Enhancement of proteasome activity by a small-molecule inhibitor of USP14


Proteasomes, the primary mediators of ubiquitin–protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that USP14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin–protein conjugates both in vitro and in cells. A catalytically inactive variant of USP14 has reduced inhibitory activity, indicating that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human USP14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. USP14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of USP14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: USP14 is an inhibitor of the proteasome.
Figure 2: IU1 inhibits human USP14 specifically and reversibly.
Figure 3: IU1 inhibits chain trimming and stimulates substrate degradation in vitro.
Figure 4: IU1 enhances proteasomal degradation in cells.
Figure 5: IU1 alleviates cytotoxicity induced by oxidative stress.


  1. 1

    Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009)

    CAS  Article  Google Scholar 

  2. 2

    Schrader, E. K., Harstad, K. G. & Matouschek, A. Targeting proteins for degradation. Nature Chem. Biol. 5, 815–822 (2009)

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    ADS  CAS  Article  Google Scholar 

  5. 5

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

    ADS  CAS  Article  Google Scholar 

  6. 6

    Lam, Y. A., Xu, W., DeMartino, G. N. & Cohen, R. E. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385, 737–740 (1997)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Koulich, E., Li, X. & DeMartino, G. N. Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol. Biol. Cell 19, 1072–1082 (2008)

    CAS  Article  Google Scholar 

  8. 8

    Jacobson, A. D. et al. The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26S proteasome. J. Biol. Chem. 284, 35485–35494 (2009)

    CAS  Article  Google Scholar 

  9. 9

    Verma, R. et al. Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439 (2000)

    CAS  Article  Google Scholar 

  10. 10

    Borodovsky, A. et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196 (2001)

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

    Wilson, S. M. et al. Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease. Nature Genet. 32, 420–425 (2002)

    CAS  Article  Google Scholar 

  13. 13

    Chernova, T. A. et al. Pleiotropic effects of Ubp6 loss on drug sensitivities and yeast prion are due to depletion of the free ubiquitin pool. J. Biol. Chem. 278, 52102–52115 (2003)

    CAS  Article  Google Scholar 

  14. 14

    Anderson, C. et al. Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice. J. Neurochem. 95, 724–731 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme Usp14. EMBO J. 24, 3747–3756 (2005)

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

    Crimmins, S. et al. Transgenic rescue of ataxia mice with neuronal-specific expression of ubiquitin-specific protease 14. J. Neurosci. 26, 11423–11431 (2006)

    CAS  Article  Google Scholar 

  19. 19

    Crimmins, S. et al. Transgenic rescue of ataxia mice reveals a male-specific sterility defect. Dev. Biol. 325, 33–42 (2009)

    CAS  Article  Google Scholar 

  20. 20

    Chen, P.-C. et al. The proteasome-associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions. J. Neurosci. 29, 10909–10919 (2009)

    CAS  Article  Google Scholar 

  21. 21

    Peth, A., Besche, H. C. & Goldberg, A. L. Ubiquitinated proteins activate the proteasome by binding to Usp14/Upb6, which cause 20S gate opening. Mol. Cell 36, 794–804 (2009)

    CAS  Article  Google Scholar 

  22. 22

    Catic, A. et al. Screen for ISG15-crossreactive deubiquitinases. PLoS ONE 2, e679 (2007)

    ADS  Article  Google Scholar 

  23. 23

    Wang, X. et al. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry 46, 3553–3565 (2007)

    CAS  Article  Google Scholar 

  24. 24

    Yao, T. et al. Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nature Cell Biol. 8, 994–1002 (2006)

    CAS  Article  Google Scholar 

  25. 25

    Spires-Jones, T. L., Stoothoff, W. H., de Calignon, A., Jones, P. B. & Hyman, B. T. Tau pathophysiology in neurodegeneration: a tangled issue. Trends Neurosci. 32, 150–159 (2009)

    CAS  Article  Google Scholar 

  26. 26

    Kwong, L. K., Uryu, K., Trojanowski, J. Q. & Lee, V. M. TDP-43 proteinopathies: neurodegenerative protein misfolding diseases without amyloidosis. Neurosignals 16, 41–51 (2008)

    CAS  Article  Google Scholar 

  27. 27

    David, D. C. et al. Proteasomal degradation of tau protein. J. Neurochem. 83, 176–185 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Petrucelli, L. et al. CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum. Mol. Genet. 13, 703–714 (2004)

