Me ino‑Cachoe al.
Cell Communica ion and Signaling (2025) 23:101
h ps://doi.o g/10.1186/s12964‑025‑02091‑5
RESEARCH Open Access
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Cell Communica ion
and Signaling
Cullin‑RING ligase BioE3 e eals
molecula ‑glue‑induced neosubs a es
and ewi ing o heendogenous Ce eblon
ubiqui ome
Lau a Me ino‑Cacho1, O hi Ba oso‑Gomila1,8, Mónica Pozo‑Rod íguez1, Ve onica Mu a o e1,9,10,
Claudia Guinea‑Pé ez1, Ál a o Se ano2,3, Co alia Pé ez1, Sand a Cano‑López1, Ainhoa U cullu1,
Mikel Azka go a1,4, Ibon Ilo o1,4, Ca les Galdeano3,5, Jo di Juá ez‑Jiménez2,3, Ugo Mayo 6,7, Felix Elo za1,4,
Rosa Ba io1* and James D. Su he land1*
Abs ac
Backg ound The speci ici y o he ubiqui ina ion p ocess is media ed by he E3 ligases. Disc imina ing genuine
subs a es o E3s om me e in e ac ing p o eins is one o he majo challenges in he ield. We p e iously de el‑
oped BioE3, a bio in‑based app oach ha uses Bi A‑E3 usions oge he wi h ubiqui in used o a low‑a ini y A iTag
o ob ain a si e‑speci ic and p oximi y‑dependen bio inyla ion o he subs a es. We p o ed he sui abili y o BioE3
o iden i y a ge s o RING and HECT‑ ype E3 ligases.
Me hods BioE3 expe imen s we e pe o med in HEK293FT and U2OS s able cell lines exp essing TRIPZ‑bioGEFUb
ansien ly ans ec ed wi h Bi A‑ce eblon (CRBN). Cells we e seeded using bio in‑ ee media, ollowed la e
by a sho ‑bio in pulse. We e alua ed he applicabili y o he BioE3 sys em o CRBN and molecula glues by Wes e n
blo and con ocal mic oscopy, blocking he p o easome wi h bo ezomib, inhibi ing NEDDyla ion wi h MLN4924
and ea ing he cells wi h pomalidomide. Fo he iden i ica ion o endogenous subs a es and neosubs a es we ana‑
lyzed he elua es o s ep a idin pull‑downs o BioE3 expe imen s by LC–MS/MS. Analysis o a ge s o which ubiqui ‑
ina ion changes signi ican ly upon ea men was done using wo‑sided S uden ’s ‑ es . O hogonal alida ions we e
pe o med by his idine pull‑down, GFP‑ ap and compu a ional modelling.
Resul s He e we demons a e ha BioE3 is sui able o he mul i‑p o ein complex Cullin‑RING E3s ligases (CRLs),
he mos u ilized E3‑ ype o a ge ed p o ein deg ada ion (TPD) s a egies. Using CRBN as p oo o concep , one
o he subs a e ecep o s o CRL4 E3 ligase, we iden i ied bo h endogenous subs a es and no el neosubs a es
upon pomalidomide ea men , including CSDE1 which con ains a G‑loop mo i po en ially in ol ed in he binding
o CRBN in p esence o pomalidomide. Impo an ly, we obse ed a majo ea angemen o he endogenous ubiqui ‑
ina ion landscape upon ea men wi h his molecula glue.
*Co espondence:
Rosa Ba io
[email p o ec ed]
James D. Su he land
[email p o ec ed]
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Page 2 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
Conclusions The abili y o BioE3 o de ec and compa e bo h subs a es and neosubs a es, as well as how sub‑
s a es change in esponse o ea men s, will acili a e bo h on‑ a ge and o ‑ a ge iden i ica ions and o e
a b oade cha ac e iza ion and alida ion o TPD compounds, like molecula glues and PROTACs.
Keywo ds Ubiqui in, Ta ge ed P o ein Deg ada ion, Molecula Glue, Immunomodula o y d ugs, E3 ligases
Backg ound
P o ein ubiqui ina ion is a pos - ansla ional modi ica-
ion in ol ed in almos all cellula p ocesses and plays a
c ucial ole in he egula ion o p o ein homeos asis [1].
Ubiqui in (Ub) is co alen ly a ached o he a ge p o ein
in an highly egula ed enzyma ic cascade ha in ol es
wo ac i a ing E1s, a ound 40 conjuga ing E2s and mo e
han 600 E3 ligases [2]. Fu he mo e, ubiqui ina ion can
be e e sed by deubiqui ina ing enzymes (DUBs) [3].
Depending on hei mechanism o Ub ans e o he sub-
s a e p o ein, E3s a e classi ied in h ee main amilies:
RING (Really In e es ing New Gene; a ound 600 mem-
be s), HECT (Homology o E6AP C Te minus, a ound 30
membe s) and RBR (RING-Be ween-RING, a ound 15
membe s) [4, 5].
Cullin-RING E3 ligases (CRLs) a e he mos abun-
dan class o RING E3s and a e composed by mul iple
subuni s [6]. These complexes con ain a cullin sca old,
a RING inge p o ein (RBX1 o RBX2) ha binds he
E2, and dis inc se s o adap o s and subs a e ecep-
o s ha speci ically ec ui a ge p o eins. CRL ac i -
i y equi es cullin NEDDyla ion and is down egula ed
by deNEDDyla ion media ed by he COP9 signalosome.
CRLs a e p esen ly he mos used sub amily o a ge ed
p o ein deg ada ion (TPD), a s a egy ha uses chemi-
cals o ec ui undesi ed p o eins-o -in e es o an E3
ligase o ubiqui ina ion and deg ada ion. CRLs based
on ce eblon (CRBN) and Von Hippel-Lindau umo sup-
p esso (VHL) a e cu en ly he mos commonly used in
TPD. Speci ically, CRBN is he subs a e ecep o o he
CUL4–RBX1–DDB1–CRBN (CRL4CRBN) E3 complex.
CRBN was iden i ied as he a ge o immunomodula-
o y imide d ugs (IMiDs) [7], which include halidomide,
pomalidomide and lenalidomide. These compounds and
hei de i a i es ha e been he basis o many desc ibed
p o ein deg ada ion d ugs, like mono alen molecula
glues (MGs) and bi alen p o eolysis a ge ing chime-
as (PROTACs) [8, 9]. IMiDs bind o CRBN and al e i s
subs a e speci ici y, ac ing as MGs and leading o ubiq-
ui ina ion o non-na i e subs a es (neosubs a es). Ubiq-
ui ina ion by a pa icula E3 o on pa icula subs a es
can occu in di e se ways (on a single si e, mul iple si es,
and wi h ex ended ubiqui in-chains o di e en linkages
and opologies). This in luences subs a e a e and o en,
bu no always, leads o p o ein deg ada ion. The e o e,
su eying endogenous ubiqui ina ed subs a es and o
neosubs a es upon cellula ea men wi h molecula
glues and PROTACs should be a c ucial s ep in alida ion
o his p omising d ug class.
Unde s anding subs a e ecogni ion by pa icula
E3 ligases is a ele an a ea o esea ch in he Ub ield,
especially in he ligh o new de elopmen s in TPD [10].
