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Arch Biochem Biophys. 2006 Sep 1;453(1):70-4. Epub 2006 Mar 20.

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

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  • 1Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

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 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.

PMID:
16620772
[PubMed - indexed for MEDLINE]
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