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USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites


USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes1,2,3,4,5,6,7,8,9. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin–cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

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Figure 1: USP14 cleaves supernumerary ubiquitin chains from substrates.
Figure 2: USP14 cleaves arrayed tetraubiquitin chains proximally, stopping at the last chain.
Figure 3: Single-molecule analysis of deubiquitination by USP14.
Figure 4: Kinetic competition between USP14 and the proteasome.

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  1. Hanna, J. et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127, 99–111 (2006)

    Article  CAS  Google Scholar 

  2. Hanna, J., Meides, A., Zhang, D. & Finley, D. A ubiquitin stress response induces altered proteasome composition. Cell 129, 747–759 (2007)

    Article  CAS  Google Scholar 

  3. Lee, B. H. et al. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184 (2010)

    Article  ADS  CAS  Google Scholar 

  4. Peth, A., Besche, H. C. & Goldberg, A. L. Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Mol. Cell 36, 794–804 (2009)

    Article  CAS  Google Scholar 

  5. Aufderheide, A. et al. Structural characterization of the interaction of Ubp6 with the 26S proteasome. Proc. Natl Acad. Sci. USA 112, 8626–8631 (2015)

    Article  ADS  CAS  Google Scholar 

  6. Bashore, C. et al. Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome. Nature Struct. Mol. Biol . 22, 712–719 (2015)

    Article  CAS  Google Scholar 

  7. Leggett, D. S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell 10, 495–507 (2002)

    Article  CAS  Google Scholar 

  8. Homma, T. et al. Ubiquitin-specific protease 14 modulates degradation of cellular prion protein. Sci. Rep. 5, 11028 (2015)

    Article  ADS  Google Scholar 

  9. Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005)

    Article  CAS  Google Scholar 

  10. Finley, D., Chen, X. & Walters, K. J. Gates, channels, and switches: elements of the proteasome machine. Trends Biochem. Sci. 41, 77–93 (2016)

    Article  CAS  Google Scholar 

  11. Kirkpatrick, D. S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nature Cell Biol. 8, 700–710 (2006)

    Article  CAS  Google Scholar 

  12. Dimova, N. V. et al. APC/C-mediated multiple monoubiquitylation provides an alternative degradation signal for cyclin B1. Nature Cell Biol. 14, 168–176 (2012)

    Article  CAS  Google Scholar 

  13. Jin, L., Williamson, A., Banerjee, S., Philipp, I. & Rape, M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133, 653–665 (2008)

    Article  CAS  Google Scholar 

  14. Thrower, J. S., Hoffman, L., Rechsteiner, M. & Pickart, C. M. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000)

    Article  CAS  Google Scholar 

  15. Lu, Y., Lee, B. H., King, R. W., Finley, D. & Kirschner, M. W. Substrate degradation by the proteasome: a single-molecule kinetic analysis. Science 348, 1250834 (2015)

    Article  CAS  Google Scholar 

  16. Mansour, W. et al. Disassembly of Lys11 and mixed linkage polyubiquitin conjugates provides insights into function of proteasomal deubiquitinases Rpn11 and Ubp6. J. Biol. Chem. 290, 4688–4704 (2015)

    Article  CAS  Google Scholar 

  17. Vaden, J. H. et al. Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction. Mol. Neurodegener. 10, 3 (2015)

    Article  CAS  Google Scholar 

  18. Xu, D. et al. Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system. eLife 4, e10510 (2015)

    Article  Google Scholar 

  19. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002)

    Article  ADS  CAS  Google Scholar 

  20. Yao, T. & Cohen, R. E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002)

    Article  ADS  CAS  Google Scholar 

  21. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011)

    Article  CAS  Google Scholar 

  22. Kravtsova-Ivantsiv, Y., Cohen, S. & Ciechanover, A. Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-κB precursor. Adv. Exp. Med. Biol. 691, 95–106 (2011)

    Article  CAS  Google Scholar 

  23. Haglund, K. et al. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nature Cell Biol. 5, 461–466 (2003)

    Article  CAS  Google Scholar 

  24. Pathare, G. R. et al. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11. Proc. Natl Acad. Sci. USA 111, 2984–2989 (2014)

