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Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

Chadchankar, J; Korboukh, V; Conway, LC; Wobst, HJ; Walker, CA; Doig, P; Jacobsen, SJ; ... Wang, Q; + view all (2019) Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells. PLOS ONE , 14 (11) , Article e0225145. 10.1371/journal.pone.0225145. Green open access

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Abstract

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated β-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect β-catenin, demonstrating USP14 but not UCHL5 has a specific effect on β-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides β-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Type: Article
Title: Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1371/journal.pone.0225145
Publisher version: https://doi.org/10.1371/journal.pone.0225145
Language: English
Additional information: Copyright © 2019 Chadchankar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: Proteasomes, Immunoprecipitation, Immunoblotting, Ubiquitination, Immune system proteins, Amyotrophic lateral sclerosis, Coomassie Blue staining, Proteases
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology
URI: https://discovery.ucl.ac.uk/id/eprint/10086267
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