A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs

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.

Resonance energy transfer sensitises and monitors in situ switching of LOV2-based optogenetic actuators

“…Excess positive charge in the spacer should favour an extended confirmation, but even a random-coil conformation (with residue spacing of 0.38 nm 10 ) would add 1.9 nm. Generously estimating initial inter-dipole distance at 5 nm, a typical value for R 0 , this equates to 1.4-fold distancing and a minimum sevenfold reduction of RET ([R/R 0 ] 6 ).…”

“…Half-maximal de-quenching took~1.3 s (5 readings, total~140 µmol m −2 photons). Such a fast de-quench half-time may estimate in situ activation rate if cell-free LOV2Jα relaxation rates (~20-80 s 6,16,17 ) are applicable and counteracting relaxation can be neglected.…”

“…Except in specific cases where protein translocation or other visualised downstream phenomena are targeted, it is hard to know whether an actuator has been switched. Relaxation rate constants are obtained by cell-free spectroscopy 3,6 and occasionally with heavily modified constructs not subsequently used for experimental actuation 7 . LOV2 designs that modify Jα, the moiety regulated by light-dependent switching, can accelerate cell-free relaxation >30-fold 8 .…”

ZLc002, a putative small-molecule inhibitor of nNOS interaction with NOS1AP, suppresses inflammatory nociception and chemotherapy-induced neuropathic pain and synergizes with paclitaxel to reduce tumor cell viability

“…HEK293T cells were cultured in Dulbecco’s minimal essential medium (with 10% fetal bovine serum, 19.4 mM supplementary glucose, 2 mM glutamine, 50 μg/ml streptomycin sulfate, and 50 U/ml penicillin) at 37°C under a 5% CO 2 humidified atmosphere. The cells were transfected by the calcium phosphate method as previously described 19 with full-length plasmid DNA pEGFP-nNOSα (human sequence fused to enhanced green fluorescent protein) and full-length pLuc-NOS1AP (human, transcript variant 1, fused to firefly luciferase) or pLuc-PSD95-PDZ2 (encoding aa 159–249 of human Dlg4 transcript variant4, fused to firefly luciferase) as indicated, or empty luciferase vector pLuc-C1 as negative control. These constructs have been previously described in the studies by Li et al.…”

Controlling Cells with Light and LOV

“…In most cases, this was accompanied by trial‐and‐error testing of LOV Jα–peptide hybrids in order to achieve successful peptide photocaging while preserving the favorable LOV photoswitching properties. Recent work by Courtney and co‐workers in their lab on kinase inhibitory peptides suggests a second, much simpler peptide photocaging approach . They fused truncated variants of a janus kinase inhibitory peptide to the unmodified As LOV2 Jα.…”