title: A novel injectable BRET-based in vivo imaging probe for detecting the activity of hypoxia-inducible factor regulated by the ubiquitin-proteasome system.
abstract: The ubiquitin-proteasome system (UPS) is a selective protein degradation system that plays a critical role in many essential biological processes by regulating the existence of various cellular proteins. The target proteins of UPS are recognized and tagged with polyubiquitin chains by E3 ubiquitin ligases, which have high substrate-specific activities. Here we present a novel injectable imaging probe POL-N that can detect the UPS-regulated hypoxia-inducible factor (HIF) activity in vivo. Because the luciferase is fused to the E3 ligase-recognition domain of the HIF-1α, POL-N is intact only in the HIFα-overexpressing cells, that is, HIF-active cells, generating signals via an intramolecular bioluminescence resonance energy transfer (BRET) between luciferase and a near-infrared (NIR) fluorescent dye at the C-terminal end of the probe. Off-target signals of the NIR-BRET were so low that we could achieve highly sensitive and fast detection of intratumoral HIF-activity. Notably, we successfully detected hypoxic liver metastasis, which is extremely difficult to detect by injectable imaging probes due to strong off-target signals, as early as 1 h after systemic injection of POL-N. Our probe design can be widely adapted to UPS-target proteins and may contribute to the exploration of their roles in animal disease models.
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, 4259-B60, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kuchimaru
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, 4259-B60, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Suka
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, 4259-B60, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Hirota
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, 4259-B60, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kadonosono
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, 4259-B60, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan.
authors.forename: Shinae
authors.initials: S
authors.lastname: Kizaka-Kondoh
mesh_terms: D000818:Animals; D004735:Energy Transfer; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D015335:Molecular Probes; D009374:Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D019265:Spectroscopy, Near-Infrared; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D051793:Hypoxia-Inducible Factor 1; D015335:Molecular Probes; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex