François-Moutal, LibertyDustrude, Erik T.Wang, YueBrustovetsky, TatianaDorame, AngieJu, WeinaMoutal, AubinPerez-Miller, SamanthaBrustovetsky, NickolayGokhale, VijayKhanna, MayKhanna, Rajesh2020-02-032020-02-032018-10François-Moutal, L., Dustrude, E. T., Wang, Y., Brustovetsky, T., Dorame, A., Ju, W., … Khanna, R. (2018). Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain. Pain, 159(10), 2115–2127. doi:10.1097/j.pain.0000000000001294https://hdl.handle.net/1805/21974We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.en-USPublisher PolicyCells, CulturedDisease Models, AnimalGanglia, SpinalGene Expression RegulationHyperalgesiaImmunoprecipitationIntercellular Signaling Peptides and ProteinsNAV1.7 Voltage-Gated Sodium ChannelNerve Tissue ProteinsNeuralgiaPatch-Clamp TechniquesRats, Sprague-DawleyRotarod Performance TestSensory Receptor CellsSodiumTransduction, GeneticUbiquitin-Conjugating EnzymesArticle