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Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode, provides clues to proteasome regulation Nitzan Shabek 1 , Yifat Herman-Bachinsky 1 , and Aaron Ciechanover 2

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Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode,

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Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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


Lisossomal dependente de c lcio ubiquitina proteassoma residual

Lisossomal

Dependente de cálcio

Ubiquitina-proteassoma

Residual

Vias proteolíticas

Mas, quem degrada os componentes

das vias???


Lisossomal dependente de c lcio ubiquitina proteassoma residual1

Lisossomal ?

Dependente de cálcio ?

Ubiquitina-proteassoma

Residual ?

Vias proteolíticas

Mas, Quem degrada os componentes

das vias???

Lisossomal

Este trabalho....


Ubiquitina

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.


Ubiquitina1

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

Ubiquitina e proteassoma


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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.

Não conjuga

Dep. ATP

Indução de formação de reticulócitos com fenilhidrazina

Dep. proteassoma


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

Ub é degradada por UPS em um mecanismo (“piggyback”) que envove seu substrato conjugado (ambos são degradados conjuntamente)


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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”)


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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

+HAr

+Tyr

> 20 resíduos


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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.


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

O proteassoma 26S pode ligar e degradar Ub monomérica independentemente de ubiquitinação


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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.


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

Ubs de cadeias curtas inibem a degradação de substratos de UPS pela sua habilidade de serem ubiquitinadas


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(D) Stabilities of transfectedFlag-ODCandFlag-AZ were monitored in cells expressing UBB1 or UBB1/K29,48R (from pcDNA3) as described in B and C.


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

O efeito inibitório de derivados com extensões curtas de Ub afeta substratos dependentes de ubiquitinação


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

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


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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.


Ubiquitin degradation with its substrate or as a monomer in a ubiquitination independent mode

(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.


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