Z-REX uncovers a bifurcation in function of Keap1 paralogs

Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-& agrave;-vis off-target, modifications. By combining bolus dosing, knockdown, and Z- REX & mdash;a tool investigating on- target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish- Keap1- paralog (zKeap1b) stimulates Nrf2driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2signaling, and itself is nonpermissive for electrophile-induced Nrf2upregulation. This behavior is recapitulated in human cells: (1) zKeap1b-expressing cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding following whole -cell electrophile treatment; (2) zKeap1a-expressing cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered upon whole -cell electrophile exposure; (3) 1:1 ZKeap1a:zKeap1b-co-expressing cells show no Nrf2release from the Keap1-complex following whole -cell electrophile administration, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1a's behavior during electrophilic stress. Human-Keap1(C273I), of known diminished Nrf2regulatory capacity, dominantly muted electrophile-induced Nrf2signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing.