MSc.Thesis Defense:Seyed Mohammad Miri

REVISITING THE IMPACT OF H₂O₂ ON INTRACELLULAR NRF2 LEVELS USING A NEWLY DEVELOPED OXYGEN-INDEPENDENT NRF2 BIOSENSOR

Seyed Mohammad Miri
Molecular Biology, Genetics and Bioengineering, MSc. Thesis, 2024

Thesis Jury

Prof. Selim Çetiner (Thesis Advisor)

Assoc. Prof. Özlem Kutlu

Assist. Prof. Esra Nur Yiğit

Date & Time: December 12th, 2024 – 1 PM

Place: FENS L047

Keywords: Nrf2, oxygen-insensitive biosensors, hydrogen peroxide (H₂O₂), chemogenetic tools, Auranofin

Abstract

Reactive oxygen species (ROS) are central to cellular communication, and Nrf2 serves as a crucial transcription factor in maintaining redox balance. Despite extensive research, the interplay between Nrf2 and hydrogen peroxide (H₂O₂) remains contentious. This study aimed to explore how varying intracellular H₂O₂ concentrations impact Nrf2 activity in brain endothelial cells under physiological oxygen conditions (5 kPa O₂), as opposed to conventional hyperoxic conditions. We developed the Pericellular Oxygen-Insensitive Nrf2 Total LEvel Reporter (POINTER), a novel biosensor for oxygen-independent Nrf2 monitoring in human cerebral microvascular endothelial cells (hCMEC/D3). By utilizing external H₂O₂, chemogenetic production of H₂O₂ via modified D-amino acid oxidase (mDAAO), and Auranofin-induced oxidative stress, we evaluated the effects of H₂O₂ on Nrf2. Our findings demonstrated significantly lower Nrf2 levels under physioxia compared to room air, with Auranofin and chemogenetically produced H₂O₂ showing distinct effects on Nrf2 modulation. POINTER revealed that while Auranofin significantly increased Nrf2 levels under physiological oxygen, exogenous and chemogenetically produced H₂O₂ did not sustain this effect. These results underscore the nuanced regulation of Nrf2 by both antioxidant inhibition and H₂O₂ elevation under physioxic conditions.