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A Generalizable Platform for Interrogating Target- and Signal-Specific Consequences of Electrophilic Modifications in Redox-Dependent Cell Signaling
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Details:
  • Pubmed ID:
    25909755
  • Pubmed Central ID:
    PMC4528680
  • Description:
    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.

  • Document Type:
  • Collection(s):
  • Funding:
    1DP2GM114850/DP/NCCDPHP CDC HHS/United States
    DP2 GM114850/GM/NIGMS NIH HHS/United States
    T32 GM008500/GM/NIGMS NIH HHS/United States
    T32 GM008500/GM/NIGMS NIH HHS/United States
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