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Extended ubiquitin species are protein-based DUB inhibitors

Abstract

A frameshift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin (Ub), UBB+1. UBB+1 has been considered to inhibit proteasomes and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrate that expression of extended Ub variants leads to accumulation of heterogeneously linked polyubiquitin conjugates, indicating a pervasive effect on Ub-dependent turnover. 20S proteasomes selectively proteolyzed Ub extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of Lys48 linkages at 26S proteasomes. Processing of Lys48 and Lys63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of Ub-dependent degradation by extended Ub variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs.

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Figure 1: Outcomes of Ubext species expression in Saccharomyces cerevisiae.
Figure 2: Ubext molecules are processed by isolated 20S CPs.
Figure 3: In vitro, Ubext molecules associate with proteasomes without inhibiting them.
Figure 4: Ubext molecules are targets of DUBs.
Figure 5: Extended Ub mutants are protein-based DUB inhibitors.
Figure 6: A model of Ubext-proteasome engagement: implications for efficient proteolysis by proteasomes.

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Acknowledgements

This research was funded by US National Institutes of Health grant GM095755 to D.F. and M.H.G. and a USA-Israel Binational Science Foundation grant (2009487) to D.F. and M.H.G.

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Contributions

D.K., M.H.G., N.R., M.A.N. and D.F. designed experiments. D.K. carried out in vivo and in vitro proteasomal experiments. S.P.G. and D.S.K. aided in aqua analysis. M.A.N. and D.Z. synthesized all of the Ub conjugates. A.B. and P.S. collected and analyzed processed Ub fragments. D.K., M.A.N. and M.G.H. prepared the manuscript.

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Correspondence to Michael H Glickman.

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The authors declare no competing financial interests.

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Krutauz, D., Reis, N., Nakasone, M. et al. Extended ubiquitin species are protein-based DUB inhibitors. Nat Chem Biol 10, 664–670 (2014). https://doi.org/10.1038/nchembio.1574

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