title: A cell-permeant peptide corresponding to the cUBP domain of USP5 reverses inflammatory and neuropathic pain.
abstract: BACKGROUND
  Cav3.2 T-type calcium currents in primary afferents are enhanced in various painful pathological conditions, whereas inhibiting Cav3.2 activity or expression offers a strategy for combating the development of pain hypersensitivity. We have shown that Cav3.2 channel surface density is strongly regulated by the ubiquitination machinery and we identified the deubiquitinase USP5 as a Cav3.2 channel interacting protein and regulator of its cell surface expression. We also reported that USP5 is upregulated in chronic pain conditions. Conversely, preventing its binding to the channel in vivo mediates analgesia in inflammatory and neuropathic pain models.
  
  
  RESULTS
  To identify which USP5 domain is responsible for the interaction, we used a series of USP5-derived peptides corresponding to different regions in nUBP, cUBP, UBA1, and UBA2 domains to outcompete full length USP5. We identified a stretch of amino acid residues within the cUBP domain of USP5 as responsible for binding to Cav3.2 calcium channels. Based on this information, we generated a TAT-cUBP1-USP5 peptide that could disrupt the Cav3.2/USP5 interaction in vitro and tested its physiological effect in well-established models of persistent inflammatory pain (CFA test) and chronic mononeuropathy and polyneuropathy in mice (partial sciatic nerve injury and the (ob/ob) diabetic spontaneous neuropathic mice). Our results reveal that the TAT-cUBP1-USP5 peptide attenuated mechanical hyperalgesia induced by both Complete Freund's Adjuvant and partial sciatic nerve injury, and thermal hyperalgesia in diabetic neuropathic animals. In contrast, Cav3.2 null mice were not affected by the peptide in the partial sciatic nerve injury model. Cav3.2 calcium channel levels in diabetic mice were reduced following the administration of the TAT-cUBP1-USP5 peptide.
  
  
  CONCLUSIONS
  Our findings reveal a crucial region in the cUBP domain of USP5 that is important for substrate recognition and binding to the III-IV linker of Cav3.2 channels. Targeting the interaction of this region with the Cav3.2 channel can be exploited as the basis for therapeutic intervention into inflammatory and neuropathic pain.
authors.affiliation: Department of Physiology and Pharmacology, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Agustin
authors.initials: A
authors.lastname: Garcia-Caballero
authors.affiliation: Department of Physiology and Pharmacology, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Vinicius M
authors.initials: VM
authors.lastname: Gadotti
authors.affiliation: Department of Physiology and Pharmacology, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Lina
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Physiology and Pharmacology, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada zamponi@ucalgary.ca.
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Zamponi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020747:Calcium Channels, T-Type / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D003921:Diabetes Mellitus, Experimental / Q000150:complications / Q000188:drug therapy / Q000473:pathology; D004195:Disease Models, Animal; D010450:Endopeptidases / Q000737:chemistry*; D006930:Hyperalgesia / Q000150:complications / Q000188:drug therapy / Q000473:pathology; D007249:Inflammation / Q000150:complications* / Q000188:drug therapy* / Q000473:pathology; D008297:Male; D008810:Mice, Inbred C57BL; D009437:Neuralgia / Q000150:complications* / Q000188:drug therapy* / Q000473:pathology; D011485:Protein Binding; D000072417:Protein Domains; D025941:Protein Interaction Mapping; D012584:Sciatic Nerve / Q000187:drug effects / Q000293:injuries / Q000473:pathology
chemical_list: C504883:Cacna1h protein, mouse; D020747:Calcium Channels, T-Type; D057846:Cell-Penetrating Peptides; D010450:Endopeptidases; C091939:ubiquitin isopeptidase
keywords: Cav3.2; Complete Freund’s Adjuvant; T-type channels; TAT-cUBP1-USP5 peptide; USP5; chronic pain; diabetic neuropathy