    Article  ADS  CAS  Google Scholar 

  25. Worden, E. J., Padovani, C. & Martin, A. Structure of the Rpn11-Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation. Nature Struct. Mol. Biol . 21, 220–227 (2014)

    Article  CAS  Google Scholar 

  26. Cox, C. J. et al. The regions of securin and cyclin B proteins recognized by the ubiquitination machinery are natively unfolded. FEBS Lett. 527, 303–308 (2002)

    Article  CAS  Google Scholar 

  27. Krude, T., Jackman, M., Pines, J. & Laskey, R. A. Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system. Cell 88, 109–119 (1997)

    Article  CAS  Google Scholar 

  28. Huttlin, E. L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010)

    Article  CAS  Google Scholar 

  29. Paulo, J. A., Gaun, A. & Gygi, S. P. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells. J. Proteome Res. 14, 4246–4256 (2015)

    Article  CAS  Google Scholar 

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We are indebted to J. Paulo for expert assistance with mass spectrometry. We thank J. Hanna and Y. Ye for comments on the manuscript, N. Dimova for NCB1 constructs, Y. Ye for USP21 protein, Y. Saeki for PY-Sic1 clones, J. Yuan for USP14 plasmids, and R.P. Fisher and E. Winter for full-length cyclin B1 baculoviruses. This study was supported by NIH grant R01GM5660052 (to D.F.), NIH grant R37-GM043601 (to D.F.), NIH grant R01GM66492-9 (to R.W.K.), NIH grant 5R01GM039023-26 (to M. W. Kirschner), and a grant from the Rainwater Foundation to D.F.

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Authors and Affiliations



B.-H.L. performed and analysed most of biochemical experiments. Y.L. performed single molecule analysis. M.A.P. performed mass spectrometric analysis. Y.S., S.S., G.T., and S.E. generated biochemical reagents. G.T. also performed structural modeling. S.P.G. guided the mass spectrometry experiments. B.-H.L., R.W.K., and D.F. supervised the study.

Corresponding authors

Correspondence to Byung-Hoon Lee, Randall W. King or Daniel Finley.

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Competing interests

USP14 assays and inhibitors of the IU1 and IU2 series are under patent (held by B.-H.L, R.W.K. and D.F.).

Extended data figures and tables

Extended Data Figure 1 USP14 is highly specific for multi-chain ubiquitin-protein conjugates.

In vitro degradation and deconjugation assays with polyubiquitinated conjugates. a, b, In vitro deubiquitination assays with Ubn–NCB1 or Ubn-1KNCB1 generated by Ubc4 in parallel. Each conjugate sample (~110 nM) was incubated with 4 nM of human proteasome in the presence or absence of USP14 (80 nM). c, This experiment tests whether the failure of USP14 to deubiquitinate a single-chain conjugate formed using K64-only NCB1 (Fig. 1d and f) is predictive of the behaviour of USP14 on other single-chain conjugates. Conjugates (~110 nM) were incubated with human proteasome (4 nM) in the presence or absence of USP14 (80 nM). d, e, In vitro degradation and deubiquitination assays with polyubiquitinated Sic1PY or polyubiquitinated K36-only Sic1PY (1KSic1PY). d, Ubn-Sic1PY (~200 nM) was incubated with human proteasome (5 nM) in the presence or absence of USP14 (100 nM). e, Assays were performed similarly as in d, except using a single-lysine variant of Sic1 (1KSic1PY) modified with wild-type Ub. IU1 (75 μM)3 was used to inhibit USP14 deubiquitinating activity. f, In vitro degradation and deconjugation assays with polyubiquitinated securin. Human proteasome (4 nM) was incubated with Ubn-securin (~100 nM) generated by UbcH10 and APC/C. Where indicated, USP14 (80 nM) was added. Samples were resolved by SDS–PAGE and immunoblotted using an antibody to the HA epitope (ac), the T7 epitope (d, e), or securin (f).

