Abstract
Cav3.2 calcium channels play a key role in nociceptive signaling in the primary afferent pain pathway. We have previously reported the regulation of Cav3.2 calcium channels by the deubiquitinase USP5 and its importance for regulating peripheral transmission of pain signals. Here we describe the regulation of the Cav3.2-USP5 interaction by SUMOylation. We show that endogenous USP5 protein expressed in dorsal root ganglia undergoes SUMOylation, and the level of USP5 SUMOylation is reduced following peripheral nerve injury. SUMO prediction software identified several putative lysines that have the propensity to be targets for SUMO conjugation. A series of single lysine substitutions in an mCherry tagged USP5 construct followed by expression in tsA-201 cells identified lysine K113 as a key target for USP5 SUMO2/3 modification. Finally, Cav3.2 calcium channel immunoprecipitates revealed a stronger interaction of Cav3.2 with a SUMO2/3 resistant USP5-K113R mutant, indicating that SUMO2/3 modification of USP5 reduces its affinity for the calcium channel Cav3.2. Collectively, our data suggest that dysregulation of USP5 SUMOylation after peripheral nerve injury may contribute to the well described alteration in Cav3.2 channel activity during neuropathic pain states.
Highlights
Ubiquitination and deubiquitination are post-translational modifications that regulate protein stability through the modification of lysine residues by attachment/removal of one or more ubiquitin groups [1]
We first examined whether ubiquitin specific peptidase 5 (USP5) endogenously expressed in dorsal root ganglia (DRG) neurons is subject to SUMOylation
DRG neurons (L4-L6) were isolated from sham operated male wild type mice and USP5 immunoprecipitates were assayed by Western blotting
Summary
Ubiquitination and deubiquitination are post-translational modifications that regulate protein stability through the modification of lysine residues by attachment/removal of one or more ubiquitin groups [1]. In rodent models of neuropathic, inflammatory, visceral and postsurgical pain, USP5 protein expression levels in dorsal root ganglia (DRG) and spinal cord are upregulated [6,7,8,9,10], leading to an increased association with Cav3.2 T-type calcium channels and their enhanced deubiquitination. SUMOylation of USP5 is dysregulated after peripheral never injury, and show that a SUMO-deficient USP5 mutant displays increased interactions with Cav3.2 calcium channels.
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