ELECTROPHILE SIGNALING IN MAMMALIAN AND ZEBRAFISH IMMUNITY AND STRESS RESPONSE

299 pagesSupplemental file(s) description: Table A2.3, Table A1.4, Table A1.2.Reactive electrophilic species (RES) have emerged in recent years as bona fide cellular signaling mediators, controlling myriad cellular processes. RES such as the lipid-derived electrophile (LDE) 4-hydroxynonenal (HNE) engage specific targets in cells featuring kinetically-privileged cysteine residues able to react rapidly with the RES. These modification events trigger signaling cascades to produce a cellular response. The growth of the RES signaling field has coincided with an explosion of interest in covalent drugs, which can target kinetically-privileged cysteines to produce a therapeutic effect. For instance, dimethyl fumarate (DMF; Tecfidera®)—a relatively simple electrophile—was approved in 2013 for the treatment of multiple sclerosis, and is now a multi-billion dollar drug. In spite of these advances, electrophile signaling and mechanisms of action of drugs that are believed to function through electrophile-signaling-like mechanisms remain particularly challenging to study. The pleiotropic nature of these molecules along with the often pleiotropic effects engendered by cellular electrophile exposure make pinning down the mechanisms by which they evoke cellular responses difficult. This is underscored by the fact that DMF was approved without a clear mechanism of action, and prior to our work there existed no identified target of DMF whose modification was fully sufficient to explain the clinical effects of this drug, namely induction of apoptosis in immune cells. Herein, we leveraged our lab’s Targetable Reactive Electrophiles and Oxidants (T-REX) electrophile delivery platform to delineate a novel signaling cascade controlling apoptosis of neutrophils and macrophages in zebrafish exposed to DMF. Unlike previous studies proposing targets of DMF, knockdown or inhibition of the protein players we identified to control the effects of DMF—Keap1, Wdr1, Cfl1, and Bax—was able to fully suppress apoptosis of immune cells, clearly demonstrating the necessity of these proteins in DMF’s mechanism of action. Moving to the realm of RES signaling in regulation of mRNA, we sought to characterize the reported electrophile sensing abilities of two key, disease-relevant mRNA binding proteins (mRBPs), HuR and AUF1. These studies led us to identify an unexpected novel regulatory axis controlling the Nrf2-driven antioxidant response (AR), a key stress response pathway upregulated in cells exposed to oxidants and RES. Testifying to the pleiotropic nature of these regulatory events, HuR regulation of Nrf2-mRNA produced divergent effects on AR under non-stimulated and HNE-stimulated conditions. Furthermore, in contrast to previous reports suggesting the electrophile-sensing abilities of both HuR and AUF1, we found that only HuR features kinetically privileged HNE-sensing ability. Taken together, these studies have revealed novel signaling events controlling diverse cellular pathways/processes engendered by RES. Additionally, identification of a sufficient mechanism by which DMF functions and the privileged RES sensing ability of HuR point to inroads for novel/improved therapeutics for these druggable pathways.2022-06-0

Electrophile Signaling and Emerging Immuno- and Neuro-modulatory Electrophilic Pharmaceuticals

With a lipid-rich environment and elevated oxygen consumption, the central nervous system (CNS) is subject to intricate regulation by lipid-derived electrophiles (LDEs). Investigations into oxidative damage and chronic LDE generation in neural disorders have spurred the development of tools that can detect and catalog the gamut of LDE-adducted proteins. Despite these advances, deconstructing the precise consequences of individual protein-specific LDE modifications remained largely impossible until recently. In this perspective, we first overview emerging toolsets that can decode electrophile-signaling events in a protein/context-specific manner, and how the accumulating mechanistic insights brought about by these tools have begun to offer new means to modulate pathways relevant to multiple sclerosis (MS). By surveying the latest data surrounding the blockbuster MS drug dimethyl fumarate that functions through LDE-signaling-like mechanisms, we further provide a vision for how chemical biology tools probing electrophile signaling may be leveraged toward novel interventions in CNS disease

Weighing up the Selenocysteome Uncovers New Sec-rets

Challenging the paradigm of SECIS-dependent selenoprotein translation, in this issue of Cell Chemical Biology Guo et al. (2018) introduce a new selenoprotein profiling platform with which they identify novel selenoproteins apparently lacking SECIS. With increased interest in covalent targeting of reactive Sec residues in drug discovery, their method adds a valuable contribution toward expanding the druggable human proteome

