A general approach for investigating enzymatic pathways and substrates for ubiquitin-like modifiers.
Ubiquitin-like modifiers (UBLs) contain ubiquitin homology domains and can covalently modify target proteins in a manner similar to ubiquitylation. In this study, we revealed a general proteomic approach to elucidate the enzymatic pathways and identify target proteins for three UBLs: SUMO-2, SUMO-3, and NEDD8. Expression plasmids containing the cDNAs of Myc/6xHis doubly-tagged processed or non-conjugatable forms of these UBLs were constructed. The constructed vectors were then used to transfect HEK 293 Tet-On cells, and stable cell lines expressing these UBLs and their mutants were established. The epitope-tagged proteins were purified by immunoprecipitation under native conditions or by affinity chromatography on nickel resin under denaturing conditions. Purified proteins were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Most of the E1-like activating enzymes, E2-like conjugating enzymes and the majorities of the known target as well as some previously unreported proteins for SUMO-2, SUMO-3, and NEDD8 pathways were identified.
Li
Tianwei
T
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Santockyte
Rasa
R
Shen
Rong-Fong
RF
Tekle
Ephrem
E
Wang
Guanghui
G
Yang
David C H
DC
Chock
P Boon
PB
D002460:Cell Line; D003198:Computer Simulation; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism*; D000075687:NEDD8 Protein; D010449:Peptide Mapping / Q000379:methods; D015398:Signal Transduction / Q000502:physiology*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D009097:Multienzyme Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D014452:Ubiquitins