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A. Yu. Khristichenko, D. M. Hushpulian, N. A. Smirnova, A. A. Zakhariants, T. A. Chubar, V. I. Tishkov, I. G. Gazaryan, A. A. Poloznikov
Optimization of Neh2-Luc
reporter for screening of activators of antioxidant program
Abstract
Reporter vectors
expressing amino acid residues 16–96 and 16–85 from Neh2-domain of Nrf2
transcription factor fused to firefly luciferase have been constructed. Cell
lines stably expressing the above reporter constructs as well as an earlier
developed Neh2-luc fusion reporter have been generated using HeLa cells. Comparative
analysis of all three reporter lines has been performed using a well-known Nrf2
activator, nordihydroguaiaretic acid. The reporter expressing the minimal
fragment of Neh2-domain containing 16–85 aa sequence
exhibits a 2-fold higher amplitude of activation compared to Neh2-luc reporter,
and thus can be recommended for high throughput screening for Nrf2 activators.
A likely reason for the improved response of the above reporter could be
ubiquitination of N-terminal lysine residues (7, 10, 11) of luciferase protein
adjacent to the ubiquitinated Neh2 sequence.
Key words: Nrf2,
Keap1, luciferase, transcription factor, fusion protein, cell reporter, nordihydroguaiaretic
acid.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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