A Generalizable
Platform for Interrogating Target-
and Signal-Specific Consequences of Electrophilic Modifications in
Redox-Dependent Cell Signaling
- Publication date
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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 (<u>t</u>argetable <u>r</u>eactive <u>e</u>lectrophiles and o<u>x</u>idants), 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