A Generalizable Platform for Interrogating Target- and Signal-Specific Consequences of Electrophilic Modifications in Redox-Dependent Cell Signaling

• Published in: Journal of the American Chemical Society Vol. 137; no. 19; pp. 6232 - 6244
• Main Authors: Lin, Hong-Yu, Haegele, Joseph A, Disare, Michael T, Lin, Qishan, Aye, Yimon
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 20.05.2015; Amer Chemical Soc
• Subjects: Alkylation; Chemistry; Chemistry, Multidisciplinary; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins - analysis; Intracellular Signaling Peptides and Proteins - metabolism; Kelch-Like ECH-Associated Protein 1; Models, Molecular; NF-E2-Related Factor 2 - analysis; NF-E2-Related Factor 2 - metabolism; Oxidation-Reduction; Physical Sciences; Science & Technology; Signal Transduction; OXIDATIVE STRESS; ACTIVATION; LIPID-PEROXIDATION; NRF2; POTENCY; ANTIOXIDANT; ENZYMES; NF-E2-RELATED FACTOR-2; TRANSDUCTION; INDUCERS
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja5132648

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 and 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.