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

Abstract

A C‐terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site‐directed probe that irreversibly modifies a subset of Ub C‐terminal hydrolases (UCHs) and Ub‐specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [125I]UbVS labeling with N‐ethylmaleimide and Ub aldehyde, but also by genetic analysis. [125I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [125I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S‐associated USP14 with [125I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.

Activity Profiling of Papain-Like Cysteine Proteases in Plants

“…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).…”

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

“…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).…”

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

“…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).…”