Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes

We have used solid-phase chemistry to synthesize proteins equivalent to a h uman ubiquitin precursor (ubiquitin-52-amino-acid ribosomal protein fusion; UBICEP52) and representative of isopeptide-linked ubiquitin-protein conjug ates [ubiquitin-(epsilon N)-lysine]; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D, Along with the previously synthesized ubiquitin-(alpha N)-valine, these syn thetic proteins were used as substrates to assess the catalytic capacities of a number of diverse DUBs expressed in Escherichia coli: human HAUSP; mou se Unp; and yeast Ubps 1p, 2p, 3p, 6p, 11p, and 15p and Yuh1p. Distinct spe cificities of these enzymes were detected; notably, in addition to UCH-D, i sopeptidase activity [ubiquitin-(epsilon N)lysine cleavage] was only associ ated with Yuh1p, Unp, Ubp1p, and Ubp2p. Additionally, human placental 26S p roteasomes were only able to cleave UBICEP52 and ubiquitin-(epsilon N)-lysi ne, suggesting that 26S proteasome-associated DUBs are class II-like. This work demonstrates that the synthetic approach offers an alternative to reco mbinant methods for the production of small proteins in vitro. (C) 1999 Aca demic Press.