Assays ha aid in he disco e y and/o cha ac e iza ion
o subs a e speci ici y o E3 ligases will be an impo an
addi ion o he chemical biology and d ug disco e y ool-
box. Di e en s a egies o iden i y a ge s o E3 ligases
ha e been de eloped. P oximi y p o eomics, which has
been applied o membe s o he mul i-subuni RING
SCF (Skp, Cullin, F-box) complex and o he s, may iden-
i y in e ac o s, some o which may be a ge s [11–13].
O he s a egies in ol e he di ec usion o E3 ligases o
Ub-like p o eins (UbLs) (UBAIT, TULIP, and SATT) o
Ub-binding domains [14–17]. O e exp ession o an E3
in combina ion wi h epi ope- agged Ub has also been
used o iden i y candida e E3 subs a es [18, 19], bu
some o hese subs a es migh no be di ec . To com-
plemen hese app oaches, we ecen ly de eloped BioE3
[20], a bio in-based s a egy based on wo elemen s: (1)
he usion o he Bi A enzyme, a bio in ligase ha labels
speci ically a bio in accep o pep ide (A iTag), o he
E3 ligase o in e es ; and (2) a UbL used o an A iTag
wi h lowe a ini y o Bi A (bioGEF) [21]. The use o
bioGEFUbLs allows a si e-speci ic and p oximi y-depend-
en bio inyla ion ha leads o he speci ic labeling o
he ubiqui ina ed subs a es. Those can be cap u ed by
s ep a idin pull-down and iden i ied using liquid ch o-
ma og aphy-mass spec ome y (LC–MS/MS) p o eom-
ics. BioE3 was applied o he RING non-associa ed o
cullins (RNF4, MIB1, MARCH5 and RNF214) and HECT
(NEDD4) E3 ligases. Simila me hods (E-STUB and Ub-
POD) ha e been desc ibed ha suppo his bioUb-based
app oach o iden i y a ge s [22, 23].
He e we demons a e how BioE3 can be used o he
iden i ica ion o bo h CRBN endogenous subs a es and
neosubs a es in p esence o iMiDs. By using Bi A o
he N- e minus o CRBN, we show speci ic bio inyla-
ion in HEK293FT- and U2OS-TRIPZ-bioGEFUb cells.
We alida ed Spal -like 4 (SALL4) as a neosubs a e upon
pomalidomide ea men , hus con i ming he capaci y o
CRBN BioE3 o iden i y neosubs a es. Ou p o eomic
s udy iden i ied known and no el endogenous sub-
s a es o CRBN, and po en ial pomalidomide-induced
Page 3 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
neosubs a es, including CSDE1 (Cold Shock Domain
Con aining E1), wi h o hogonal alida ion and compu-
a ional modelling o explo e binding si es. Impo an ly,
we disco e ed global di e ences in he ubiqui ina ion o
endogenous subs a es upon pomalidomide ea men .
By e ealing changes in bo h endogenous subs a es
and neosubs a es o pa icula E3-d ug combina ions,
we an icipa e ha BioE3 will be a e y use ul ool in he
u u e de elopmen o TPD.
Me hods
Cell cul u e
U2OS (ATCC HTB-96) and HEK293FT (In i ogen) we e
cul u ed a 37ºC and 5% CO2 in Dulbecco’s Modi ied
Eagle Medium (DMEM) supplemen ed wi h 10% e al
bo ine se um (FBS, Biowes ) and 1% penicillin/s ep o-
mycin (Gibco). HEK293FT cells we e used o Wes e n
blo and mass spec ome y expe imen s, whe eas U2OS
cells we e used o con ocal mic oscopy. Fo all BioE3
expe imen s, cells we e p e-cul u ed o 24h in bio in-
ee media supplemen ed wi h 10% dialyzed FBS (3.5kDa
MWCO; 150mM NaCl; il e -s e ilized) p io o ans-
ec ions, o allow obus labelling du ing bio in pulses.
Cul u ed cells we e main ained o a maximum o 20 pas-
sages and es ed nega i e o mycoplasma.
Cloning
Plasmids we e gene a ed by s anda d cloning o Gib-
son Assembly (NEBuilde HiFi Assembly, NEB). XL10-
Gold bac e ia (Agilen ) we e used. Depending on he
cons uc ion, we used plasmid backbones de i ed om
TRIPZ (Open Biosys ems/Ho izon) o Len i-Cas9-
blas (Addgene #52,962, kindly p o ided by F. Zhang).
TRIPZ-bioGEFUb and TRIPZ-bioGEFUbnc we e p e i-
ously desc ibed (Addgene #208,045, 208,044) [20]. CRBN
ORF was ampli ied om hTERT-RPE1 cell cDNA by
high- ideli y PCR (Pla inum Supe Fi DNA Polyme ase;
In i ogen #12,351,010) and was inse ed in o he EcoR1-
No 1 si es o Len i-EFS-Bi Aop -GSQ-RBXN-P2A-blas
(Addgene #208,048) [20]. CRBN mu a ion desc ibed
in he ex was in oduced by 2- agmen o e lap PCR
and Gibson assembly o using p ime s: CRBN.W386A.
qc. o (agc gg cc ggg a gccGCTac g gcccag g aaga c)
and CRBN.W386A.qc. e (ga c acac gggcaacag AGCg-
gca acccaggaaaccagc ). Cons uc ions we e alida ed by
Sange sequencing. Fu he cons uc de ails a e a ail-
able upon eques .
Len i i al ansduc ion
Packaging o len i i al exp ession cons uc s was done
in HEK293FT cells by ans ec ing psPAX2 and pMD2.G
(kindly p o ided by D. T ono; Addgene #12,260, #12,259)
and pTAT (kindly p o ided by P. Fo es; o TRIPZ-based
ec o s) using calcium phospha e. A e 12–18h ans-
ec ion media we e emo ed and eplaced wi h esh
media. Len i i al supe na an s we e collec ed wice (24h
each), pooled, il e ed (0.45 µm), supplemen ed wi h
s e ile 8.5% PEG6000, 0.3 M NaCl, and incuba ed o
12–18h a 4°C. Len i i al pa icles we e concen a ed by
cen i uga ion (1, 500 × g, 45min, 4°C). HEK293FT and
U2OS cells we e ansduced wi h non-concen a ed o
5 × concen a ed i us, espec i ely. D ug selec ion was
pe o med wi h 1µg/ml pu omycin (ChemC uz).
T ans ec ions andd ug ea men s
HEK293FT and U2OS cells we e ans ec ed using cal-
cium phospha e o Lipo ec amine 3000 (The mo Fishe ),
espec i ely. TRIPZ cell lines s ably ansduced we e
induced wi h DOX (doxycycline hycla e 1µg/ml; 24h;
Sigma-Ald ich) p io o bio in ea men (50µM; 2h;
Sigma-Ald ich). BTZ (200 nM; MedChemExp ess),
MLN4924 (1µM, MedChemExp ess) and pomalidomide
(10µM, MedChemExp ess) ea men s we e pe o med
wi hou bio in p io o cell lysis o immunos aining a he
indica ed ime-poin s.
