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

• Published in: The EMBO journal Vol. 20; no. 18; pp. 5187 - 5196
• Main Authors: Borodovsky, Anna, Kessler, Benedikt M., Casagrande, Rocco, Overkleeft, Herman S., Wilkinson, Keith D., Ploegh, Hidde L.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 17-09-2001; Blackwell Publishing Ltd; Oxford University Press
• Subjects: 3T3 Cells; active site-directed probe; Animals; Binding Sites; Cell Extracts - chemistry; Cell Line; deubiquitylating enzyme; Endopeptidases - metabolism; Enzyme Inhibitors - pharmacology; Fungal Proteins - analysis; Fungal Proteins - genetics; Gene Deletion; hydrolase; Iodine Radioisotopes; Macromolecular Substances; Mammals; Mice; Oligopeptides - pharmacology; Peptide Hydrolases - metabolism; proteasome; Proteasome Endopeptidase Complex; proteasomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sulfones - chemical synthesis; Sulfones - chemistry; Sulfones - pharmacology; Thiolester Hydrolases - analysis; ubiquitin; Ubiquitin Thiolesterase; ubiquitin vinyl sulfone; Ubiquitins - analogs & derivatives; Ubiquitins - chemical synthesis; Ubiquitins - chemistry; Ubiquitins - metabolism; Ubp1 protein; Ubp12 protein; Ubp15 protein; Ubp2 protein; Ubp6 protein; USP14 gene; vinyl sulfone; Yeasts; Yeasts - enzymology; Yuh1 protein
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1093/emboj/20.18.5187

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.