The PDZ Protein Mupp1 Promotes G i Coupling and Signaling of the Mt 1 Melatonin Receptor

International audienceIntracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT 1 melatonin receptor and directly regulates its G i-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT 1 carboxyl terminus. This high affinity interaction (K d ϳ 4 nM), which is independent of MT 1 activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT 1 /MUPP1 interaction has no effect on the subcel-lular localization, trafficking, or degradation of MT 1 , it destabilizes the interaction between MT 1 and G i and abolishes G i-mediated signaling of MT 1. Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs

Consequences of reduced production of NO on vascular reactivity of porcine coronary arteries after angioplasty: importance of EDHF

1. The consequences of the reduced production of nitric oxide (NO) by cells from regenerated endothelium were investigated by measuring membrane potential of smooth muscle cells (SMCs), isometric tension and cyclic nucleotides content in porcine coronary arteries with intimal thickening, four weeks following angioplasty. 2. Under basal conditions, SMCs of coronary arteries with regenerated endothelium were depolarized by 10 mV. This depolarization was associated with 82% decreased level of cGMP without alteration in cAMP. 3. Sodium nitroprusside (SNP, 1 μM) repolarized SMCs of the previously denuded coronary arteries. This repolarization was abolished by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) and not suppressed by glibenclamide (10 μM), iberiotoxin (IbTX, 100 nM) and the combination of charybdotoxin (ChTX, 40 nM) plus apamin (100 nM). 4. Four-aminopyridine (4-AP, 1-5 mM) generated spontaneous rhythmic activities only in coronary arteries with regenerated endothelium which were abolished by SNP. Nevertheless, 4-AP did not suppress the repolarization induced by SNP. 5. In vascular segments with regenerated endothelium, contracted with prostaglandin F(2α) (PGF(2α)), relaxation to bradykinin (BK, 30 nM) was unaltered despite a reduced production of cGMP (−70%). Indomethacin (10 μM) plus N(ω)-nitro-L-arginine (L-NA, 30 μM) reduced relaxation (−12% and −35% for native and regenerated endothelium, respectively) but did not abolish it. 6. The hyperpolarizations induced by BK were not altered by the presence of indomethacin and L-NA and were unchanged in segments with regenerated endothelium. 7. These data are consistent with a contribution of impairment in NO production to the depolarization of SMCs. Nevertheless, EDHF responses to BK are sufficient to maintain a normal relaxation after angioplasty