A novel injectable BRET-based in vivo imaging probe for detecting the activity of hypoxia-inducible factor regulated by the ubiquitin-proteasome system.

Takahiro Kuchimaru,Keisuke Hirota,Tetsuya Kadonosono,Tomoya Suka,Shinae Kizaka-Kondoh

doi:10.1038/srep34311

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.

Highlights

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
Design of injectable bioluminescence resonance energy transfer (BRET) probe regulated by hypoxia-inducible factor (HIF)-1α-specific UPS
The recombinant proteins containing ODD548–603 would be under the same UPS regulation as HIF-1α:They were stabilized in hypoxic cells and degraded in a VHL-dependent manner under normoxic conditions[14,15]

Summary

Introduction

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. We present a novel injectable imaging probe POL-N that can detect the UPS-regulated hypoxia-inducible factor (HIF) activity in vivo. In spite of the benefits of these reporter genes, a transgenic reporter system has limited applications; the signals from fluorescence proteins and luciferases are not long enough (λmax < 600 nm) to noninvasively detect targets in deep tissues of living animals; reporter genes have to www.nature.com/scientificreports/. BRET is a promising technology that needs no excitation light and minimizes tissue autofluorescence[9,10,11,12], achieving a high target-to-background (T/B) ratio[13]

Methods

Results

Conclusion