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Molecular and Cellular Biology, January 2004, p. 84-95, Vol. 24, No. 1
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.1.84-95.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins

Joris Hemelaar,1 Anna Borodovsky,1 Benedikt M. Kessler,1 David Reverter,2 Julie Cook,1 Nagamallesawari Kolli,3 Tudev Gan-Erdene,3 Keith D. Wilkinson,3 Grace Gill,1 Christopher D. Lima,2 Hidde L. Ploegh,1* and Huib Ovaa1

Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115,1 Structural Biology Program, Biochemistry Department, Weill Medical College, Cornell University, New York, New York 10021,2 Department of Biochemistry, Emory University, Atlanta, Georgia 303223

Received 11 July 2003/ Returned for modification 25 August 2003/ Accepted 18 September 2003

Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.

* Corresponding author. Mailing address: Harvard Medical School, Department of Pathology, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-4777. Fax: (617) 432-4775. E-mail: ploegh{at}hms.harvard.edu.

Molecular and Cellular Biology, January 2004, p. 84-95, Vol. 24, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.1.84-95.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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