title: Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins.
abstract: 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.
authors.affiliation: Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Joris
authors.initials: J
authors.lastname: Hemelaar
authors.forename: Anna
authors.initials: A
authors.lastname: Borodovsky
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.forename: David
authors.initials: D
authors.lastname: Reverter
authors.forename: Julie
authors.initials: J
authors.lastname: Cook
authors.forename: Nagamallesawari
authors.initials: N
authors.lastname: Kolli
authors.forename: Tudev
authors.initials: T
authors.lastname: Gan-Erdene
authors.forename: Keith D
authors.initials: KD
authors.lastname: Wilkinson
authors.forename: Grace
authors.initials: G
authors.lastname: Gill
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Lima
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D000818:Animals; D010450:Endopeptidases / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D051379:Mice; D015335:Molecular Probes; D025842:SUMO-1 Protein / Q000378:metabolism; D013450:Sulfones / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D004798:Enzymes; D015335:Molecular Probes; D025842:SUMO-1 Protein; D013450:Sulfones; D014452:Ubiquitins; C009873:divinyl sulfone; D010450:Endopeptidases