Extended Data Figure 2 Preparation of ATP-depleted proteasome for uncoupling degradation from deubiquitination.

a, For ATP-depletion, purified human proteasomes were treated with hexokinase or mock-treated with vehicle for hexokinase (that is, H2O). Native gel analysis of untreated proteasome (ATP-Ptsm) or hexokinase-treated ADP-proteasome (ADP-Ptsm) with suc-LLVY-AMC and Coomassie blue (CBB) staining. Each lane was loaded with 5 μg of protein. Note that there was essentially no change in proteasome integrity upon hexokinase treatment. b, In vitro Ubn–NCB1 degradation assays with ATP–Ptsm or ADP–Ptsm (4 nM each). Assays were performed and analysed as in Fig. 1b or e.

Extended Data Figure 3 The substrate specificity of USP14 is evolutionarily conserved.

In vitro degradation and deconjugation assays by yeast proteasome and Ubp6 with NCB1 conjugate. a, Purified ubp6Δ yeast proteasome (50 nM) was incubated with Ubn–NCB1 or Ubn-1KNCB1 (~110 nM) in the presence or absence of Ubp6 (200 nM). b, Assays as in a, except yeast proteasome was pre-incubated with ADP and other inhibitors of the proteasome (ATPγS, o-phenanthroline, PS-341, and MG-262) to suppress the degradation. c, Assays with human proteasome, USP14, and polyubiquitinated conjugates (NCB1 or 1KNCB1) were carried out as in Figs 1b and d, and compared side-by-side in one gel. These data serve as a control for Extended Data Fig. 3a, allowing Ubp6 and USP14 to be compared directly.

Extended Data Figure 4 USP14 deubiquitinates singleton chains poorly, regardless of length or linkage type.

In vitro deubiquitination assays are shown. a, b, ADP–Ptsm (4 nM) was incubated with K63-linked octa-Ub conjugates of K64-only NCB1 (~110 nM) in the presence or absence of USP14 (80 nM). c, Single chain conjugates were generated with Ube2S/UbcH10/APC and K11-only Ub to achieve a homogeneously K11-linked singleton species. Assays were done as in Fig. 1f. d, e, UbcH10/APC-mediated conjugation reactions were performed on the K64-only substrate 1KNCB1 using K48-only Ub (d) or K63-only Ub (e). The conjugation reaction formed rather short chains containing no more than three ubiquitin groups. In vitro degradation and deubiquitination assays were performed as described above. All panels show samples resolved by SDS–PAGE and immunoblotted with HA antibody (a, ce) or Ub antibody (b).

Extended Data Figure 5 Free chains in trans do not stimulate USP14 activity for singleton conjugates.

In vitro deubiquitination assays with single chain conjugates in the presence of free chains. ADP–Ptsm (4 nM) was incubated with Ubn–1KNCB1 (~110 nM) in the presence or absence of USP14 (80 nM), and with K48Ub4 (a), K48Ub2 (b), or free ubiquitin (c) at the indicated concentrations. Samples were analysed by SDS–PAGE and immunoblotted with an antibody to the HA epitope.

Extended Data Figure 6 A phosphomimetic mutant of USP14 that mimics AKT-phosphorylated enzyme does not promote cleavage of singleton ubiquitin chains.

a, Free USP14 wild-type (400 nM) does not cleave multi-chain cyclin B1 conjugates (~110 nM). b, High concentration of free USP14 does not deubiquitinate Ubn–NCB1. USP14 (15 μM) activity on Ubn–NCB1 (~0.45 μM) in the absence of proteasome was tested by incubating at room temperature for time periods as indicated. Note that there is no obvious deubiquitination of the conjugate by free USP14, although high levels of USP14 were tested (>1,000-fold over that of activated, proteasome-associated USP14 in our standard assay). This experiment confirms that deubiquitination of Ubn–NCB1 by USP14 occurs on and requires the proteasome. c, Free USP14(S432E) (400 nM) does not cleave single chain cyclin B1 conjugates (~110 nM). d, Proteasome-associated USP14(S432E) (4 nM proteasome and 80 nM USP14) does not cleave single chain cyclin B1 conjugates (~110 nM). Assay in d was performed in the presence of ATP under conditions permissive for degradation. Samples were resolved by SDS–PAGE and immunoblotted using an antibody to the HA epitope.

