., V. Wong, K., S. Ng, Y., Yao, D., and Poh, A., G. Low, P. G., Koh, Smith, D. E., A. Park, D’Andrea, and A., H. L. Kim, Moreau, S., Chang, M., Huang, Y., Kee, K., Sasaki, M., Shimada, C., Guo, T., D. Pilz, Y., Nakazawa, K., Kashiyama, P. E. Candido, and A. T. Stevens, E., Crowe, D., Jones, li .F,Pc,P . au .M n ae,K J. K. Patel, and M. M. Babu, E., P. Pace, F., A. Alpi, J. K. Patel, and A. Dutta, J., Y. Machida, G., Mosedale, F., Langevin, S. A., Alpi, Polo, and S. Sigismund, W. F., Gu, Acconcia, and W. Zhang, W., Zhao, A., Nikolaev, M., W. Abida, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.150706/-/DC1 online available material Supplementary material Supplementary the and X.R. Cancer. Cancer; ANR-08-GENO-0013]. Canceropo le number by Contre [grant supported Ligue Recherche was La la from de grants Nationale by Agence supported was work This Funding the wrote and data S.A. analyzed and study, J.-H.G. the paper. paper. designed the F.R. wrote research. and the data designed analyzed research, performed X.R. contributions Author interests. competing no declare authors The interests Competing uril,J-. ead . aaa .adRsel,F. Rosselli, and M. Takata, E., Renaud, J.-H., Guervilly, T. E. Yeh, and T. Shi, J., Cheng, F., Gao, Fo C. J. J. Belmonte, L. Izpisua Sun, and and H. J. G. Z. Liu, Chen, J., Qu, M., Li, N., Cai, A., Raams, Mace Y., D., Briot, Su, B., Derkunt, C., Stoepker, B., P., Schuster, Grange, M., la Bogliolo, de Brunet D., Lewandowski, A., M. Mouthon, V., Barroca, adk,S,Ol .adFo and L. Ohl, S., Hardtke, ehis .J n ozio .A. C. Joazeiro, and Urbe J. and R. M. Deshaies, J. Coulson, J., M. Clague, se,R,Mti,A . rme,E,Wl,E,Be,G n ip M. Lipp, and G. Brem, E., Wolf, E., Kremmer, E., A. Mattis, R., rster, ¨ ucinlcasfcto fCtp lectins. C-type of Y. classification D. functional Lee, and P. K. pathway. anemia/BRCA Fanconi agents. cross-linking interstrand D. anemia. A. D’Andrea, Fanconi causes and and al. manifestations pigmentosum, Genet. et Hum. clinical xeroderma L. J. diverse Carr, syndrome, O., in S. Cockayne results Lewin, ERCC1-XPF F., J. nuclease Wing, H., Fawcett, elegans: ubiquitin-like Caenorhabditis and in enzymes, proteins. system ubiquitin-activating ubiquitylation enzymes, the ubiquitin-conjugating of analysis phylogenetic nost-etitdmnuiutnto fFND yUet AC,and FANCL, Ube2t, by FANCD2 FANCD2 of of regulation monoubiquitination the site-restricted for into basis a recruited are FANCD2 chromatin: and monoubiquitination. complex, to core anemia independently Fanconi the UBE2T, (2007). work. at network activity. transcriptional its inhibits and p53 Chem. of Neddylation the promotes ebqiiaemitispopoyae H1b iiigisDDB1- its limiting by represses CHK1 that degradation. phosphorylated dependent deacetylase maintains histone deubiquitinase III class a defined transcription. recruits NFkappaB-dependent to protein migration associated spleen. cell the of B compartments directs anatomic BLR1, specific 1037-1047. and receptor, organs chemokine lymphoid putative A pluripotency. of modulation translational Fanconi cause XPF, endonuclease DNA-repair the encoding anemia. ERCC4, in Minguillo P., J. Trujillo, I. Allemand, L., cells. Riou, stem F., hematopoietic Arwert, D., al. F. Boussin, F., et Pflumio, R., L. Gauthier, FANCI. oeflilsadi seta o fiin -elhelp. B-cell lymph efficient to for positioning essential transient is their and determines cells follicles helper node T follicular on CCR7 proteins. of nu