Z-REX uncovers a bifurcation in function of Keap1 paralogs

• DOI: 10.7554/elife.83373
• Publication Date: 2022-10-27T10:00:20Z
• Authors: Alexandra Van Hal...; Jesse R Poganik; Kuan-Ting Huang; Saba Parvez; Yi Zhao; Hong-Yu Lin; Xuyu Liu; Marcus John Curti...; Yimon Aye
• Keywords: KEAP1

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