Wdr1 and cofilin are necessary mediators of immune-cell-specific apoptosis triggered by Tecfidera

Abstract

Despite the emerging importance of reactive electrophilic drugs, deconvolution of their principal targets remains difficult. The lack of genetic tractability/interventions and reliance on secondary validation using other non-specific compounds frequently complicate the earmarking of individual binders as functionally- or phenotypically-sufficient pathway regulators. Using a redox-targeting approach to interrogate how on-target binding of pleiotropic electrophiles translates to a phenotypic output in vivo, we here systematically track the molecular components attributable to innate immune cell toxicity of the electrophilic-drug dimethyl fumarate (Tecfidera®). In a process largely independent of canonical Keap1/Nrf2-signaling, Keap1-specific modification triggers mitochondrial-targeted neutrophil/macrophage apoptosis. On-target Keap1–ligand-engagement is accompanied by dissociation of Wdr1 from Keap1 and subsequent coordination with cofilin, intercepting Bax. This phagocytic-specific cell-killing program is recapitulated by whole-animal administration of dimethyl fumarate, where individual depletions of the players identified above robustly suppress apoptosis.

Hitting the Bullseye: Endogenous Electrophiles Show Remarkable Nuance in Signaling Regulation

“…Nevertheless, using T-REX TM we have validated multiple novel electrophile sensing proteins (>15, so far) 5,6,[12][13][14][15] . T-REX TM is also able to target specific proteins in specific subcellular environments with specific electrophiles as well as functioning in a range of model organisms 12,[16][17][18][19][20] . T-REX TM has proven to be particularly insightful at assaying specific signaling consequences of targetspecific electrophile protein labeling 5,12,17,19,[21][22][23] .…”

“…T-REX TM is also able to target specific proteins in specific subcellular environments with specific electrophiles as well as functioning in a range of model organisms 12,[16][17][18][19][20] . T-REX TM has proven to be particularly insightful at assaying specific signaling consequences of targetspecific electrophile protein labeling 5,12,17,19,[21][22][23] . T-REX TM is a very precise method, which is both its greatest strength and source of uniqueness but it is also its ultimate weakness.…”

“…We used Z-REX TM 8,12,19 , a derivative of T-REX TM performed in live zebrafish (Figure 2B), together with RNA-seq to examine how global mRNA pools were changed upon calibrated, sub-stoichiometric electrophile engagement of Keap1 in the vast majority of cells in zebrafish embryos 19 . Given that we were effectively performing the same process in each cell of the embryo, we were intrigued to find that under these conditions, selective loss in mRNAs specific to neutrophils and macrophages was observed 19 . We proceeded to show that upon Z-REX TM targeting Keap1 in zebrafish and several mammalian cultured cells, upregulation of apoptosis occurs.…”

“…Consistent with the RNA-seq data, the impact of targeting Keap1 is particularly elevated in innate immune cells, such as macrophages and neutrophils. Indeed, when we examined fish embryos that had undergone Z-REX TM targeting Keap1 which expressed fluorescent proteins within their macrophages or neutrophils, we showed selective depletion of these cells that was suppressed by caspase inhibitors 19 . This depletion did not happen when the same experiment was repeated, but the electrophile was not delivered to Keap1, or when a Keap1 mutant was used that cannot sense electrophiles 19 .…”

“…Indeed, when we examined fish embryos that had undergone Z-REX TM targeting Keap1 which expressed fluorescent proteins within their macrophages or neutrophils, we showed selective depletion of these cells that was suppressed by caspase inhibitors 19 . This depletion did not happen when the same experiment was repeated, but the electrophile was not delivered to Keap1, or when a Keap1 mutant was used that cannot sense electrophiles 19 . Our further examination uncovered that Keap1 can bind to Wdr1 42,43 , a protein linked to regulation of actin, and this binding is inhibited upon electrophile labeling of Keap1 19 .…”