Chemically Synthesized Ubiquitin Extension Proteins Detect Distinct Catalytic Capacities of Deubiquitinating Enzymes

Abstract

We have used solid-phase chemistry to synthesize proteins equivalent to a human ubiquitin precursor (ubiquitin–52-amino-acid ribosomal protein fusion; UBICEP52) and representative of isopeptide-linked ubiquitin–protein conjugates [ubiquitin–(ϵN)-lysine]; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D. Along with the previously synthesized ubiquitin–(αN)-valine, these synthetic proteins were used as substrates to assess the catalytic capacities of a number of diverse DUBs expressed in Escherichia coli: human HAUSP; mouse Unp; and yeast Ubps 1p, 2p, 3p, 6p, 11p, and 15p and Yuh1p. Distinct specificities of these enzymes were detected; notably, in addition to UCH-D, isopeptidase activity [ubiquitin–(ϵN)-lysine cleavage] was only associated with Yuh1p, Unp, Ubp1p, and Ubp2p. Additionally, human placental 26S proteasomes were only able to cleave UBICEP52 and ubiquitin–(ϵN)-lysine, suggesting that 26S proteasome-associated DUBs are class II-like. This work demonstrates that the synthetic approach offers an alternative to recombinant methods for the production of small proteins in vitro.