In silico modeling of the Menkes copper-translocating P-type ATPase 3rd metal binding domain predicts that phosphorylation regulates copper-binding.

The Menkes (ATP7A) P(1B)-type ATPase is a transmembrane copper-translocating protein. It contains six similar high-affinity metal-binding domains (MBDs) in the N-terminal cytoplasmic tail that are important for sensing intracellular copper and regulating ATPase function through the transfer of copper between domains. Molecular characterization of copper-binding and transfer is predominantly dependent on NMR structures derived from E. coli expression systems. A limitation of these models is the exclusion of post-translational modifications. We have previously shown that the third copper-binding domain, MBD3, uniquely contains two phosphorylated residues: Thr-327, which is phosphorylated only in the presence of elevated copper; and Ser-339, which is constitutively phosphorylated independent of copper levels. Here, using molecular dynamic simulations, we have incorporated these phosphorylated residues into a model based on the NMR structures of MBD3. Our data suggests that constitutively phosphorylated Ser-339, which is in a loop facing the copper-binding site, may facilitate the copper transfer process by exposing the CxxC copper-binding region of MBD3. Copper-induced phosphorylation of Thr327 is predicted to stabilize this change in conformation. This offers new molecular insights into how cell signaling (phosphorylation) can affect MBD structure and dynamics and how this may in turn affect copper-binding and thus copper-translocation functions of ATP7A.

Transient receptor potential vanilloid 4 inhibits mouse colonic motility by activating NO-dependent enteric neurotransmission.

UNLABELLED: Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca(2+) release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. KEY MESSAGES: • Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. • Our aim was to characterize the role of TRPV4 in the control of GI motility. • We found that TRPV4 activation reduced colonic contractility. • Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. • TRPV4 may be a novel pharmacological target in functional GI diseases.

Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe.

BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.

The tyrosine kinase inhibitor bafetinib inhibits PAR2-induced activation of TRPV4 channels in vitro and pain in vivo.

BACKGROUND AND PURPOSE: Protease-activated receptor 2 (PAR2) is expressed on nociceptive neurons, and can sensitize transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain. EXPERIMENTAL APPROACH: Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice. KEY RESULTS: In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 channels caused a sustained increase of [Ca(2+) ]i , inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo. CONCLUSIONS AND IMPLICATIONS: This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy.

Protein kinase-dependent phosphorylation of the Menkes copper P-type ATPase.

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a key regulator of copper homeostasis in humans. It has a dual role in supplying copper to essential cuproenzymes in the trans-Golgi network (TGN) and effluxing copper from the cell. These functions are achieved through copper-regulated trafficking of MNK between the TGN and the plasma membrane. However, the exact mechanism(s) which regulate the localisation and biochemical functions of MNK are still unknown. Here we investigated copper-dependent phosphorylation of MNK by a putative protein kinase(s). We found that in the presence of elevated copper there was a substantial increase in phosphorylation of the wild-type MNK in vivo. The majority of copper-dependent phosphorylation was on serine residues in two phosphopeptides. In contrast, there was no up-regulation of phosphorylation of a non-trafficking MNK mutant with mutated cytosolic copper-binding sites. Our findings suggest a potentially important role of kinase-dependent phosphorylation in the regulation of function of the MNK protein.