Redox Signaling by Reactive Electrophiles and Oxidants

The concept of cell signaling in the context of nonenzyme-assisted protein modifications by reactive electrophilic and oxidative species, broadly known as redox signaling, is a uniquely complex topic that has been approached from numerous different and multidisciplinary angles. Our Review reflects on five aspects critical for understanding how nature harnesses these noncanonical post-translational modifications to coordinate distinct cellular activities: (1) specific players and their generation, (2) physicochemical properties, (3) mechanisms of action, (4) methods of interrogation, and (5) functional roles in health and disease. Emphasis is primarily placed on the latest progress in the field, but several aspects of classical work likely forgotten/lost are also recollected. For researchers with interests in getting into the field, our Review is anticipated to function as a primer. For the expert, we aim to stimulate thought and discussion about fundamentals of redox signaling mechanisms and nuances of specificity/selectivity and timing in this sophisticated yet fascinating arena at the crossroads of chemistry and biology

On-Demand Targeting: Investigating Biology with Proximity-Directed Chemistry

Proximity enhancement is a central chemical tenet underpinning an exciting suite of small-molecule toolsets that have allowed us to unravel many biological complexities. The leitmotif of this opus is “tethering”a strategy in which a multifunctional small molecule serves as a template to bring proteins/biomolecules together. Scaffolding approaches have been powerfully applied to control diverse biological outcomes such as protein–protein association, protein stability, activity, and improve imaging capabilities. A new twist on this strategy has recently appeared, in which the small-molecule probe is engineered to unleash controlled amounts of reactive chemical signals within the microenvironment of a target protein. Modification of a specific target elicits a precisely timed and spatially controlled gain-of-function (or dominant loss-of-function) signaling response. Presented herein is a unique personal outlook conceptualizing the powerful proximity-enhanced chemical biology toolsets into two paradigms: “multifunctional scaffolding” versus “on-demand targeting”. By addressing the latest advances and challenges in the established yet constantly evolving multifunctional scaffolding strategies as well as in the emerging on-demand precision targeting (and related) systems, this Perspective is aimed at choosing when it is best to employ each of the two strategies, with an emphasis toward further promoting novel applications and discoveries stemming from these innovative chemical biology platforms

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

The mechanism-of-action of many electrohilic drugs remains poorly understood. Here, the authors use a redox-targeting approach to elucidate the basis for the innate immune cell toxicity of dimethyl fumarate, showing that it modifies Keap1 to trigger mitochondrial-targeted neutrophil/macrophage apoptosis

The mRNA-Binding Protein HuR Is a Kinetically-Privileged Electrophile Sensor

The key mRNA-binding proteins HuR and AUF1 are reported stress sensors in mammals. Intrigued by recent reports of sensitivity of these proteins to the electrophilic lipid prostaglandin A2 and other redox signals, we here examined their sensing abilities to a prototypical redox-linked lipid-derived electrophile, 4-hydroxynonenal (HNE). Leveraging our T-REX electrophile delivery platform, we found that only HuR, and not AUF1, is a kinetically-privileged sensor of HNE in HEK293T cells, and sensing functions through a specific cysteine, C13. Cells depleted of HuR, upon treatment with HNE, manifest unique alterations in cell viability and Nrf2-transcription-factor-driven antioxidant response (AR), which our recent work shows is regulated by HuR at the Nrf2-mRNA level. Mutagenesis studies showed that C13-specific sensing alone is not sufficient to explain HuR-dependent stress responsivities, further highlighting a complex context-dependent layer of Nrf2/AR regulation through HuR

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1

The nuclear factor erythroid 2 related factor 2 (Nrf2) signaling axis is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of Nrf2 regulation has been focused at the protein level. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These RNA-sequencing data and subsequent mechanistic analyses led us to discover a novel role of HuR in regulating Nrf2 activity, and in the process, we further identified the related mRBP AUF1 as an additional novel Nrf2 regulator. Both mRBPs regulate Nrf2 activity by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export, whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using a Nrf2 activity-reporter zebrafish strain, we document that this post-transcriptional control of Nrf2 activity is conserved at the whole-vertebrate level