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

Journal name:
Nature
Volume:
467,
Pages:
179–184
Date published:
DOI:
doi:10.1038/nature09299
Received
Accepted

Abstract

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.

At a glance

Figures

  1. USP14 is an inhibitor of the proteasome.
    Figure 1: USP14 is an inhibitor of the proteasome.

    a, Ub–AMC hydrolysis assay of USP14 activity in the presence or absence of Ub–VS-treated human proteasome (VS-proteasome; 1nM). RFU, relative fluorescence units. Ptsm, 26S proteasome. b, In vitro degradation assay with polyubiquitinated CCNB (Ubn–CCNB), human proteasome (4nM), and wild-type (WT) USP14 (USP14) or mutant USP14(C114A) (USP14(CA); 60nM). Samples in b, c and eh were analysed by SDS–PAGE/immunoblotting (IB). c, Plasmids expressing tau, Flag–TDP-43, or V5–LacZ were co-transfected into Usp14−/− MEFs with variants of Flag–USP14 as indicated. Samples were collected 2days after transfection. Actin, loading control. d, Diagram of human USP14, showing ubiquitin-like (UBL) and catalytic (CAT) domains. C114, active site cysteine. Splice variant USP14(SF) is produced from an mRNA lacking exon 4 (ref. 12). e, Flag-tagged ATXN3(Q22) or ATXN3(Q80) was co-expressed with USP14 variants in Usp14−/− MEFs and detected with anti-Flag antibodies. f, Arg–GFP or control Met–GFP co-expressed with USP14 variants in Usp14−/− MEFs. g, As c except HEK293 cells were used. h, USP14(SF) associates with but is not activated by proteasomes. Each variant of Flag–USP14 was expressed in HEK293T cells containing tagged hRPN11, and proteasomes were affinity purified. Where indicated, Ub–VS was incubated with lysate before proteasome purification. Extract samples represent 5% of total. Asterisks indicate nonspecific signals. Proteasome subunit hRPN13, loading control. Control samples, empty vector. Equal cell numbers were used for each lane.

  2. IU1 inhibits human USP14 specifically and reversibly.
    Figure 2: IU1 inhibits human USP14 specifically and reversibly.

    a, Chemical structures of IU1 and IU1C. Analytical data shown in Supplementary Fig. 16. b, IC50 determination for IU1 inhibition of Ub–AMC hydrolysis by proteasome-bound USP14 (4.7±0.7μM), IsoT (100±0.4μM) and UCH37 (700 ± 300 μM). c, Ub–AMC (1μM) hydrolysis assays showing specificity of IU1 for USP14 in comparison to eight other DUB enzymes. d, Reversibility of USP14 inhibition. 60nM USP14 and 5nM human proteasome were treated with vehicle (DMSO) or 100μM IU1 for 2h. After rapid spin gel-filtration, proteins were assayed for Ub–AMC hydrolysis. All values are presented as mean±s.d. (n = 3).

  3. IU1 inhibits chain trimming and stimulates substrate degradation in vitro.
    Figure 3: IU1 inhibits chain trimming and stimulates substrate degradation in vitro.

    a, Chain-trimming assays. Samples contained 4nM proteasome, and USP14 was added at 15-fold molar excess over proteasome. IU1 was added at 50μM and proteasome inhibitors (PI) at 5μM (PS-341, epoxomicin). Asterisk indicates CCNB species derived from residual thrombin from USP14 preparation16. All panels, SDS–PAGE/immunoblot analysis. b, In vitro Ubn–CCNB degradation assay (IU1 at 34μM). c, In vitro degradation assay with polyubiquitinated, T7-tagged Sic1PY, human proteasome (5nM) and wild-type USP14 (75nM) in the absence or presence of IU1 (75μM).

