Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized “electrophile toolbox” with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology—T-REX (targetable reactive electrophiles & oxidants)—is established by: (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein—one of several redox-sensitive regulators of the Nrf2–ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2–ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

2 Materials and Methods Statistics and data presentation. For experiments involving cultured cells, samples generated from individual wells or plates were considered biological replicates. For zebrafish experiments, each larval fish was considered a biological replicate. In the figure legend for each experiment, the number of independent biological replicates and how the data are presented in the figure (typically mean ± SEM) are clearly indicated. P-values calculated with Student's unpaired t-test are clearly indicated within datafigures. Data were plotted/fit and statistics generated using GraphPad Prism 7 or 8.

Dedicated to career of and contributions by Prof. Dr. Michael Graetzel (ISIC, EPFL) on his 75 th birthdayThe 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 Nrf2transcription-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 HuRdependent stress responsivities, further highlighting a complex context-dependent layer of Nrf2/AR regulation through HuR. Figure 1. HuR, but not AUF1, is a sensor of HNE in HEK293T cells. A) Structures of the native RES prostaglandin A2 (PGA2) and alkyne-functionalized HNE. B) HEK293T cells were treated with HNE(alkyne) (20 μM, 18 h). Click chemistry was used to biotinylate HNE-modified proteins, which were then enriched with streptavidin resin. Shown is a representative blot from four independent experiments. Inset at right: Quantification (mean � SEM) of western blot data from n = 4 independent replicates. ND: not detected.

We dedicate this work to the memory of Professor ThomasA.S teitz, whose work on the biophysics and structure of T7 RNAP provided key insights to our evolutions.[a] Dr.

Each flask in this phage‐assisted continuous evolution (PACE) experiment contains a replicating population of millions of M13 phage carrying a target protein of interest, which is evolving to have desired new properties. In this case, PACE was used to evolve T7 RNA polymerase (RNAP)‐based biosensors to tolerate C‐terminal fusions, a biophysical limitation integrally linked to T7 RNAP function. After evolution, the structural requirements of the enzyme are reprogramed and the limitation is lifted. More information can be found in the full paper by B. C. Dickinson et al. on page 1547 in Issue 12, 2019 (DOI: 10.1002/cbic.201800707).