Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes.
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-(epsilonN)-lysine]; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D. Along with the previously synthesized ubiquitin-(alphaN)-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-(epsilonN)-lysine cleavage] was only associated with Yuh1p, Unp, Ubp1p, and Ubp2p. Additionally, human placental 26S proteasomes were only able to cleave UBICEP52 and ubiquitin-(epsilonN)-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.
Layfield
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R
Laboratory of Intracellular Proteolysis, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom. Robert.Layfield@nottingham.ac.uk
Franklin
K
K
Landon
M
M
Walker
G
G
Wang
P
P
Ramage
R
R
Brown
A
A
Love
S
S
Urquhart
K
K
Muir
T
T
Baker
R
R
Mayer
R J
RJ
D000818:Animals; D004330:Drosophila; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000138:chemical synthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014452:Ubiquitins / Q000378:metabolism*
D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D014452:Ubiquitins; D010450:Endopeptidases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease