Dataset Information

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

ABSTRACT: The removal of (poly)ubiquitin (Ub) chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Specific polyubiquitin linkages may lead to distinct cellular effects, with several linkage types associated with proteasomal degradation and others with lysosomal degradation, protein cellular localization or signaling events. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. The functional roles and specificities of these proteasomal DUBs remains elusive. To reveal the specificities of these proteasome associated DUBs, we use SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. 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 removal of both simultaneously suggests less redundancy for these DUBs 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 in wild-type versus double-knockout cells. Strikingly, we observed broad and severe off-target effects, questioning the alleged specificity of the 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jeroen Demmers

LAB HEAD: Jeroen Demmers

PROVIDER: PXD030714 | Pride | 2022-06-30

REPOSITORIES: Pride

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			Action	DRS
	1757_L_LennartvdWal_FW_Hilic_P1.raw	Raw
	1757_L_LennartvdWal_FW_Hilic_P1_least.raw	Raw
	1757_L_LennartvdWal_FW_Hilic_P2.raw	Raw
	1757_L_LennartvdWal_FW_Hilic_P2_least.raw	Raw
	1757_L_LennartvdWal_FW_Hilic_P3.raw	Raw

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

van der Wal Lennart L Bezstarosti Karel K Demmers Jeroen A A JAA

Journal of proteomics 20220427

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 ubiqui ...[more]

PMID: 35489684

Similar Datasets

Project description:In eukaryotic cells, short-lived, regulatory and misfolded or denatured proteins are degraded by the ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins for proteasomal degradation by ubiquitin. The UPS is critical in regulating proteostasis and dysfunctioning of this system has been implicated in diseases such as cancer and neurodegenerative disorders. We have investigated the effects of proteasome malfunctioning in Drosophila by monitoring the global proteome and ubiquitinome using SILAC proteomics. The proteasome was inactivated by either chemical inhibitors or by dsRNA mediated knockdown of specific proteasome subunit constituents. Upon proteasome perturbation both the global proteome and the ubiquitinome were remodeled to a great extent, albeit that the overall impact on the ubiquitinome was much more dramatic. The abundances of ~10% of all observable proteins were increased as a result of both de novo synthesis and of accumulation of ubiquitinated proteins. In the ubiquitinome analysis ~14,000 diGly peptides were identified and most of these could also be quantified. The far majority of diGly peptides showed increased levels, indicating that the pool of ubiquitinated proteins is highly dynamic. Interestingly, because of the high level of detail the ubiquitination dynamics of individual proteins could be explored. Several examples (e.g., Rps3 and H2B) will be discussed, in which different target lysine residues show either increased or decreased ubiquitination on the same protein, suggesting the occurrence of simultaneous and possibly functionally different ubiquitination events. In conclusion, the combination of quantitative proteomic methods with dsRNA mediated knockdown of individual subunits of the 26S proteasome offers a powerful tool to study the dynamics of the (modified) proteome upon perturbation of the UPS. This strategy opens up new avenues for the dissection of the mode of action of this cellular machinery, both under proteostasis and proteotoxic conditions.