Wes e n blo analysis
To emo e excess bio in, we washed cells wi h 1 × PBS
and hen we lysed hem in highly s ingen washing bu e
(WB) 5 (WB5: 8M u ea, 1% SDS in 1 × PBS) con aining
1 × p o ease inhibi o cock ail (Roche) and 50µM N-E h-
ylmaleimide (NEM, Al a Aesa ). Samples we e sonica ed
and cen i uged (16,000 × g, 30min a oom empe a u e,
RT). P o ein concen a ion was de e mined by he BCA
P o ein Assay (Pie ce) ollowing manu ac u e ’s ins uc-
ions. Fo SDS-PAGE, 20µg o p o ein was loaded and
ans e ed o ni ocellulose memb anes. PBT (1 × PBS,
0.1% Tween-20) was used o blocking, excep o an i-
bio in blo s, whe e casein-based blocking solu ion
(Sigma) was used. P ima y an ibodies we e incuba ed o
2h a RT o o e nigh a 4ºC and seconda y an ibodies o
45min a RT. An ibodies we e used as ollows: an i-bio-
in-HRP (1/1,000, Cell Signaling Technology Ca #7075S);
an i-Bi A (1/1,000, SinoBiological Ca #11,582-T16); an i-
A iTag (1/1,000, GenSc ip Ca #A00674); an i-NEDD8
(1/1,000, Abcam Ca # ab81264); an i-GAPDH (1/5,000,
P o ein ech Ca # 60,004–1-Ig); an i-CSDE1 (1/1,000,
P o ein ech Ca #13,319–1-AP); an i-GFP (1/1,000, Roche
Ca #11,814,460,001); an i-GFP (1/2,000, abbi poly-
clonal se um; gene a ed in-house agains ecombinan
GFP p o ein); an i-HA- ag (1/2,000, Cell Signaling Tech-
nology Ca #3724); an i-Mouse-HRP (1/5,000, Jackson
ImmunoResea ch Ca #115–035–062); an i-Rabbi -HRP
(1/5,000, Jackson ImmunoResea ch Ca #111–035–045).
We used Supe Signal Wes Fem o (The moFishe ) o
Cla i y ECL (BioRad) o de ec he p o eins using an
Page 4 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
iB igh CL1500 (The mo Fishe ). Unc opped blo s a e
p o ided as Supplemen a y Ma e ial 12.
Immunos aining andcon ocal mic oscopy
U2OS cells we e seeded on 11 mm co e slips (20,000
cells pe well; 24 well pla e). A e washing he cells wi h
1 × PBS hey we e ixed wi h 4% PFA supplemen ed wi h
0.1% T i on X-100 in 1 × PBS o 20min a RT. Then,
co e slips we e washed 3 imes wi h 1 × PBS and incu-
ba ed in blocking bu e (2% e al cal se um, 1% BSA
in 1 × PBS) o 30min a RT. P ima y an ibodies we e
incuba ed o 1h a 37ºC and cells we e washed 3 imes
wi h 1 × PBS. P ima y an ibodies we e used as ollow:
an i-Bi A (1/200, No us Biologicals Ca #NBP2-59,939);
an i-NEDD8 (1/100, Abcam Ca # ab81264). Seconda y
an ibodies and luo escen s ep a idin we e incuba ed
o 30min a 37ºC. A e ha , nuclei we e s ained wi h
DAPI (300ng/ml in 1 × PBS, Sigma Ald ich) o 10min.
Seconda y an ibodies (The moFishe ) we e all used a
1/200: an i-Mouse Alexa Fluo 488 (Ca #A-11029), an i-
Rabbi Alexa Fluo 647 (Ca #A-21244), an i-Mouse Alexa
Fluo 647 (Ca #A-31571). S ep a idin Alexa Fluo 594
(1/200, Ca #016–290-084, Jackson ImmunoResea ch)
was also used. Images we e aken wi h a con ocal mic o-
scope (Leica SP8 Ligh ning) using 63 × Plan ApoCh oma
NA1.4 objec i e.
Pull‑down o bio inyla ed p o eins
The lysa es clea ed in WB5 we e no malized o he same
p o ein concen a ion and incuba ed o e nigh a RT
wi h equilib a ed Neu A idin-aga ose beads (The -
moFishe ) a a a io o 1/50 (Vbeads/ Vlysa e). The high a in-
i y be ween bio in and s ep a idin allows s ingen se ies
o washes, as ollows (VWB/2Vlysa e): 2 × WB1 (8M u ea,
0.25% SDS); 3 × WB2 (6 M Guanidine-HCl); 1 × WB3
(6.4M u ea, 1M NaCl, 0.2% SDS); 3 × WB4 (4M u ea,
1M NaCl, 10% isop opanol, 10% e hanol and 0.2% SDS);
1 × WB1; 1 × WB5; and 3 × WB6 (2% SDS; WB1-6 p e-
pa ed in 1 × PBS). Bio inyla ed p o eins we e elu ed om
he beads using 1 olume o Elu ion Bu e (4 × Laemmli
bu e , 100mM DTT; 80µl o LC–MS/MS expe imen s)
by hea ing a 99°C o 5min wice, ollowed by o ex-
ing. Beads we e sepa a ed using 0.8µm cla i ying il e s
(2,000 × g, 2min; Vi aclea Mini, Sa o ius).
Liquid Ch oma og aphy Mass Spec ome y (LC–MS/MS)
Pull-down expe imen s o mass-spec ome y we e
pe o med independen ly in iplica es. Fo each ep-
lica e, ou con luen 15cm dishes (8 × 107 cells, 2ml
o lysis pe pla e; 8ml o al) we e analyzed by LC–MS/
MS. Samples elu ed om he Neu A idin beads we e
sepa a ed in SDS-PAGE and s ained wi h Syp o Ruby
(In i ogen) ollowing manu ac u e ’s ins uc ions. Gel
lanes we e ca e ully cu o ensu e consis ency and ep o-
ducibili y. Slices we e subsequen ly washed in milli-Q
wa e . Reduc ion and alkyla ion we e pe o med (10mM
DTT in 50mM ammonium bica bona e, 56°C, 20min,
ollowed by 50mM chlo oace amide in 50mM ammo-
nium bica bona e, 20 min, p o ec ed om ligh ). Gel
pieces we e d ied and incuba ed wi h ypsin (12.5µg/
ml in 50mM ammonium bica bona e, 20min, ice-cold).
A e ehyd a ion, he ypsin supe na an was disca ded.
A e hyd a ion wi h 50 mM ammonium bica bona e,
gel pieces we e incuba ed a 37°C o e nigh . Following
diges ion, 0.1% TFA was used o clean acidic pep ides,
which we e d ied in a RVC2 25 SpeedVac concen a o
(Ch is ). Pep ides we e esuspended in 10µl 0.1% o mic
acid (FA) and sonica ed o 5min p io o analysis.
Samples we e analyzed using a imsTOF P o mass
spec ome e ( apped ion mobili y spec ome y/quad-
upole ime o ligh hyb id, B uke Dal onics) coupled
online o a EVOSEP ONE (E osep), which uses pa allel
accumula ion–se ial agmen a ion (PASEF), a he p o-
eomics pla o m o CIC bioGUNE. Sample (200ng) was
di ec ly loaded in a 15cm pe o mance column (E osep)
applying a 30 samples pe day me hod and da a depend-
en acquisi ion mode.
