A general approach for investigating enzymatic pathways and substrates for ubiquitin-like modifiers.

摘要: Abstract 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 general proteomic approach elucidate the enzymatic pathways identify for three UBLs: SUMO-2, SUMO-3, NEDD8. Expression plasmids containing cDNAs of Myc/6× His doubly-tagged processed or non-conjugatable forms these UBLs were constructed. The constructed vectors then used transfect HEK 293 Tet-On cells, stable cell lines expressing their mutants established. epitope-tagged purified by immunoprecipitation under native conditions affinity chromatography on nickel resin denaturing conditions. Purified analyzed using liquid coupled with mass spectrometry (LC–MS/MS). Most E1-like activating enzymes, E2-like conjugating enzymes majorities known as well some previously unreported NEDD8 identified.