Development of Neh2-luciferase Reporter and Its Application for High Throughput Screening and Real-time Monitoring of Nrf2 Activators

Overview

Journal Chem Biol

Publisher Elsevier

Specialties Biochemistry
Biology
Chemistry

Date 2011 Jun 25

PMID 21700211

Citations 58

Authors

Natalya A Smirnova

Renee E Haskew-Layton

Manuela Basso

Dmitry M Hushpulian

Jimmy B Payappilly

Rachel E Speer

Young-Hoon Ahn

Ilay Rakhman

Philip A Cole

John T Pinto

Rajiv R Ratan

Irina G Gazaryan

Affiliations

Soon will be listed here.

Abstract

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found--nordihydroguaiaretic acid, fisetin, and gedunin--induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

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