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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 1696 and 1685 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 1685 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.
Moscow University Chemistry Bulletin.
2020, Vol. 61, No. 3, P. 214
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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