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

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.

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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.

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Acknowledgements

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.).

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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.

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Correspondence to Randall W. King or Daniel Finley.

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

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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

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