Midori-ishi Cyan/monomeric Kusabira-Orange-based fluorescence resonance energy transfer assay for characterization of various E3 ligases.
Many bacterial pathogens hijack the host ubiquitin system for their own benefit by delivering effectors with ubiquitin ligase (E3) into host cells via the type III secretion system. Therefore, screening for small compounds that selectively inhibit bacterial but not mammalian E3 ligases is a promising strategy for identifying molecules that could substitute for antibiotics. To facilitate high-throughput screening for bacterial E3 ligase inhibitors, we developed a MiCy/mKO (Midori-ishi Cyan/monomeric Kusabira-Orange)-based FRET (fluorescence resonance energy transfer) assay and validated it on Shigella IpaH E3 ligase effectors. We showed the feasibility of using the MiCy/mKO-based FRET assay to identify the most appropriate ubiquitin-conjugating enzymes (E2s) and determine the lysine specificity of a given E3, both hallmarks of E3 activity. Furthermore, we showed the usefulness of the FRET assay in characterizing mammalian E3 ligases, such as TNF receptor-associated factor 6 (TRAF6) and mouse double minute 2 homologue (MDM2). In addition, we confirmed the feasibility of determining the efficiency of inhibition of E3 ligase activity using inhibitors of E1 ubiquitin-activating enzymes, such as UBE1-41, by measuring the IC50 . Based on these results, we concluded that the MiCy/mKO-based FRET assay is useful for characterizing E3 enzyme activity, as well as for high-throughput E3 inhibitor screening.
Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan.
Ryota
http://orcid.org/0000-0002-1152-4198
R
Otsubo
Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan.|The Hakubi Center for Advanced Research, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto-shi, Kyoto, 606-8501, Japan.|Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto-shi, Kyoto, 606-8501, Japan.
Minsoo
M
Kim
Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan.
Jihye
J
Lee
Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan.|Nippon Institute for Biological Science, 9-2221-1 Shinmachi, Ome, Tokyo, Japan.|Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan.
Chihiro
C
Sasakawa
D000818:Animals; D004405:Dysentery, Bacillary / Q000201:enzymology / Q000382:microbiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis*; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D008164:Luminescent Proteins / Q000032:analysis; D008239:Lysine / Q000378:metabolism; D051379:Mice; D012763:Shigella flexneri / Q000201:enzymology*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008239:Lysine; D044767:Ubiquitin-Protein Ligases; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein