A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14

Borodovsky, Anna; Kessler, Benedikt M.; Casagrande, Rocco; Overkleeft, Herman S.; Wilkinson, Keith D.; Ploegh, Hidde L.

doi:10.1093/emboj/20.18.5187

Cited by 457 publications

(450 citation statements)

References 56 publications

Supporting

Mentioning

433

Contrasting

Order By: Relevance

“…Third, use of probes different from DCG-04 (used in this study) can reveal activities of other proteases. There are probes with a broad specificity described for Ser proteases (Liu et al, 1999), and deubuiqitinating enzymes (Borodovsky et al, 2001), whereas many other probes for other proteases are under development (Campbell and Szardenings, 2003).…”

Section: Discussionmentioning

confidence: 99%

Activity Profiling of Papain-Like Cysteine Proteases in Plants

et al. 2004

View full text Add to dashboard Cite

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanismbased manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B-and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

show abstract

Section: Discussionmentioning

confidence: 99%

Activity Profiling of Papain-Like Cysteine Proteases in Plants

et al. 2004

View full text Add to dashboard Cite

show abstract

“…Ubiquitin-specific protease 14 (USP14), like its yeast ortholog Ubp6, is a proteasome-associated deubiquitinating enzyme (DUB) that is activated upon binding to the proteasome and catalyzes the cleavage of ubiquitin subunits from substrates before degradation by the proteasome ( Borodovsky et al , 2001; Hanna et al , 2006; Hu et al , 2005). By releasing ubiquitin molecules from the substrate, USP14/Ubp6 helps to prevent the rapid degradation of ubiquitin molecules together with the substrate protein ( Hanna et al , 2007).…”

Section: Introductionmentioning

confidence: 99%

Does inactivation of USP14 enhance degradation of proteasomal substrates that are associated with neurodegenerative diseases?

2016

View full text Add to dashboard Cite

A common pathological hallmark of age-related neurodegenerative diseases is the intracellular accumulation of protein aggregates such as α-synuclein in Parkinson’s disease, TDP-43 in ALS, and tau in Alzheimer’s disease. Enhancing intracellular clearance of aggregation-prone proteins is a plausible strategy for slowing progression of neurodegenerative diseases and there is great interest in identifying molecular targets that control protein turnover. One of the main routes for protein degradation is through the proteasome, a multisubunit protease that degrades proteins that have been tagged with a polyubiquitin chain by ubiquitin activating and conjugating enzymes. Published data from cellular models indicate that Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme (DUB), slows the degradation of tau and TDP-43 by the proteasome and that an inhibitor of USP14 increases the degradation of these substrates. We conducted similar experiments designed to evaluate tau, TDP-43, or α-synuclein levels in cells after overexpressing USP14 or knocking down endogenous expression by siRNA.

show abstract

“…Deubiquitination of substrates before association with the proteasome can rescue proteins from irreversible degradation, whereas deubiquitination of proteasome-associated substrates can either promote or prevent degradation, depending on the timing and extent of deubiquitination in relation to substrate release or proteolysis (9 -12). Multiple deubiquitinases are associated with eukaryotic proteasomes, including Poh1/Rpn11 (5, 10, 11), Usp14/Ubp6 (13,14), and Uch37 (9,15,16).…”

Section: Introductionmentioning

confidence: 99%

The JAMM motif of human deubiquitinase Poh1 is essential for cell viability

Gallery

Blank

Lin

et al. 2007

Molecular Cancer Therapeutics

View full text Add to dashboard Cite

Poh1 deubiquitinase activity is required for proteolytic processing of polyubiquitinated substrates by the 26S proteasome, linking deubiquitination to complete substrate degradation. Poh1 RNA interference (RNAi) in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels, supporting the link between Poh1 and the ubiquitin proteasome pathway. To more specifically test for any requirement of the zinc metalloproteinase motif of Poh1 to support cell viability and proteasome function, we developed a RNAi complementation strategy. Effects on cell viability and proteasome activity were assessed in cells with RNAi of endogenous Poh1 and induced expression of wild-type Poh1 or a mutant form of Poh1, in which two conserved histidines of the proposed catalytic site were replaced with alanines. We show that an intact zinc metalloproteinase motif is essential for cell viability and 26S proteasome function. As a required enzymatic component of the proteasome, Poh1 is an intriguing therapeutic drug target for cancer. [Mol Cancer Ther 2007;6(1):262 -8]

show abstract

A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14

Cited by 457 publications

References 56 publications

Activity Profiling of Papain-Like Cysteine Proteases in Plants

Activity Profiling of Papain-Like Cysteine Proteases in Plants

Does inactivation of USP14 enhance degradation of proteasomal substrates that are associated with neurodegenerative diseases?

The JAMM motif of human deubiquitinase Poh1 is essential for cell viability

Contact Info

Product

Resources

About