DUBs (deubiquitinating enzymes) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity towards small substrates that constitute the P4–P1 LRGG (Lys-Arg-Gly-Gly) C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs, we synthesized positional scanning libraries of fluorigenic tetrapeptides and tested them on three examples of human DUBs [OTU-1 (ovarian tumour 1), Iso-T (isopeptidase T) and UCH-L3 (ubiquitin C-terminal hydrolase L3)] and one viral ubiquitin-specific protease, namely PLpro (papain-like protease) from SARS (severe acute respiratory syndrome) virus. In most cases the results show flexibility in the P4 position, very high specificity for arginine in the P3 position and glycine in the P2 position, in accord with the sequence of the natural substrate, ubiquitin. Surprisingly, screening of the P2 position revealed that UCH-L3, in contrast with all the other tested DUBs, demonstrates substantial tolerance of alanine and valine at P2, and a parallel analysis using the appropriate mutation of the full-length ubiquitin confirms this. We have also used an optimal tetrapeptide substrate, acetyl-Lys-Arg-Gly-Gly-7-amino-4-methylcoumarin, to investigate the activation mechanism of DUBs by ubiquitin and elevated salt concentration. Together, our results reveal the importance of the dual features of (1) substrate specificity and (2) the mechanism of ubiquitin binding in determining deubiquitination by this group of proteases.
Skip Nav Destination
Article navigation
November 2008
- Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
October 15 2008
Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes)
Marcin Drag;
Marcin Drag
*Apoptosis and Cell Death Research, Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
†Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
Search for other works by this author on:
Jowita Mikolajczyk;
Jowita Mikolajczyk
*Apoptosis and Cell Death Research, Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
Search for other works by this author on:
Miklos Bekes;
Miklos Bekes
*Apoptosis and Cell Death Research, Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
‡Graduate Program in Molecular Pathology, University of California San Diego, La Jolla, CA 92093, U.S.A.
Search for other works by this author on:
Francisca E. Reyes-Turcu;
Francisca E. Reyes-Turcu
§Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, U.S.A.
Search for other works by this author on:
Jonathan A. Ellman;
Jonathan A. Ellman
¶Department of Chemistry, University of California Berkeley, Berkeley, CA 94720, U.S.A.
Search for other works by this author on:
Keith D. Wilkinson;
Keith D. Wilkinson
§Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, U.S.A.
Search for other works by this author on:
Guy S. Salvesen
Guy S. Salvesen
1
*Apoptosis and Cell Death Research, Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
1 To whom correspondence should be addressed (email gsalvesen@burnham.org).
Search for other works by this author on:
Biochem J (2008) 415 (3): 367–375.
Article history
Received:
April 15 2008
Revision Received:
June 26 2008
Accepted:
July 04 2008
Accepted Manuscript online:
July 04 2008
Citation
Marcin Drag, Jowita Mikolajczyk, Miklos Bekes, Francisca E. Reyes-Turcu, Jonathan A. Ellman, Keith D. Wilkinson, Guy S. Salvesen; Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes). Biochem J 1 November 2008; 415 (3): 367–375. doi: https://doi.org/10.1042/BJ20080779
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.