Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins
AUTOR(ES)
Hemelaar, Joris
FONTE
American Society for Microbiology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=303361Documentos Relacionados
- Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins
- Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1)
- Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
- Structure and Evolution of Genes Encoding Polyubiquitin and Ubiquitin-like Proteins in Arabidopsis Thaliana Ecotype Columbia
- Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1