e.Biochem. Rev. Annu. DUBs. signaling. Fanconi in oversecretion TNF-alpha to leads pathways anemia. stress-response of ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal .Cl Sci. Cell J. 282 o.Cell Mol. 21) mardfntoaiyadhmn fFancg-deficient of homing and functionality Impaired (2012). m .Hm Genet. Hum. J. Am. Blood eoeBiol. Genome o.Cell Mol. 1797-1804. , n.J Biochem. J. Int. ´-Aime 111 92 21) niiino h ed ytmsniie el oDNA to cells sensitizes system Nedd8 the of Inhibition (2012). x.Cl Res. Cell Exp. 32 807-819. , 1913-1923. , ,G,Sba .adRsel,F. Rosselli, and F. Subra, G., ´, 125 33 767-777. , o.Cl.Biol. Cell. Mol. 21) nertv nlsswrfo o h tutrland structural the for workflow analysis Integrative (2011). 78 275-286. , 277-286. , ,J,Ramı J., n, ´ 3 u.Ml Genet. Mol. Hum. 399-434. , se,R. rster, ¨ RESEARCH0002. , u.Ml Genet. Mol. Hum. l l-eFac;adL iu otele Contre Ligue La and Ile-de-France; ˆle 20) opoeltcfntoso bqii ncell in ubiquitin of functions Nonproteolytic (2009). 21) euaino N rs-ikrpi ythe by repair cross-link DNA of Regulation (2012). 24 19) ottasainlceia modification(s) chemical Post-translational (1992). 92 19-28. , 800-806. , 315 o.Cne Res. Cancer Mol. ee Dev. Genes rz .J,Pjl .e al. et R. Pujol, J., M. ´rez, 20) aacdepeso fCC5and CXCR5 of expression Balanced (2005). 27 20) IGdmi 3uiutnligases. ubiquitin E3 domain RING (2009). a.Cl Biol. Cell Nat. 1610-1618. , 8421-8430. , 20) edlto fabes cancer- breast a of Neddylation (2006). ,S. ´, .Ml elBiol. Cell Mol. J. M Bioinformatics BMC 20 20) bqii ntafcig the trafficking: in Ubiquitin (2009). 21 2171-2181. , 21) ellrfntoso the of functions Cellular (2012). 26 121-135. , 1393-1408. , 8 10 20) ehnsi insight Mechanistic (2008). 20) bratactivation Aberrant (2008). 1171-1177. , 369-377. , Blood 20) ucinland Functional (2002). 21) afnto of Malfunction (2013). 4 262-265. , 106 2Spl 14 Suppl. 12 21) Mutations (2013). 20) FBXO11 (2007). 21) USP1 (2011). 1924-1931. , 21) Post- (2012). Cell (1996). .Biol. J. 3553 S5. , Am. 87 ,

Journal of Cell Science eze,V,Wl,I,Brsalr . Fo R., Burgstahler, I., Wolf, V., Pevzner, Rathbun, S., Fagerlie, A., T. Christianson, J., Diaz, K. W., Wu, and Keeble, K. Q., Yamoah, C., Pang, D. Dias, A., Kentsis, Q., Z. Pan, Pallardo G., Castello, A., A. Talamanca, G., Pagano, Mu vd . oesn . wn,Y,Snoio . higa,K,Marmor, K., Shtiegman, E., Santonico, Y., Zwang, Y., Mosesson, S., Oved, de C., Fontaine, M., Ferrer, J., Steltenpool, H., Laghmani, L., A. Medhurst, ahd,Y . ahd,Y,Ce,Y,Gra,A . ufr .M,D’Andrea, M., G. Kupfer, M., A. Gurtan, Y., Chen, Y., Machida, J., Y. Machida, M. Rape, and D. Komander, ARTICLE RESEARCH 3554 lr . Ho G., ller, ¨ euaino xrsino hmkn eetrBR/XR uigBcell B during BLR1/CXCR5 receptor chemokine of gamma, maturation. expression interferon of RNA. to Regulation cells double-stranded C Jr and C., group factor-alpha, 1652. G. complementation necrosis hypersensitivity Bagby, anemia mediating tumor and in Fanconi M. kinase of O’Dwyer, protein R., RNA-dependent double-stranded G. Faulkner, K., destruction. protein to expressway an building cullin: management. clinical in prospects towards molecules P. Degan, 2072-2080. .D,Kcuuakl .S,Kt,M,Lv,S,Csrn,G tal. et G. Cesareni, activated of S., kinases. down-regulation Lavi, tyrosine and M., ubiquitylation receptor instigates Katz, Nedd8 S., to B. Conjugation Kochupurakkal, D., M. immunity. systemic and development 117-135. organ al. et lymphoid W. on Wang, R., A. pathway. anemia Meetei, Fanconi J., the R. in subcomplexes Scheper, for A., Evidence M. (2006). Rooimans, J., Groot, n nege eaieautoregulation. negative undergoes A. and Dutta, and D. A. 203-229. kn .E n ip M. Lipp, and E. U. pken, ¨ ur o.Mcoil Immunol. Microbiol. Top. Curr. 21) xdtv tesi acn nei:fo el and cells from anaemia: Fanconi in stress Oxidative (2012). 20) B2 steE nteFnoiaei pathway anemia Fanconi the in E2 the is UBE2T (2006). .Bo.Chem. Biol. J. 21) h bqii code. ubiquitin The (2012). 20) h mato C7adCXCR5 and CCR7 of impact The (2003). o.Cell Mol. 281 246 se,R n ip M. Lipp, and R. rster, ¨ 21640-21651. , 98,dsuso 85. discussion 79-84, , 23 .V,Zteae .and A. Zatterale, V., F. , ´ Oncogene 589-596. , nu e.Biochem. Rev. Annu. il Chem. Biol. muo.Rev. Immunol. 20) ed on Nedd8 (2004). Blood 20) oeof Role (2001). 23 1985-1997. , 393 Blood 97 11-21. , ,1644- (1999). (2006). 108 195 81 , , , tu,T,Guin,S,Y,T,Bnt . em,C,Kb,D,L rs S., Smeitink, and F. Trijbels, Gras, R., Smeets, J., Loeffen, Le L., Heuvel, den D., van R., Triepels, Kobi, C., Keime, C., Bonet, T., Ye, S., Giuliano, T., Strub, cmd,T . anr,O,Ga,E .adCse,J G. J. Cyster, and E. E. J. Soulier, Gray, O., Bannard, H., T. Schmidt, osli . aca,J,Wezri,J n osaci E. Moustacchi, and J. Wietzerbin, J., Sanceau, F., Rosselli, u . hn .H,Za,X . ilas .S,Cry .B,Sih . Scott, E., Smith, B., G. Carey, S., M. Williams, F., X. Zhao, H., X. Zhan, D., Yu, G., P. P. Smith, S., D. T. Xirodimas, Lawrence, L., Zhao, T., J. Sebolt, J., Yu, H., Li, D., Wei, ag . a,M,Wn,G n u,Y. Sun, and G. P. Wang, D. M., Lane, Tan, and T. D., R. Hay, Yang, J.-C., Bourdon, K., M. Saville, P., D. Xirodimas, DF8sbnt DAcoig hoooa oaiain isedistribution, tissue localization, complex-I-deficient patients. chromosomal in detection mutation cloning, and cDNA subunit: NDUFA8 genomic J. and al. mitosis et replication, C. DNA melanoma. in for Bertolotto, stability factor R., transcription Ballotti, microphthalmia M., Cormont, XR rmtsBcl gesfo ee’ patches. Peyer’s from egress cell B 1099-1107. promotes CXCR4 ypoiepouto:anvlfaueo h eei ies acn anemia. Fanconi disease interleukin-6. genetic the of of Involvement feature I. novel a production: lymphokine . h,J,Go . hrkr,S tal. et S. Cherukuri, lymphomagenesis. Y., to predisposed Guo, are J., expression Zhu, D., enzyme NEDD8-activating investigational an MLN4924, by Y.inhibitor. Sun, cells and cancer A. M. Morgan, aisniiaino ua ratcne el yMN94 ninvestigational an enzyme. MLN4924, activating by NEDD8 cells of cancer inhibitor breast human of transcriptional radiosensitization its inhibits p53 of conjugation activity. NEDD8 Mdm2-mediated (2004). NEDD8. molecule 19) h ula-noe ua AHuiunn oxidoreductase NADH:ubiquinone human nuclear-encoded The (1998). ora fCl cec 21)17 5635 doi:10.1242/jcs.150706 3546–3554 127, (2014) Science Cell of Journal Cell acrRes. Cancer 21) acn anemia. Fanconi (2011). 118 83-97. , 20) oe usrtsadfntosfrteubiquitin-like the for functions and substrates Novel (2008). ice.Sc Trans. Soc. Biochem. 72 Oncogene 282-293. , 21) aisniiaino ua pancreatic human of Radiosensitization (2012). u.Genet. Hum. 30 Hematology 2319-2332. , LSONE PLoS 36 802-806. , 89 2011 42-48. , 21) iedfceti MIM in deficient Mice (2012). 21) h p21-dependent The (2012). 7 Oncogene e34079. , 492-497. , u.Genet. Hum. 21) seta oeof role Essential (2011). .Ep Med. Exp. J. 19) Abnormal (1992). 31 3561-3568. , 103 557-563. , (2013). 210 ,

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