Mass spec ome y da a analysis
DIA da a was p ocessed wi h DIA-NN so wa e o
p o ein iden i ica ion and quan i ica ion using de aul
pa ame e s. Sea ches we e ca ied ou agains a da abase
consis ing o Homo sapiens p o ein en ies om Unip o
in lib a y- ee mode. We conside ed ca bamidome hyla-
ion o cys eines and oxida ion o me hionines as ixed
and a iable modi ica ions, espec i ely. Ma ch be ween
uns was applied and p ecu so FDR was se a 1%. Da a
was p ocessed and analyzed by Pe seus ( e sion 1.6.15)
[24]. P o eins iden i ied by a leas 2 pep ides and p e-
sen in a leas 2 ou o 3 eplica es in a leas one g oup
we e included in he analysis. S a is ical signi icance was
assessed using a wo-sided S uden ’s - es . Da a we e
loaded in o G aphPad P ism 10 e sion 10.0.2 o build
he co esponding olcano-plo s.
Ne wo k analysis was conduc ed using STRING
e sion 1.4.2 in Cy oscape e sion 3.9.1, applying a
high-con idence in e ac ion sco e o 0.7 [25, 26]. Size,
anspa ency and wid h o he edges we e con inuously
mapped o he Log2 old change. The Molecula COm-
plex DE ec ion (MCODE) plug-in e sion 1.5.1 was
used o iden i y highly connec ed subclus e s o p o eins
(deg ee cu o o 2; Clus e inding: Hai cu ; Node sco e
cu o o 0.2; K-Co e o 2; Max. Dep h o 100) [27]. Gene
on ology analysis was pe o med using g:P o ile web
se e e sion e108_eg55_p17_0254 b and REVIGO [28,
Page 5 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
29]. Venn diag ams we e d awn using In e ac iVenn web
ool [30].
Immobilized me al a ini y ch oma og aphy o alida ions
Fo o hogonal alida ions, HEK293FT cells we e
co- ans ec ed wi h he indica ed cons uc s and
pcDNA3-6xHIS-Ub (gi om M. Rod iguez, CRNS-
LCC, Toulouse), lysed in lysis bu e (8M u ea, 0.1M
Na2HPO4/NaH2PO4 pH 8.0, 0.01M T is–HCl pH 8.0,
20 mM imidazole pH 8.0, 5 mM β-me cap oe hanol,
and 0.1% T i on X-100), supplemen ed wi h 1 × p o ease
inhibi o cock ail (Roche) and 50µM PR619 DUB inhibi-
o (Me ck). Samples we e hen sonica ed and clea ed
by cen i uga ion (25, 000 × g, 30 min a RT). Clea ed
lysa es we e adjus ed o he same p o ein concen a ion
be o e incuba ing hem wi h 1/50 ( olbeads/ ollysa e) equil-
ib a ed Ni–NTA aga ose beads (In i ogen) o e nigh
a RT. Beads we e hen washed h ee imes using WBB
(8M u ea, 0.1M Na2HPO4/NaH2PO4 pH 8.0, 0.01M
T is–HCl pH 8.0, 20 mM imidazole pH 8.0, 2.5 mM
β-me cap oe hanol, and 0.1% T i on X-100), and wo
imes using WBC (8M u ea, 0.1M Na2HPO4/NaH2PO4
pH 6.3, 0.01M T is–HCl pH 6.3, 10mM imidazole pH
7.0, 2.5mM β-me cap oe hanol, and 0.1% T i on X-100).
P o eins we e elu ed wi h 1 olbeads o Elu ion Bu e (4M
u ea, 50mM NaH2PO4/Na2HPO4, 5mM T is/HCl pH 8,
500mM, imidazole pH 7.0, 1.25mM β-me cap oe hanol,
and 0.05% T i on X-100). Elua es we e analyzed by wes -
e n blo ing wi h app op ia e an ibodies.
GFP‑ ap pull‑down
All s eps we e pe o med a 4ºC. HEK293FT cells we e
collec ed 48h a e ans ec ion, washed 3 imes wi h
1 × PBS and lysed in RIPA lysis bu e (50mM T is–HCl
pH 8, 150mM NaCl, 1% IGEPAL CA-630, 0.5% sodium
deoxychola e, and 0.1% SDS) supplemen ed wi h 1 × p o-
ease inhibi o cock ail (Roche), BTZ (MedChemEx-
p ess) and 50µM PR619 (Me ck). Lysa es we e kep on
ice o 30min and clea ed by cen i uga ion (25,000 × g,
30 min a 4ºC). Clea ed lysa es we e incuba ed wi h
15µl o equilib a ed GFP-Selec o beads (Nano ag Bio-
echnologies) o e nigh a 4ºC in a o a ing wheel. Beads
we e washed 4 imes wi h RIPA lysis bu e , wice wi h
RIPA/4M u ea, and once mo e wi h RIPA. The samples
we e elu ed in 2 × Laemmli bu e by boiling o 5min
a 95ºC. Elua es we e analyzed by wes e n blo ing wi h
app op ia e an ibodies.
Molecula modelling
The G-loop egions o CSDE1 we e iden i ied by com-
pa ing he posi ion o α-ca bon a oms be ween he
CSDE1 AlphaFold model AF-O75534-F1 and he c ys-
allog aphic s uc u e o SALL4 wi h PDB id: 7BQU.
The s uc u e o he i s CSDE1 domain was ob ained
om he AlphaFold model AF-O75534-F1 and he sec-
ond domain was ex ac ed om he NMR model wi h
PDB id: 2YTV. The e na y complexes we e modeled by
supe imposing each o he G-loop sequences o CSDE1
o he s uc u e o CK1α in complex wi h CRBN (PDB
id 5FQD) as e e ence and he pomalidomide-binding
mode in he halidomide-binding domain was aken
om he c ys allog aphic s uc u e wi h PDB id: 6H0F.
The AMBER 14SB and GAFF2 o ce ields we e used o
assign a om ypes o he p o eins and pomalidomide,
espec i ely. The pa ial cha ges o pomalidomide we e
de i ed using he RESP p o ocol a he HF/6-31G(d)
le el o heo y, calcula ed using Gaussian16 [31]. The
Zn2 + ca ion coo dina ed wi h CRBN was modeled wi h
he bound model, using he ZAFF pa ame e s [32]. The
sys ems we e sol a ed on a unca ed oc ahed al box o
TIP3P wa e molecules, and neu alized wi h sal coun-
e ions. Following he p o ocol we ha e ecen ly epo ed
[33], each sys em was minimized, hea ed o 298K, and
equilib a ed o 1ba . Each H-bond in he e na y com-
plex in e ace was e alua ed using 100 independen
s ee ed molecula dynamics ajec o ies. S a ing posi-
ions and eloci ies o each sys em we e sampled om
independen classical MD 10ns ajec o ies, using a la -
bo om es ain o keep he H-bonds be ween 2.5 and
3.5 Å. Then he H-bonds we e b ough o a 2.5 Å dis ance
o s a a cons an speed s ee ing a a speed o 0.5 Å/
ns, using he s i sp ing app oxima ion. All simula ions
we e pe o med wi h he CUDA accele a ed e sion o
PMEMD om he Ambe 23 package [34]. The po en ial
mean o ce (PMF) o each H-bond was compu ed using
he Ja zynski equali y on he esul ing wo k p o iles [35].