    CAS  Article  Google Scholar 

  29. 29

    Todi, S. V. et al. Cellular turnover of the polyglutamine disease protein ataxin-3 is regulated by its catalytic activity. J. Biol. Chem. 282, 29348–29358 (2007)

    CAS  Article  Google Scholar 

  30. 30

    Varshavsky, A., Turner, G., Du, F. & Xie, Y. The ubiquitin system and the N-end rule pathway. Biol. Chem. 381, 779–789 (2000)

    CAS  Article  Google Scholar 

  31. 31

    Dantuma, N. P., Lindsten, K., Glas, R., Jellne, M. & Masucci, M. G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nature Biotechnol. 18, 538–543 (2000)

    CAS  Article  Google Scholar 

  32. 32

    Saeki, Y., Isono, E. & Toh-E, A. Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring proteasome activity. Methods Enzymol. 399, 215–227 (2005)

    CAS  Article  Google Scholar 

  33. 33

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

    CAS  Article  Google Scholar 

  34. 34

    Amerik, A. Y., Li, S. J. & Hochstrasser, M. Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae . Biol. Chem. 381, 981–992 (2000)

    CAS  Article  Google Scholar 

  35. 35

    Hanna, J., Leggett, D. S. & Finley, D. Ubiquitin depletion as a key mediator of toxicity by translational inhibitors. Mol. Cell. Biol. 23, 9251–9261 (2003)

    CAS  Article  Google Scholar 

  36. 36

    Shabek, N., Herman-Bachinsky, Y. & Ciechanover, A. Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode, provides clues to proteasome regulation. Proc. Natl Acad. Sci. USA 106, 11907–11912 (2009)

    ADS  CAS  Article  Google Scholar 

  37. 37

    Hoyt, M. A., Zhang, M. & Coffino, P. Probing the ubiquitin/proteasome system with ornithine decarboxylase, a ubiquitin-independent substrate. Methods Enzymol. 398, 399–413 (2005)

    CAS  Article  Google Scholar 

  38. 38

    Stadtman, E. R. Protein oxidation and aging. Free Radic. Res. 40, 1250–1258 (2006)

    CAS  Article  Google Scholar 

  39. 39

    Ahmed, E. K., Picot, C. R., Bulteau, A. L. & Friguet, B. Protein oxidative modifications and replicative senescence of WI-38 human embryonic fibroblasts. Ann. NY Acad. Sci. 1119, 88–96 (2007)

    ADS  CAS  Article  Google Scholar 

  40. 40

    Hamazaki, J. et al. A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 25, 4524–4536 (2006)

    CAS  Article  Google Scholar 

  41. 41

    Qiu, X. B. et al. hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J. 25, 5742–5753 (2006)

    CAS  Article  Google Scholar 

  42. 42

    Husnjak, K. et al. Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453, 481–488 (2008)

    ADS  CAS  Article  Google Scholar 

  43. 43

    Chauhan, D., Bianchi, G. & Anderson, K. C. Targeting the UPS as therapy in multiple myeloma. BMC Biochem. 9 (Suppl. 1). S1 (2008)

    Article  Google Scholar 

  44. 44

    Muchamuel, T. et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nature Med. 15, 781–787 (2009)

    CAS  Article  Google Scholar 

  45. 45

    Chondrogianni, N. et al. Overexpression of proteasome β5 subunit increases the amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates. J. Biol. Chem. 280, 11840–11850 (2005)

    CAS  Article  Google Scholar 

  46. 46

    Tonoki, A. et al. Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process. Mol. Cell. Biol. 29, 1095–1106 (2009)

    CAS  Article  Google Scholar 

  47. 47

    Lehman, N. L. The ubiquitin proteasome system in neuropathology. Acta Neuropathol. 118, 329–347 (2009)

    CAS  Article  Google Scholar 

  48. 48

    Hinault, M. P., Ben-Zvi, A. & Goloubinoff, P. Chaperones and proteases: cellular fold-controlling factors of proteins in neurodegenerative diseases and aging. J. Mol. Neurosci. 30, 249–265 (2006)

    CAS  Article  Google Scholar 

  49. 49

    Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008)

    ADS  CAS  Article  Google Scholar 

  50. 50

    Goldberg, A. L. Protein degradation and protection against misfolded and damaged proteins. Nature 426, 895–899 (2003)

    ADS  CAS  Article  Google Scholar 

  51. 51

    Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009)

    CAS  Article  Google Scholar 

  52. 52

    Elsasser, S., Schmidt, M. & Finley, D. Characterization of the proteasome using native gel electrophoresis. Methods Enzymol. 398, 353–363 (2005)

