Z-REX uncovers a bifurcation in function of Keap1 paralogs

Abstract

Studying electrophile signaling is marred by difficulties in parsing changes in 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 electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2- driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells, wherein following electrophile treatment: (1) zKeap1b-transfected cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding; (2) zKeap1a-transfected cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered; (3) 1:1 ZKeap1a:zKeap1b-transfected cells show no Nrf2-release from the Keap1-complex, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1a's behavior. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing.

Climbing into their Skin to Understand Contextual Protein–Protein Associations and Localizations: Functional Investigations in Transgenic Live Model Organisms

“…Using the technologies we have developed (Figure 4A) that are applicable to cells, [34] fish, [35] and worms, [36] these reactive small molecules can be photocaged and released on demand with rapid kinetics, [37] similar to biotinylation methods. However, unlike the small-molecule species used in biotinylation experiments that are all non-drug-like in nature, and indiscriminately reactive, and often inherently unstable, reactive electrophiles are relatively stable, [38] often affect specific functions of the proteins they label, [39][40][41][42][43][44][45][46] and critically, can Figure 3. A) Expression of TURBO-ID in all CamK2a neurons followed by excision of different regions of the brain allows mapping of tissue-specific expression patterns.…”

Keap It in the Family: How to Fish out New Paradigms in Keap1‐Mediated Cell Signaling

“…In our recent work, [5] we investigated the Keap1/Nrf2 system in zebrafish D. rerio . Zebrafish is a particularly interesting model system in which to study protein function due to a genome duplication that occurred in teleost fish, meaning that zebrafish contain two copies of many of their genes.…”

“…Nonetheless, zKeap1b's function is regulated by RES, similarly as in wt hKeap1. The detailed mechanism of action (MoA) of the two zKeap1 paralogs and their similarities in electrophile sensing versus differences in electrophile signaling have been reported recently [5] …”

Structural Insights into Electrophiles and Electrophilic Metabolites Sensing and Signaling: The Missing Information

“…Following wash‐out of the excess unbound probe, light exposure releases a brief burst of the designated electrophile in the vicinity of HaloTag, at a preordained time. We refer interested readers to technological reviews and protocols focused on REX technologies [15–21] . These methods, equipped with rigorous technical and biological controls, led us to several key findings, including: 1 ) low occupancy, i. e ., substoichiometric modification of a RES on a given protein, is often sufficient to drive functional signaling outcomes; 2 ) many electrophile‐sensor proteins identified by REX technologies display enhanced RES adduction rates, i. e ., are kinetically‐privileged sensor (KPS) proteins, compared to others identified using the bolus RES treatment underpinning all state‐of‐the‐art profiling methods; 3 ) KPS proteins identified and validated by REX technologies have often not been previously profiled by existing chemoproteomics methods (fundamental underlying differences leading to this outcome have been comprehensively reviewed elsewhere); 4 ) nuanced regulatory pathways involving RES are often masked by using bolus dosing conditions; and 5 ) applying REX technologies in zebrafish ( D. rerio ) and worms ( C. elegans ) reveals that mechanisms of electrophile sensing and signaling are largely conserved throughout evolution [1,9,13,15–20,22–30] …”

“…Despite the myriad profiling tools (spearheaded by the innovative activity‐based protein profiling (ABPP) method and its derivatives) and emerging function‐guided proximity mapping‐based REX technologies (T‐REX, Z‐REX, G‐REX, and more recently, Localis‐REX), several fundamental questions remain unaddressed, particularly with regard to structure/function relationship principles underlying electrophile‐sensor protein regulation ( Figure 2). [15,16,18–20] For instance, what makes a protein a KPS, given that a two units change in thiol pKa can only increase the reaction rate with peroxide by no more than 20‐fold and that full cysteine thiol deprotonation only enhances the Michael addition reaction rate with the electrophile acrolein by a maximum of 10‐fold, i. e ., how is the more functionally relevant kinetic selectivity, such as transition state stabilization, achieved? [1] How does RES modification of a single site affect the sensor protein's activity/function, or how does it modulate the function of a binding partner, the interactome or downstream signal transduction? Insights into such fundamental mechanisms, especially from structure/function perspectives, and generalizable principles — should there be any — of electrophile sensing and signaling remain hugely limited, particularly for pleiotropic native electrophiles such as LDEs and TCA cycle metabolites, and related low molecular weight (<300 Da) covalent drugs.…”