Extended Data Figure 7 USP14 deubiquitinates free chains poorly, regardless of linkage type.

In vitro deubiquitination assays with free chains. ad, Each linkage-specific free chain (200 nM) was incubated with human proteasome (12 nM) in the presence or absence of USP14 (240 nM). The sample was treated with pan-specific Ub isopeptidase USP21 (400 nM) for 20 min as a positive control showing that chain disassembly is readily detected. e, f, K48-linked Ub3–7 free chains (125 nM) or K63-linked octa-Ub free chains (300 nM) were incubated with human proteasomes (5 nM for e and 10 nM for f) in the presence or absence of USP14 (20-fold excess over proteasome). Samples were resolved by SDS–PAGE and immunoblotted using an antibody to ubiquitin.

Extended Data Figure 8 USP14 cleaves ubiquitin chains at the proximal site.

In vitro deubiquitination assays with multi-chain conjugates. a, Multi-chain K63Ub4 conjugates on NCB1 were prepared using pre-formed K63Ub4 free chains as described in Methods. Assays were performed as in Fig. 2a. Arrow indicates accumulated K63Ub4–NCB1 as major deubiquitinated species generated by USP14 cleavage. Asterisks indicate presumptive deconjugated K63Ub5- or other multi-chain K63Ubn–NCB1 species originating from other K63Ubn contaminants in the commercial preparation of K63Ub4. b, Ubn–NCB1 was generated using UbcH10, APC/C, and wild type ubiquitin, and purified as described in Methods. Note that this conjugate may be enriched with short multi-ubiquitin chains with different linkages, based on previous reports12. Deubiquitination assays were performed as in Fig. 2c. Arrowhead indicates di-Ub chains as major products of USP14 activity, suggesting that Ubn–NCB1 in this sample was rich in diubiquitin chains. Samples were resolved by SDS–PAGE and immunoblotted with antibody against the HA epitope (a) or Ub (b). c, TMT labelling and quantitative mass spectrometry. Purified Ubn–NCB1 was assayed in biological duplicates for each condition, and the resulting samples were analysed for specific ubiquitin chain linkages by mass spectrometry as in Fig. 2d. The experiment was repeated in biological duplicates at a 4-min point with equivalent results (data not shown).

Extended Data Figure 9 Modelling of free ubiquitin chain cleavage by proteasome-activated Ubp6 or USP14.

Possible steric conflict between the proximal ubiquitin and proteasome-associated Ubp6 or USP14 was assessed by molecular modelling. a, As in Fig. 2f, except employing K48- or K63-linked proximal ubiquitin. b, Proximal ubiquitin with each different linkage was fitted in USP14 structure with minimal steric hindrance, and then docked onto the proteasome electron microscopy structure. Structures were obtained from the PDB database: Ubp6 and proteasome (5A5B), USP14 (2AYO), K63 di-Ub (2RR9), and ubiquitin (1UBQ). Note that for K6, 27, 29, and 33 linkages, modelling on the proteasome was not attempted because of extensive steric occlusion between proximal ubiquitin and USP14.

Extended Data Figure 10 Single-molecule studies of RPN11 show patterns of chain removal similar to those of USP14.

These experiments serve as controls for Fig. 3. Since RPN11 is known to work as a proximal-ubiquitin-specific DUB, its similarity to USP14 in these particular chain removal assays supports the hypothesis that USP14 is a proximal-specific deubiquitinating enzyme. Single-molecule assays were performed and analysed as described in Fig. 3, and were carried out using the same samples of proteasomes and ubiquitinated securin. The data shown are examples of step-trace segmentation graphs from a single-molecule analysis of purified Dylight550-labelled Ubn–securin conjugates incubated with human proteasome lacking USP14 in the presence of ATP. These deubiquitination events represent RPN11-mediated activity as previously reported15.

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Lee, BH., Lu, Y., Prado, M. et al. USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites. Nature 532, 398–401 (2016).

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