    CAS  Article  Google Scholar 

  53. 53

    Kleijnen, M. F. et al. Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nature Struct. Mol. Biol. 14, 1180–1188 (2007)

    CAS  Article  Google Scholar 

  54. 54

    Kusmierczyk, A. R., Kunjappu, M. J., Funakoshi, M. & Hochstrasser, M. A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nature Struct. Mol. Biol. 15, 237–244 (2008)

    CAS  Article  Google Scholar 

  55. 55

    Park, S. et al. Hexameric assembly of the proteasomal ATPases is templated through their C termini. Nature 459, 866–870 (2009)

    ADS  CAS  Article  Google Scholar 

  56. 56

    Malo, N., Hanley, J., Cerquozzi, S., Pelletier, J. & Nadon, R. Statistical practice in high-throughput screening data analysis. Nature Biotechnol. 24, 167–175 (2006)

    CAS  Article  Google Scholar 

  57. 57

    Kobayashi, H. et al. Hrs, a mammalian master molecule in vesicular transport and protein sorting, suppresses the degradation of ESCRT proteins signal transducing adaptor molecule 1 and 2. J. Biol. Chem. 280, 10468–10477 (2005)

    CAS  Article  Google Scholar 

  58. 58

    Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004)

    ADS  CAS  Article  Google Scholar 

  59. 59

    Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010)

    CAS  Article  Google Scholar 

  60. 60

    Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983)

    CAS  Article  Google Scholar 

  61. 61

    Li, X., Traganos, F., Melamed, M. R. & Darzynkiewicz, Z. Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation. Cytometry 20, 172–180 (1995)

    CAS  Article  Google Scholar 

  62. 62

    Jordan, M. A., Thrower, D. & Wilson, L. Mechanism of inhibition of cell proliferation by Vinca Alkaloids. Cancer Res. 51, 2212–2222 (1991)

    CAS  PubMed  Google Scholar 

  63. 63

    Chondrogianni, N. et al. Central role of the proteasome in senescence and survival of human fibroblasts. J. Biol. Chem. 278, 28026–28037 (2003)

    CAS  Article  Google Scholar 

  64. 64

    Kwak, M. K. et al. Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway. Mol. Cell. Biol. 23, 8786–8794 (2003)

    CAS  Article  Google Scholar 

Download references


We thank K. Gordon, J. Y. Suk and N. Bays for advice and assistance, and members of the Finley laboratory for comments on the manuscript. We thank C. Shamu and the staff of the ICCB facility at Harvard Medical School, where the HT screen was carried out. We also thank N. Hathaway for ubiquitinated cyclin B, L. Huang for the tagged proteasome cell line, R. Baker for anti-USP14 antibody, G. DeMartino for anti-UCH37 antibody, K. Wilkinson and K. Walters for DUB enzymes, as well as C. Seong, M. Kim, S. M. Lim and D. Waterman for assistance in some experiments. For plasmids, we thank K. Walters, M. Sowa, W. Harper, V. Lee, F. Baralle, H. Paulson, Y. T. Kwon, M. Masucci, M.-K. Kwak, P. Coffino and C. Kahana. This work was supported by grants from the National Institutes of Health (DK082906 to D.F., GM65592 to D.F., GM66492 to R.W.K. and NS047533 to S.M.W.); the Harvard Technology Development Accelerator Fund (D.F.); Merck & Co. (D.F. and R.W.K.); and Johnson & Johnson (D.F. and R.W.K.).

Author information




B.-H.L. carried out screening and most in vitro studies, and M.J.L. chemical analysis and most cell-based assays. R.W.K. and D.F. were responsible for overall design and oversight of the project. S.P., S.E. and N.D. provided skilled assistance in proteasome biochemistry and assays. D.-C.O., C.G. and S.P.G. designed and carried out chemistry studies. P.-C.C., S.M.W. and J.H. provided key reagents and intellectual input. Many authors contributed to preparation of the manuscript.

Corresponding authors

Correspondence to Randall W. King or Daniel Finley.

Ethics declarations

Competing interests

There is a patent application on this work, filed by Harvard University on behalf of the authors.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-29 with legends and additional references. (PDF 4052 kb)

Supplementary Table

This table contains a summary of nonspecific and weak hits. (PDF 907 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, B., Lee, M., Park, S. et al. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184 (2010). https://doi.org/10.1038/nature09299

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing