Ubiquitin degradation with its substrate, or as amonomer 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

LisossomalDependente de cálcio

Ubiquitina-proteassomaResidual

Mas, quem degrada os componentes das vias???

Vias proteolíticas

Lisossomal ?Dependente de cálcio ?

Ubiquitina-proteassomaResidual ?

Mas, Quem degrada os componentes das vias???

LisossomalEste trabalho....

Ubiquitina

Regulação dos níveis em diferentes condições é desconhecidaEstudos anteriores: Degradação não lisossomal e dependente de ATPMeia-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

Impede que seja usada como Ub marcadora e que seja clivada no c-terminal por Proteases específicas para Ub

Nitzan Shabek, Kazuhiro Iwai, Aaron Ciechanover Ubiquitin is degraded by the ubiquitin system as a monomerand as part of its conjugated target Biochemical and Biophysical Research Communications 363 (2007) 425–431

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.

Indução de formação de reticulócitos com fenilhidrazina

Dep. ATP

Dep. proteassoma

Não conjuga

(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

Sem lisinas (“lisineless”)

Não podem ser ubiquitinados

(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 suficientemente 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

Lys29 e Lys48 → Arg +Tyr +HAr

> 20 resíduos

Não ubiquitináveis

(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

ConclusõesUb 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) 35S-labeled UPS substrates

were subjected to ATP-dependent

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.