Midori‐ishi Cyan/monomeric Kusabira‐Orange‐based fluorescence resonance energy transfer assay for characterization of various E3 ligases

Abstract

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.

Selective Ablation of Tumorigenic Cells Following Human Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cell Transplantation in Spinal Cord Injury

“…253G1‐hiPSCs (provided by Prof. Shinya Yamanaka at CiRA, Kyoto University) were transduced with the Tet‐inducible HSVtk lentiviral vector at a multiplicity of infection (MOI) of 2–10. Almost 100% transduction efficiency was observed based on examining humanized Kusabira‐Orange 1 fluorescent protein (hKO1) expression under a fluorescence microscope. Single hKO1‐positive iPSCs were sorted using the FACSAria III system (BD Biosciences, San Jose, CA) and then expanded.…”

“…These secondary neurospheres were passaged to fourth‐passage neurospheres and used for transplantation. Almost 100% transduction efficiency of HSVtk was observed based on hKO1 expression under a fluorescence microscope when observed at the time of transduction with the lentiviral vector expressing ffLuc.…”

Shigellaeffector IpaH4.5 targets 19S regulatory particle subunit RPN13 in the 26S proteasome to dampen cytotoxic T lymphocyte activation

“…His6‐E1 and UBC5A were used as described previously (Otsubo et al, ). GST‐V5‐IpaH4.5, GST‐RPN13, GST‐UCH37, and GST‐ubiquitin were expressed in BL21 (DE3) E .…”

“…His6-E1 and UBC5A were used as described previously (Otsubo et al, 2016). GST-V5-IpaH4.5, GST-RPN13, GST-UCH37, and GST-ubiquitin were expressed in BL21 (DE3) E. coli cells and purified on glutathione-Sepharose 4B; GST was removed with PreScission™ protease (GE Healthcare, Piscataway, NJ, USA).…”

“…Shigella possesses 12 ipaH genes, which encode IpaH proteins that injected into host cells via the T3SS (Otsubo, Kim, Lee, & Sasakawa, ; Rohde, Breitkreutz, Chenal, Sansonetti, & Parsot, ; Singer et al, ; Zhu et al, ). IpaH family proteins contain N‐terminal leucine‐rich repeats (LRRs) and have E3 ubiquitin ligase activity in their conserved C‐terminal regions (CTRs; de Jong, Liu, Chen, & Alto, ; Li et al, ; Wandel et al, ; Zheng et al, ).…”