The e o es ima ions o he PMF p o iles we e ob ained
by boo s apping 25 imes wi h 25 eplica subsamples.
S a is ical analysis
S a is ical analysis was pe o med wi h G aphPad P ism
10 using wo- ailed unpai ed S uden ’s - es . Di e -
ences we e conside ed signi ican when p- alue < 0.05 (*).
Quan i ica ion o p o ein le els in Wes e n blo s and s a-
is ical analysis a e shown in Supplemen a y Ma e ial 13.
Resul s
Applying BioE3 os udy Cullin‑RING E3 ligases (CRL)
We p e iously used BioE3 o de ec ing speci ic a ge s
o RING and HECT ype E3 ligases [20], bu he po en-
ial o BioE3 o de ec a ge s o E3s mul i-p o ein com-
plexes like he CRLs was no es ed. Fi s , we conside ed
he o ien a ion o he Bi A enzyme wi hin he usion
p o ein, which could p oduce s e ic p oblems. We pe -
o med BioE3 expe imen s wi h Bi A used o he N- o
C- e minus o CRBN and we obse ed a simila pa e n
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Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
o bio inyla ed p o eins (Fig. S1a). Based on his and p e-
ious epo s [36, 37], we con inued using a usion o
Bi A o he N- e minus o CRBN (Fig.1a, b) and an-
sien ly ans ec ed i in o s able cell lines exp essing he
bioGEFUb in a doxycycline (DOX)-dependen manne
(TRIPZ-bioGEFUb). The low-a ini y bioGEF A iTag ena-
bles si e-speci ic and p oximi y-dependen bio inyla-
ion [21]. In his way, a e DOX induc ion o 24h and
con olled bio in pulses, subs a es modi ied by he
CRL4Bi A−CRBN and labelled wi h bio inyla ed bioGEFUb
can be pu i ied using s ep a idin pull-down o iden i-
ica ion by LC–MS/MS, o imaged by immuno luo es-
cence and con ocal mic oscopy (Fig.1a, b).
To es he speci ici y o he sys em, we pe o med
BioE3 using Bi A-CRBN and HEK293FT-TRIPZ-bioGE-
FUb cells, inducing he exp ession o bioGEFUb wi h DOX
and pe o ming a 2-h bio in pulse. We obse ed bio i-
nyla ed p o eins, indica ing he ac i i y o Bi A-CRBN
usion (Fig.1c), simila ly o wha was p e iously epo ed
by Huang and collabo a o s o CRBN-Bi A [22]. Addi-
ionally, when p o easome ac i i y was blocked wi h
bo ezomib (BTZ) we obse ed a u he accumula ion o
bio inyla ed p o eins ha was e e sed upon inhibi ion
o NEDDyla ion wi h MLN4924 (Fig.1c).
We hen con i med co-localiza ion o he Bi A-CRBN
usion p o ein wi h NEDD8 in he cy oplasm by con o-
cal mic oscopy in U2OS-TRIPZ-bioGEFUb cells (Fig.1d),
which sugges s ha he usion was co ec ly inco po a ed
in o he CRL complex. Upon p o easomal inhibi ion we
obse ed an inc ease in bio inyla ed p o eins (Fig. 1d,
S ep panel) ha co-localized wi h he Bi A enzyme,
indica ing speci ic bio inyla ion. MLN4924 ea men
educed he bio in labeling and dispe sed he NEDD8
signal. Taken oge he , hese da a sugges ha he BioE3
sys em is bio inyla ing CRBN subs a es in a CRL-
dependen manne .
Nex , we es ed wild ype (WT) bioGEFUb o i s use in
CRBN-BioE3. In p e ious expe imen s [20], we used a
non-clea able e sion o Ub bea ing he L73P mu a ion
(Ubnc) o p e en he ecycling o bio inyla ed bioGEFUb
by supp essing access o DUBs. Howe e , o analyze
HECT- ype E3s (e.g. NEDD4), we ound ha i is neces-
sa y o use he bioGEFUb, since bioGEFUbnc was no e i-
cien ly passed om E2 o E3 [20]. We pe o med BioE3
expe imen s bo h in HEK293FT-TRIPZ-bioGEFUb and
HEK293FT-TRIPZ-bioGEFUbnc s able cell lines (Fig. S1b)
and obse ed highe abundance o bio inyla ed p o eins
when using bioGEFUb. Impo an ly, as shown in Fig.1d,
bio inyla ed ma e ial was es ic ed o Bi A-CRBN locali-
za ion, indica ing he speci ici y o labelling in ou condi-
ions, so bioGEFUb was used o he es o expe imen s
shown he e.
BioE3 iden i ies SALL4 asaneosubs a e o CRBN
The ansc ip ion ac o SALL4 is one o he bes -known
neosubs a es o CRBN induced by pomalidomide [38–
40], so we aimed o iden i y SALL4 by BioE3 upon ea -
men wi h pomalidomide as a p oo o concep . We used
a CRBN mu an de icien in IMiD-binding (Bi A-CRB-
NW386A) [41] ha should ac as a nega i e con ol o he
iden i ica ion o neosubs a es upon pomalidomide ea -
men . We pe o med BioE3 expe imen s in HEK293FT-
and U2OS-TRIPZ-bioGEFUb cell lines ansien ly
ans ec ed wi h he WT o mu an e sions o Bi A-
CRBN, blocking he p o easome wi h BTZ and ea ing
he cells wi h he IMiD d ug pomalidomide (Fig. S1c).
By Wes e n blo we no iced a change in he bio inyla ion
pa e n when ea ing he samples wi h pomalidomide,
sugges ing ha BioE3 is sensi i e o molecula glues and
can di e en ia e he endogenous a ge s om he neo-
subs a es. Bi A-CRBNW386A also p oduced bio inyla ion
o p o eins, bu wi hou changing he pa e n obse ed
a e adding he IMiD (Fig. S1c). By con ocal mic oscopy,
we could alida e he sui abili y o he mu an o BioE3
expe imen s: in e es ingly, Bi A-CRBNWT displayed a
nuclea localiza ion a e pomalidomide ea men ha
was no obse ed wi h Bi A-CRBNW386A, oge he wi h
an inc ease in he nuclea bio inyla ed ma e ial (Fig.2a,
S ep panel).
(See igu e on nex page.)
