Ubiquitin degradation with its substrate, or as a
monomer in a ubiquitination-independent mode,
provides clues to proteasome regulation
Nitzan Shabek1, Yifat Herman-Bachinsky1, and Aaron Ciechanover2
Cancer and Vascular Biology Research Center, Rappaport Faculty of Medicine and Research Institute, Technion-Israel
Institute of Technology,Haifa 31096, Israel
Contributed by Aaron Ciechanover, May 27, 2009 (sent for review April 21, 2009)
www.pnas.org PNAS July 21, 2009 vol. 106 no. 29 11907–11912
Vias proteolíticas
Lisossomal
Dependente de cálcio
Ubiquitina-proteassoma
Residual

Mas, quem degrada os componentes
das vias???
Vias proteolíticas
Mas, Quem degrada os componentes
das vias???
Lisossomal ?
Dependente de cálcio ?

Ubiquitina-proteassoma

Residual ?

Este trabalho....
Lisossomal
Ubiquitina
Regulação dos níveis em diferentes condições é desconhecida
Estudos anteriores: Degradação não lisossomal e dependente de
ATP
Meia-vida em torno de 10 hs.
Mal funcionamento das DUBS aumentam a degradação de Ub
juntamente com seu substrato-alvo.

Ubiquitina
Como ocorre a degradação de Ub pelo proteassoma (Ub livre ou
conjugada com seus substratos-alvo?)
 Investigar mecanismos de ligação entre substratos e componentes do
proteassoma

Nitzan Shabek, Kazuhiro Iwai, Aaron Ciechanover Ubiquitin is degraded by the ubiquitin system as a monomer
and as part of its conjugated target Biochemical and Biophysical Research Communications 363 (2007) 425–431
Impede que seja usada como Ub
marcadora e que seja clivada no c-terminal por
Proteases específicas para Ub
Ubiquitina e proteassoma
ATP- and proteasome-dependent degradation of WT (UbGG) and inert (UbVV) Ubs. (i and ii)
125I- UbGG was subjected to in vitro degradation in reticulocyte fraction II (FrII) in the
presence or absence of ATP (i) and in the presence or absence of the proteasome inhibitors
MG132 (MG) or lactacystin (LC)(ii) (iii) Degradation of 125I-UbGG was compared with that of
125I-UbVV. Bars
in ii and iii represent net ATP-dependent values.
Dep. ATP
Não
conjuga
Dep. proteassoma
Indução de formação de
reticulócitos com
fenilhidrazina
(B) Ubiquitination and degradation of 125I- UbGG in the presence and absence of added MyoD. (i)
125I- UbGG was subjected to in vitro degradation t with or without MyoD (5 g). (ii) Purified MyoD was
subjected to degradation in vitro . (iii) 125I-UbGG and 125I-UbVV were subjected to in vitro
ubiquitination in the presence of HeLa cell extract or reticulocyte FrII, ATP, and purified MyoD (5 g) as
indicated.
Estimula
Ub é
degradada
como parte
de seu
substrato
conjugado
(C) ATP-dependent degradation (i) and ubiquitination (ii) of 125I-UbGG in the presence of FrII.
Purified MyoD and unlabeled iodine-modified BSA and RNase A were added as indicated (5 g each).
Degradation of labeled Ub was monitored by using release of TCA-soluble radioactivity, and
degradation of MyoD was monitored after SDS/PAGE and Western blot analysis . Conjugation of
labeled Ub was carried out as described in Materials and Methods. All degradation values of Ub
represent the average of 5 independent experiments (SD is shown). Ub conj. denotes Ub conjugates.
Ub é
degradada
como parte
de seu
substrato
conjugado
Ub é degradada por UPS em um
mecanismo (“piggyback”) que
envove seu substrato conjugado
(ambos são degradados
conjuntamente)
Fig. 2. Ubiquitination-independent degradation of extended Ubs by the
26S proteasome
(A) cDNAs coding for the indicated UbVV variants was expressed
in HEK-293 cells, and the stability of the proteins was monitored after the
addition of CHX. Proteins were visualized by using anti-Ub (i) or anti-RGSHis
(ii–vii).
> 20 resíduos
N-terminal
bloqueado
Não podem ser
ubiquitinados
Sem lisinas
(“lisineless”)
(B) 35S-labeled UbVV variants were translated in S30 bacterial extract
(except for 35S-UbVVHisHA shown in viii that was translated in wheat germ
extract) and subjected to in vitro ATP-dependent degradation in FrII without
Ub. MG132 was added as indicated. The asterisk in viii marks monoubiquitinated
UbVVHisHA that was generated during translation.
Sem
Ub
(C) His-tagged bacterially expressed and purified UbVVHisHA (i) and UbVVHis
(ii) were subjected to in vitro ATP-dependent degradation in the presence of FrII
(lanes 1–3) or purified 26S proteasome (lanes 4–6). MG132 and Ub were added
as indicated.Reaction mixtures were incubated for 2 h. Proteins were detected
by using anti-RGSHis (Upper) or anti-Ub (Lower; membranes were reblotted).
Ubs com
extensões
suficientemen
te longas
podem ser
degradadas
sem
ubiquitinação
adicional
(D) HEK-293 cells were transfected with cDNAs (in pCS2) coding for
UBB1/K29,48R, UBB1YK29,48R, andUBB1HAK29,48R, and their stability
was monitored after the addition of CHX.
UBB: gene com extensão de 19 resíduos, implicado na patogênese do mal de Alzheimer
e outras doenças neurodegenerativas
Não ubiquitináveis
Lys29 e Lys48 → Arg
+Tyr
+HAr
> 20 resíduos
(E) HEK-293 cells were transfected with cDNAs coding for UBB1HA,
UBB1HAK29,48R, and empty vector (EV). HA-tagged proteins were
immunoprecipitated as described in Materials and Methods, and the association
with the proteasome was detected with the indicated antibodies. In all
experiments, proteins were resolved via SDS/PAGE.
O proteassoma 26S pode ligar e
degradar Ub monomérica
independentemente de
ubiquitinação
Fig. 3. Inhibition of degradation of UPS substrates depends on ubiquitination of
short, C-terminally extended nondegradable Ub variants.
(A) (i) HEK-293 cells were cotransfected with cDNAs coding for the indicated UbVV variants and UPS
substrates (MyoD, Mdm2, or c-Myc), and the stability of the substrates was monitored after the
addition of CHX for the indicated times. (ii) Quantitative representation of MyoD degradation as shown
in i. (iii) Effect of the different UbVV variants on the degradation of endogenous p21. The experiment
was carried out in a similar manner as described in i.
(B) HEK-293 cells were transfected with cDNAs coding for UbVVHis or
UbVVHisHA, or their LL species. The stability of endogenous p21 (i) or
cotransfected c-MycHA (ii) was monitored as described in A.
Sem efeito
inibitório
(C) (i) cDNAs (in pcDNA3) coding for UBB1 or UBB1/K29,48R were cotransfected along with a cDNA
coding for c-MycHA, and the stabilities of endogenous p21 and the expressed Myc were monitored as
described in A. (ii) HEK-293 cells were transfected with cDNAs coding for UBB1 and UBB1/K29,48R.
Thirty hours after transfection, the total cell proteins were resolved via SDS/PAGE, and Ub conjugates
were visualized after Western blot analysis using an anti-Ub conjugates antibody. Gels described in Ai
and Cii were 10%. MG132 was added as indicated
Ubs de cadeias curtas inibem a
degradação de substratos de UPS pela sua
habilidade de serem ubiquitinadas
Fig. 4. Proteasome inhibition by extended Ub variants affects only
ubiquitination-dependent substrates.
(A)UbVVHis and LLUbVVHis interact with the 26S proteasome. Bacterially expressed and
purified UbVVHis (i) and LLUbVVHis (ii) were incubated in the presence of HeLa cell extract.
26S proteasomes were precipitated by using anti-6 (20S subunit) as described in Materials
and Methods, and the Ub derivatives and proteasome subunits were detected after
SDS/PAGE and immunoblotting. The input represents 10% of the lysate used for
immunoprecipitation (IP)
Precipitam com
o proteassoma
(B) HEK-293 cells were cotransfected with cDNAs coding for UbVVHis and
Flag-ODC as indicated. The level of ODC was monitored after the addition
of CHX and MG132 at the indicated times.
ODC= Ornitina descarboxilase, degradada pelo
proteassoma sem ubiquitinação
Não inibiu a
degradação
C)HEK-293 cells were cotransfected with cDNAs coding for Flag-AZ and UbVVHis
or LLUbVVHis as indicated, and the stability of AZ was monitored after the addition
(
ofCHXfor the indicated times.
AZ = antizima, regulador da ODC que a marca para
degradação . AZ é degradada pelo proteassoma por
ubiquitinação
(D) Stabilities of transfectedFlag-ODCandFlag-AZ were monitored in cells
expressing UBB1 or UBB1/K29,48R (from pcDNA3) as described in B and
C.
O efeito inibitório de derivados com extensões
curtas de Ub afeta substratos dependentes de
ubiquitinação
Conclusões

