Sensor Systems of KEAP1 Uniquely Detecting Oxidative and Electrophilic Stresses Separately In Vivo

In the KEAP1-NRF2 stress response system, KEAP1 acts as a sensor for oxidative and electrophilic stresses through formation of S-S bond and C-S bond, respectively. Of the many questions left related to the sensor activity, following three appear important; whether these KEAP1 sensor systems are operating in vivo, whether oxidative and electrophilic stresses are sensed by the similar or distinct systems, and how KEAP1 equips highly sensitive mechanisms detecting oxidative and electrophilic stresses in vivo. To address these questions, we conducted a series of analyses utilizing KEAP1-cysteine substitution mutant mice, conditional selenocysteine-tRNA (Trsp) knockout mice, and human cohort whole genome sequence (WGS) data. The Trsp-knockout provokes severe deficiency of selenoproteins and compensatory activation of NRF2. However, mice lacking homozygously a pair of critical oxidative stress sensor cysteine residues of KEAP1 fail to activate NRF2 in the Trsp-knockout livers. Secondly, this study provides evidence for the differential utilization of KEAP1 sensors for oxidative and electrophilic stresses in vivo. Thirdly, theoretical calculations show that the KEAP1 dimer equips quite sensitive sensor machinery in which modification of a single molecule of KEAP1 within the dimer is sufficient to affect the activity. WGS examinations of rare variants identified seven non-synonymous variants in the oxidative stress sensors in human KEAP1, while no variant was found in electrophilic sensor cysteine residues, supporting the fail-safe nature of the KEAP1 oxidative stress sensor activity. These results provide valuable information for our understanding how mammals respond to oxidative and electrophilic stresses efficiently. To examine the contribution of KEAP1-Cys226/613 to the NRF2 activation in selenoprotein deficient livers, we generated compound mutant mice by crossing conditional liver-specific Trsp knockout (Trspfl/fl::AlbCre) mice and KEAP1C226S&C613S mutant mice. We performed RNA-sequence analysis using RNA samples from the livers. We utilized RNA samples from livers of Trspfl/fl, Trspfl/fl::AlbCre, KEAP1C226S&C613S/C226S&C613S::Trspfl/fl and KEAP1C226S&C613S/C226S&C613S::Trspfl/fl::AlbCre mice, in age of 20 day-old.