Abstract
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor’s subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.
Highlights
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge
While evaluating strategies to circumvent limitations of previous optogenetic designs, we unexpectedly found that AsLOV2 (404–546) (Fig. 1c) conferred lit/dark-state dependence of Jun N-terminal kinase (JNK) binding to fused peptides
We used AsLOV2Ja-JIP11 fusion hereon for JNK inhibition, and refer to it as OptoJNKi after the cell-permeant D-form peptidic JNK inhibitor based on MAPK8IP1, D-JNKi25
Summary
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. An alternate strategy, developed to impose light-dependent steric hindrance to short effector peptides[8,9], used sequences homologous to the Ja to be interwoven within the Ja sequence generating a chimera exhibiting light regulation[9] This strategy is useful for sequences of sufficient Ja homology that their introduction prevents neither photoregulation nor peptide-target interaction. Molecular dynamics simulation suggests that interaction of JIP inhibitor peptide C-terminal phenylalanine with a non-polar pocket between the LOV2 domain Ab/Bb loop and Ib strand might stabilize the dark state, enhancing light regulation of JIP peptide availability We derive from this a design strategy we implement for another mitogen-activated protein kinase (MAPK), p38. We propose our design framework could help rapidly generate optogenetic as well as optopharmacological inhibitors of new targets of interest, perhaps even beyond MAPKs
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