Fig. 1 BioE3 labels CRL‑dependen ubiqui ina ed subs a es o CRBN. a, b Schema ic ep esen a ion o he BioE3 s a egy adap ed o he subs a e
ecep o CRBN (a) and he cons uc s used in his wo k (b). TRIPZ, all‑in‑one inducible len i i al ec o ; bioGEF, low a ini y A iTag (see ex ); DOX,
doxycycline; Te ON, e acycline inducible p omo e ; PUROR, pu omycin esis an casse e; EFS, elonga ion ac o 1α sho p omo e . c Wes e n
blo o BioE3 expe imen pe o med on HEK293FT s able cell line exp essing TRIPZ‑bioGEFUb and ans ec ed wi h EFS‑Bi A‑CRBN. Indica ed
samples we e ea ed wi h 100 nM bo ezomib (BTZ) o 24 h, 200 nM BTZ o 6 h and/o 1 µM MLN4924 o 6 o 24 h. Molecula weigh ma ke s
a e shown o he le o he blo s in kDa, an ibodies used a e indica ed o he igh . A iTag an ibodies highligh all he ubiqui ina ed p o eins,
while bio in shows hose ubiqui ina ed by CRBN. d Con ocal mic oscopy images o BioE3 expe imen pe o med on U2OS s able cell line exp essing
TRIPZ‑bioGEFUb ans ec ed wi h Bi A‑CRBN. Indica ed samples we e ea ed wi h 200 nM BTZ o 6 h and/o 1 µM MLN4924 o 24 h. Bio inyla ed
ma e ial is s ained wi h luo escen s ep a idin (S ep, magen a) and Bi A (g een) and NEDD8 (blue) wi h speci ic an ibodies. Scale ba : 8 µm. All
BioE3 expe imen s we e pe o med by p e‑incuba ing he cells in dialyzed FBS‑con aining media p io o ans ec ions, doxycycline (DOX) induc ion
a 1 µg/ml o 24 h and bio in supplemen a ion a 50 µM o 2 h
Page 7 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
Fig. 1 (See legend on p e ious page.)
Page 8 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
Conside ing ha he endogenous exp ession le el o
SALL4 in HEK293FT cells is low [42], we decided o pe -
o m he BioE3 expe imen wi h exogenously exp essed
SALL4-YFP and ea ing he cells wi h BTZ and/o
pomalidomide. A e isola ing he bio inyla ed p o eins
by s ep a idin pull-down, we obse ed an en ichmen o
polyubiqui ina ed SALL4-YFP upon pomalidomide ea -
men wi h he CRBNWT e sion bu no wi h he IMiD-
binding mu an CRBNW386A (Fig.2b). We con i med his
esul using con ocal mic oscopy, obse ing co-localiza-
ion o Bi A-CRBNWT and bio in signal a nuclea bod-
ies o med by SALL4-YFP only in hose cells ea ed wi h
pomalidomide (Fig.2c). Fu he mo e, Bi A-CRBNW386A
did no co-localize o he SALL4 nuclea bodies, nei he
bio inyla ed SALL4-YFP, e en a e ea men wi h he
IMiD. Al oge he , we concluded ha BioE3 is sensi i e
o he esponses induced by molecula glues and can be
used o disc imina e be ween endogenous subs a es and
neosubs a es.
BioE3 iden i ies endogenous a ge s o CRBN
Once we con i med he sui abili y o BioE3 o bio inyla e
bo h pu a i e subs a es and neosubs a es a e molecu-
la glue ea men , we pe o med a la ge-scale expe i-
men in iplica es, ea ing he cells wi h BTZ only, BTZ
and MLN4924 o BTZ and pomalidomide (Figs.3and4;
Supplemen a y Tables1–3). We isola ed he bio inyla ed
p o eins by s ep a idin pull-downs and con i med by
Wes e n blo he en ichmen o he elua es in bio i-
nyla ed p o eins when he p o easome was blocked wi h
BTZ (Fig.3a). By inhibi ing NEDDyla ion he amoun o
bio inyla ed p o eins was educed, suppo ing MLN4924
as a use ul nega i e con ol. We also obse ed a educ-
ion in bio inyla ed p o eins a e pomalidomide ea -
men . The elua es we e hen analyzed by LC–MS/MS o
iden i y speci ic ubiqui ina ed a ge s o CRBN. Fi s , by
compa ing he samples ea ed o no wi h BTZ (BTZ
e sus DMSO), we iden i ied 376 pu a i e a ge s o
CRBN ha a e p o easome- a ge ed (Fig. 3b; Supple-
men a y Table1). Among hem, we ound glu amine syn-
he ase (GLUL), an endogenous a ge o he subs a e
ecep o p e iously desc ibed [43].
We also in e oga ed he e ec o inhibi ing NED-
Dyla ion in he iden i ica ion o endogenous subs a es
o CRBN, by compa ing BTZ e sus BTZ and MLN4924
ea ed cells, and iden i ied 267 pu a i e a ge s o CRBN
ha equi e NEDD8 ac i a ion o he CRL complex
(Fig.3c; Supplemen a y Table2). Among hem, 177 (66%)
we e also p esen in he CRBN BioE3 BTZ e sus DMSO
compa ison, including GLUL (Fig. 3d). In e es ingly,
NEDD8 was signi ican ly en iched when inhibi ing NAE1
(le quad an o Fig.3c), indica ing ha mixed NEDD8-
Ub conjuga es we e accumula ing in ha sample. This
was also con i med by Wes e n blo , showing an en ich-
men in high molecula weigh NEDDyla ed p o eins
(Fig.3e). These esul s a e in line wi h p e ious epo s
showing he use o NEDD8 by he Ub machine y when
he NEDDyla ion machine y is blocked [44, 45].
In addi ion o he a o emen ioned hi s, we iden i ied
p o eins ela ed o he UbL machine y, including ubiq-
ui ina ion (E3s and DUBs), SUMOyla ion (SUMO1,
SUMO2, PIAS1, PIAS4), NEDDyla ion (NEDD8,
COPS6) and p o easome componen s. We also ound
componen s o he cAMP signaling pa hway like MTOR,
PRKAR1A and CREB3 [46], componen s o he Wn
signaling pa hway such as CSNK1E and CTNNB1 [47],
chlo ide channels as CLCC1 and CLCN3 [48], BSG o
which a ubiqui ina ion-independen , chape one-like
unc ion o CRBN was desc ibed [49], and componen s
o CRLs (DDB1) and he COP9 signalosome (COPS6,
COPS7A, COPS3) [50].
To de e mine he unc ional ole o CRBN we pe -
o med a STRING ne wo k analysis o he po en ial sub-
s a es (Figs. S2 and S4). The ne wo k showed a majo
in e connec ed co e-clus e composed o 71% and 64%
o he iden i ied subs a es espec i ely in BTZ e sus
Fig. 2 Iden i ica ion o SALL4 as a neosubs a e o CRBN upon pomalidomide ea men . a Con ocal mic oscopy o BioE3 expe imen pe o med
in U2OS s able cell line s ably exp essing TRIPZ‑bioGEFUb. Cells we e ans ec ed wi h EFS‑Bi A‑CRBNWT o EFS‑Bi A‑CRBNW386A (IMiD‑binding
de icien mu an ). Indica ed samples we e ea ed wi h 10 µM pomalidomide (POM) and/o wi h 200 nM bo ezomib (BTZ) o 6 h. Bio inyla ed
ma e ial is s ained wi h luo escen s ep a idin (S ep, magen a) and Bi A (g een) wi h speci ic an ibodies. b, c BioE3 expe imen pe o med
in HEK293FT‑TRIPZ‑bioGEFUb (b) o U2OS‑TRIPZ‑bioGEFUb (c) s able cell lines ansien ly ans ec ed wi h CMV‑SALL4‑YFP and EFS‑Bi A‑CRBNWT
o EFS‑Bi A‑CRBNW386A and ea ed wi h 10 µM POM and/o 200 nM BTZ o 6 h. b Wes e n blo alida ion o SALL4 as a neosubs a e
upon pomalidomide ea men . The do ep esen s he possible monoubiqui ina ed p o ein, whe eas he ba ep esen s he polyUb‑modi ied
SALL4‑YFP. S ep PD: s ep a idin pull‑down. Molecula weigh ma ke s a e shown o he le o he blo s in kDa, an ibodies used a e indica ed
o he igh . Quan i ica ion g aph o he ubiqui ina ed GFP‑SALL4 le el in he GFP panel is shown unde he Wes e n blo s; *: p‑ alue < 0.05.
c) Bio inyla ed ma e ial is s ained wi h luo escen s ep a idin (S ep, magen a) and Bi A (blue) wi h a speci ic an ibody. SALL4‑YFP can be
ound in g een. Scale ba : 8 µm. Yellow do ed lines indica e he nuclei. Inse s show he ampli ica ion o he a ea indica ed by a whi e do ed
squa e in each panel. All BioE3 expe imen s we e pe o med by p e‑incuba ing he cells in dialyzed FBS‑con aining media p io o ans ec ions,
doxycycline (DOX) induc ion a 1 µg/ml o 24 h and bio in supplemen a ion a 50 µM o 2 h
(See igu e on nex page.)
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Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
Fig. 2 (See legend on p e ious page.)
Page 16 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
Abb e ia ions
bioGEF Low a ini y A iTag
BTZ Bo ezomib
CRBN Ce eblon
CRL Cullin‑RING E3 ligase
CRL4CRBN CUL4–RBX1–DDB1–CRBN E3 complex
CSDE1 Cold Shock Domain Con aining E1
DOX Doxycycline
DUB Deubiqui ina ing enzyme
FBS Fe al Bo ine Se um
GLUL Glu amine Syn he ase
HECT Homology o E6AP C Te minus
IMiD Immunomodula o y Imide D ug
LC–MS/MS Liquid ch oma og aphy‑mass spec ome y
MG Molecula Glue
PMF Po en ial Mean Fo ce
POM Pomalidomide
PROTAC P o eolysis Ta ge ing Chime as
RBR RING‑Be ween‑RING
RING Really In e es ing New Gene
SALL4 Spal ‑like 4
TPD Ta ge ed P o ein Deg ada ion
Ub Ubiqui in
UbL Ubiqui in‑like
UPS Ubiqui in P o easome Sys em
Supplemen a y In o ma ion
The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi.
o g/ 10. 1186/ s12964‑ 025‑ 02091‑5.
Supplemen a y Ma e ial 1. Fig. S1. Op imiza ion o he expe imen al
condi ions o CRBN BioE3. a, b, c) Wes e n blo o BioE3 expe imen s
pe o med on HEK293FT s able cell lines exp essing TRIPZ‑bioGEFUb o
TRIPZ‑bioGEFUbnc and ans ec ed wi h EFS‑Bi A‑CRBN, EFS‑CRBN‑Bi A o
EFS‑Bi A‑CRBNW386A. Indica ed samples we e induced wi h doxycycline a
1 µg/ml o 24 hou s, ea ed wi h 200 nM bo ezomib o 6 hou s, 1 µM
MLN4924 o 24 hou s o 10 µM pomalidomide o 6 hou s and sup‑
plemen ed wi h 50 µM bio in o 2 hou s. Molecula weigh ma ke s a e
shown o he le o he blo s in kDa, an ibodies used a e indica ed o he
igh . All BioE3 expe imen s we e pe o med by p e‑incuba ing he cells in
dialyzed FBS‑con aining media p io o ans ec ions.
Supplemen a y Ma e ial 2. Fig. S2. STRING ne wo k analysis o CRBN
ubiqui ina ed subs a es. Subs a es de ined in Figu e 3b show a highly
in e connec ed ne wo k composed o 71% o he p o eins. Highly in e ‑
connec ed sub‑clus e s we e de i ed and cha ac e ized using MCODE.
Colo , anspa ency and size o he nodes we e disc e ely mapped o he
Log2 en ichmen alue as indica ed.
Supplemen a y Ma e ial 3. Fig. S3. Gene on ology sca e plo o he CRBN
ubiqui ina ed subs a es. REVIGO plo s we e gene a ed o a ge s de ined
in Figu e 3b. Colo s indica e he ‑Log10 p‑ alue as shown in he Figu e,
and size o he bubble indica es he size o each e m. Only e ms wi h a
p‑ alue < 0.05 a e ep esen ed.
Supplemen a y Ma e ial 4. Fig. S4. STRING ne wo k analysis o CRBN
NEDDyla ion‑dependen subs a es. Subs a es de ined in Figu e 3c
show a highly in e connec ed ne wo k composed o 64% o he p o eins.
Highly in e connec ed sub‑clus e s we e de i ed and cha ac e ized
using MCODE. Colo , anspa ency and size o he nodes we e disc e ely
mapped o he Log2 en ichmen alue as indica ed.
Supplemen a y Ma e ial 5. Fig. S5. Gene on ology sca e plo o CRBN
NEDDyla ion‑dependen subs a es. REVIGO plo s we e gene a ed o
a ge s de ined in Figu e 3c. Colo s indica e he ‑Log10 p‑ alue as shown
in he Figu e, and size o he bubble indica es he size o each e m. Only
e ms wi h a p‑ alue < 0.05 a e ep esen ed.
Supplemen a y Ma e ial 6. Fig. S6. STRING ne wo k analysis o CRBN
neosubs a es. The neosubs a es upon pomalidomide ea men de ined
in Figu e 4a show a highly in e connec ed ne wo k composed o 71%
o he p o eins. Highly in e connec ed sub‑clus e s we e de i ed and
cha ac e ized using MCODE. Colo , anspa ency and size o he nodes
we e disc e ely mapped o he Log2 en ichmen alue as indica ed.
Supplemen a y Ma e ial 7. Fig. S7. Gene on ology sca e plo o he
neosubs a es o CRBN. REVIGO plo s we e gene a ed o a ge s de ined
in Figu e 4a. Colo s indica e he ‑Log10 p‑ alue as shown in he Figu e,
and size o he bubble indica es he size o each e m. Only e ms wi h a
p‑ alue < 0.05 a e ep esen ed.
Supplemen a y Ma e ial 8. Fig. S8. E ec o pomalidomide on endogenous
p o ein le els o CSDE1. a) Endogenous CSDE1 le els in HEK293FT cells
upon bo ezomib, MLN4924 and pomalidomide ea men s. b) Quan i a‑
i e da a and s a is ical analysis o h ee independen expe imen s as
in Figu e S8a. CSDE1 le els in cells ea ed wi h DMSO o pomalidomide
10 µM we e aken in conside a ion o he analysis. c) Endogenous CSDE1
le els in HEK293FT cells ansien ly ans ec ed wi h EFS ‑Bi A o EFS‑Bi A‑
CRBN. d) Quan i a i e da a and s a is ical analysis o ou independen
expe imen s o e exp essing Bi A‑CRBN as in Figu e S8c. CSDE1 le els
we e no malized o Bi A‑CRBN exp ession. e) BioE3 expe imen pe o med
using HEK293FT s able cells ha exp ess TRIPZ‑bioGEFUb ansien ly
ans ec ed wi h Bi A‑CRBN. BTZ and POM ea men s we e pe o med as
indica ed. Bio inyla ed p o eins we e pu i ied using s ep a idin beads.
