title: A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.
abstract: 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.
authors.affiliation: †Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Lin
authors.affiliation: †Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Haegele
authors.affiliation: †Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Disare
authors.affiliation: §Proteomics/Mass Spectrometry Facility, Center for Functional Genomics, University of Albany, Rensselaer, New York 12144, United States.
authors.forename: Qishan
authors.initials: Q
authors.lastname: Lin
authors.affiliation: †Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.|‡Department of Biochemistry, Weill Cornell Medical College, New York, New York 10065, United States.
authors.forename: Yimon
authors.initials: Y
authors.lastname: Aye
mesh_terms: D000478:Alkylation; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008958:Models, Molecular; D051267:NF-E2-Related Factor 2 / Q000032:analysis / Q000378:metabolism*; D010084:Oxidation-Reduction; D015398:Signal Transduction
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human