  4. IU1 enhances proteasomal degradation in cells.
    Figure 4: IU1 enhances proteasomal degradation in cells.

    All panels show SDS–PAGE/immunoblot data. a, Thirty-six hours after co-transfecting wild-type MEFs with plasmids expressing tau and V5–LacZ, cells were incubated with 0, 25, 50, 75, or 100μM of IU1 for 6 h. LacZ, transfection control. Actin, loading control. b, As in a except that MEFs were Usp14−/− and IU1 was at 0, 10, 50, or 100 μM. c, Tau and Ub-independent proteasome substrate cODC–EGFP were co-expressed in wild-type MEFs and incubated with 50 μM IU1 for 6h. Proteasome inhibitors were MG132 (30μM) and PS-341 (10 μM). d, As b except with ATXN3(Q80) and ATXN3(Q22), and IU1 at 0, 50 and 100μM. e, Flag–TDP-43 was co-transfected with a LacZ-expressing plasmid into either wild-type or Usp14−/− MEFs, then treated with IU1 (75μM) for the indicated time. Asterisk, nonspecific signal. f, HA-tagged Ub and/or Flag-tagged TDP-43 were transiently overexpressed in wild-type MEFs with 50μM IU1 incubation for 6h. Proteasome inhibitors (20μM MG132, 10μM PS-341) were added 4h before lysis. Lysates were subjected to immunoprecipitation with anti-HA or anti-Flag. Arrows indicate likely ubiquitinated TDP-43 species. HC, heavy chain. g, Wild-type MEFs and Usp14−/− MEFs were treated with IU1 (0, 25, 50, 75, or 100 μM) for 6h, followed by analysis for ubiquitin, actin, CP subunit α7 and RP subunit mRpt5.

  5. IU1 alleviates cytotoxicity induced by oxidative stress.
    Figure 5: IU1 alleviates cytotoxicity induced by oxidative stress.

    a, HEK293 cells were pre-incubated with IU1 (75μM) or proteasome inhibitors (20μM MG132, 10 μM PS-341) for 4h, then treated with menadione (300μM) for 60min. Lysates were treated with DNPH and immunoblotted with anti-DNP antibody to assay oxidized proteins. b, Cell survival under oxidative stress measured using the MTT assay. HEK293 cells were pre-treated with 50μM IU1 for 2h. Menadione was added, followed by 4 h incubation. IU1 effects comparable to those of panels a and b were obtained in wild-type but not Usp14−/− MEFs (data not shown). Values are represented as mean±s.d. (n = 3).

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Author information

  1. These authors contributed equally to this work.

    • Byung-Hoon Lee &
    • Min Jae Lee

Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Byung-Hoon Lee,
    • Min Jae Lee,
    • Soyeon Park,
    • Suzanne Elsasser,
    • Carlos Gartner,
    • Nevena Dimova,
    • John Hanna,
    • Steven P. Gygi,
    • Randall W. King &
    • Daniel Finley
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Dong-Chan Oh
  3. Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Seoul 151-742, Republic of Korea

    • Dong-Chan Oh
  4. Department of Neurobiology, Evelyn F. McKnight Brain Institute, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

    • Ping-Chung Chen &
    • Scott M. Wilson
  5. Present addresses: Department of Biological Sciences, 193 Galvin Life Sciences Center, Notre Dame, Indiana 46556, USA (C.G.); Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115, USA (J.H.).

    • Carlos Gartner &
    • John Hanna

Contributions

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.

Competing financial interests

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

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Supplementary information

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  1. Supplementary Information (3.9M)

    This file contains Supplementary Figures 1-29 with legends and additional references.

  2. Supplementary Table (907K)

    This table contains a summary of nonspecific and weak hits.

Comments

  1. Report this comment #13707

    eunsoo kwon said:

    I can't imagine that sort of screening could been done. This is just incredible and earth-quaking. I really believe they can contribute to improvement of patients who suffer from proteasome involved diseases. And if they keep setting up that large scale of chemical screening, they will be the pioneer of anything.

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