Ub é degradada por UPS em um mecanismo (“piggyback”)
que envove seu substrato conjugado (ambos são
degradados conjuntamente)

O proteassoma 26S pode ligar e degradar Ub monomérica
independentemente de ubiquitinação
Ubs de cadeias curtas inibem a degradação de
substratos de UPS pela sua habilidade de serem
ubiquitinadas


O efeito inibitório de derivados com extensões curtas de Ub
afeta substratos dependentes de ubiquitinação
Fig. S1. UbVV variants have no effect on DUB
activity and the 20S catalytic activity. (A) 35Slabeled UPS substrates were subjected to ATPdependent ubiquitination in a cell-free system.
Reaction mixtures were incubated for 1 h at 37
°C (in a volume of 10 L) in the presence of E1
and E2 (UbcH5c for Ring1B and XIAP-1, and
Ubc7 for MyoD), and then for an additional hour
in the presence of HeLa cell extract (50 g in a
total volume of 15 L; as source for DUBs for
all substrates and MyoD’s E3). Ubal or 5 g of
purified UbVV variants was added as indicated.
Proteins were resolved by SDS/PAGE (10%)
and visualized by using PhosphoImager
(B) 26S proteasomes were isolated via immunoprecipitation from HeLa cell
extract, and the DUB-associated activity was monitored by measuring
the cleavage of Ub-AMC after preincubation with UbVVHis, LLUbVVHis, or
Ubal as described in Materials and Methods, Fig. 4A, and SI Text.
C) 20S proteasomal activity was monitored by using purified 26S
proteasome and measuring the release ofAMCfromLLVY-AMCin the
presence of ATP and increasing concentrations of MG132, purified
UbVVHis, or WT Ub as indicated. Activity was measured as described in
SI Text.
(