A ubiquitinome analysis to study the functional roles of the proteasome associated deubiquitinating enzymes USP14 and UCH37

Lennart van der Wal, Karel Bezstarosti, Jeroen A.A. Demmers*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)
2 Downloads (Pure)

Abstract

The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14 (RPN11), USP14 and UCH37 (UCHL5). However, the functional roles and specificities of these proteasomal DUBs remain elusive. To reveal the specificities of proteasome associated DUBs, we used SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. We observed distinct effects on the global ubiquitinome upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggested less functional redundancy than previously anticipated. We also investigated whether the small molecule inhibitor b-AP15 has the potential to specifically target USP14 and UCH37 by comparing treatment of wild-type versus USP14/UCH37 double-knockout cells with this drug. Strikingly, broad and severe off-target effects were observed, questioning the alleged specificity of this inhibitor. In conclusion, this work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds. Significance: Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive. Materials & Methods: We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37. Results: We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor. Conclusions: This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.

Original languageEnglish
Article number104592
JournalJournal of Proteomics
Volume262
Early online date2 May 2022
DOIs
Publication statusPublished - 30 Jun 2022

Bibliographical note

Funding Information:
This work was funded by the Dutch Organization for Scientific Research (NWO, Proteins At Work consortium, # 184032201 ). All raw mass spectrometry data were uploaded to the PRIDE-EBI repository under submission number PXD030714.

Publisher Copyright:
© 2022

Fingerprint

Dive into the research topics of 'A ubiquitinome analysis to study the functional roles of the proteasome associated deubiquitinating enzymes USP14 and UCH37'. Together they form a unique fingerprint.

Cite this