Molecula weigh ma ke s a e shown o he le o he blo s in kDa, an i‑
bodies used a e indica ed o he igh
Supplemen a y Ma e ial 9. Supplemen a y Table 1. LC‑MS/MS p ocessed
da a o CRBN BioE3 ea ed o no wi h p o easomal inhibi o bo ezomib.
Gene On ology analysis o CRBN a ge s and selec ed GO e ms by Re igo
Supplemen a y Ma e ial 10. Supplemen a y Table 2. LC‑MS/MS p ocessed
da a o CRBN BioE3 wi h p o easomal inhibi o bo ezomib, wi h o
wi hou NEDDyla ion inhibi o MLN4924. Gene On ology analysis o CRBN
a ge s and selec ed GO e ms by Re igo
Supplemen a y Ma e ial 11. Supplemen a y Table 3. LC‑MS/MS p ocessed
da a o CRBN BioE3 wi h p o easomal inhibi o bo ezomib, wi h o
wi hou pomalidomide ea men . Gene On ology analysis o CRBN
a ge s and selec ed GO e ms by Re igo
Supplemen a y Ma e ial 12. Unc opped Wes e n blo s. Images wi h he
same exposu es as used in he main Figu es a e shown o he le . Me ged
images show he posi ioning o he molecula weigh ma ke s and migh
show di e en exposu e imes.
Supplemen a y Ma e ial 13. Quan i ica ion o p o ein le els in Wes e n
blo s and s a is ical analysis
Acknowledgemen s
R.B., J.D.S., U.M. and C.G. acknowledge ne wo king suppo om he Eu opean
Coope a ion o Science & Technology P o eoCu e COST Ac ion (CA20113).
C.G. and J.J.‑J. a e membe s o he Compu a ional Biology D ug Design
Consolida ed Resea ch G oup suppo ed by he Gene ali a de Ca alunya
(2021SGR00671).
Au ho s’ con ibu ions
L.M.‑C., O.B.‑G., R.B. and J.D.S. concei ed and designed he wo k; L.M.‑C., O.B.‑
G., M.P.‑R., V.M., C.G.‑P., A.S., C.P., S.C.‑L., A.U., M.A., I.I., F.E. and J.D.S. acqui ed and
analyzed he da a; L.M.‑C., O.B.‑G., C.G., J.J.‑J., U.M., R.B. and J.D.S. in e p e ed
he da a; L.M.‑C., M.P.‑R. and R.B. d a ed he manusc ip ; and J.D.S., O.B.‑G.
and C.G., J.J.‑J. subs an i ely e ised i . All au ho s ead and app o ed he inal
manusc ip .
Funding
R.B. and J.D.S. we e unded by MCIN/AEI/h ps://doi.
o g/10.13039/501100011033, p ojec s PID2023‑147399NB‑I00 and PID2020‑
114178 GB‑I00 and CEX2021‑001136‑S and CEX2021‑001202‑M Se e o Ochoa
Excellence P og am and addi ional suppo p o ided by he Dipu ación
Fo al de Bizkaia, P og ama T ans e encia Tecnológica 2023, p ojec 6/12/
TT/2023 /00001, and he Depa men o Indus y, Tou ism, and T ade o
he Basque Coun y Go e nmen (Elka ek Resea ch P og ams); J.J.‑J. was
unded by g an CNS2022‑135307 unded by he MCIN/AEI/h ps://doi.
o g/10.13039/501100011033and he Eu opean Union Nex Gene a ion EU/
PRTR; V.M., C.G.‑P., M.P.‑R. and S.C.‑L. we e unded by FPI g an s unded by
Page 17 o 18
Me ino‑Cachoe al. Cell Communica ion and Signaling (2025) 23:101
MCIN/AEI /h ps://doi.o g/10.13039/501100011033and FSE, PRE2018‑086230,
PRE2021‑099359, PRE2022‑104553 and PREP2023‑000294, espec i ely; L.M.‑C.
was unded by FPU g an FPU20/05282 ( unded by Minis e io de Uni e si‑
dades pa a la Fo mación de P o eso ado Uni e si a io). O.B.‑G., J.D.S. and R.B.
acknowledge unding by he g an 765445‑EU (UbiCODE P og am). U.M. was
unded by he Spanish MCIU (PID2020‑117333 GB‑I00 (FEDER/EU)). Access o
HPC acili ies we e g an ed h ough he Red Española de Supe compu ación
(BCV‑2021–3‑0006 and BCV‑2021–2‑0005).
Da a a ailabili y
The da ase s gene a ed and/o analyzed du ing he cu en s udy a e a ail‑
able in he P o eomeXchange Conso ium ia he PRIDE pa ne eposi o y
[57] wi h he da ase iden i ie PXD055877. All he o he da a gene a ed o
analyzed du ing his s udy a e included in his published a icle [and i s sup‑
plemen a y in o ma ion iles].
Decla a ions
E hics app o al and consen o pa icipa e
No applicable.
Consen o publica ion
No applicable.
Compe ing in e es s
C.G. is co‑ ounde and D ug Disco e y Scien i ic Ad iso o Oni ia The apeu‑
ics. The o he au ho s decla e ha hey ha e no compe ing in e es s.
Au ho de ails
1 Cen e o Coope a i e Resea ch in Biosciences (CIC Biogune), Basque
Resea ch and Technology Alliance (BRTA), 48160 De io, Spain. 2 Depa ‑
men o Pha macy and Pha maceu ical Technology and Physical‑Chemis y,
School o Pha macy, Uni e si y o Ba celona, 08028 Ba celona, Spain.
3 Ins i u e o Theo e ical and Compu a ional Chemis y (IQTC), School
o Chemis y and Physics, Uni e si y o Ba celona, 08028 Ba celona, Spain.
4 Ins i u o de Salud Ca los III, C/ Mon o e de Lemos 3‑5, Pabellón 11, Plan a 0,
28029 Mad id, Spain. 5 Ins i u e o Biomedicine (IBUB), Uni e si y o Ba celona,
08028 Ba celona, Spain. 6 Biochemis y and Molecula Biology Depa men ,
Uni e si y o he Basque Coun y, Leioa, Spain. 7 Ike basque ‑Basque Founda‑
ion o Science, Bilbao, Spain. 8 P esen add ess: Biobizkaia Heal h Resea ch
Ins i u e, Ba akaldo 48903, Spain. 9 P esen add ess: Tele hon Ins i u e o Gene ‑
ics and Medicine (TIGEM), Pozzuoli, I aly. 10 Depa men o Clinical Medicine
and Su ge y, Fede ico II Uni e si y, Naples, I aly.
Recei ed: 25 Oc obe 2024 Accep ed: 8 Feb ua y 2025
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Publishe ’s No e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub‑
lished maps and ins i u ional a ilia ions.
