Functional role of HSP90 complexes with endothelial nitric-oxide synthase (eNOS) and calpain on nitric oxide generation in endothelial cells.
Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.
Averna
Monica
M
Department of Experimental Medicine (DIMES), Biochemistry Section, and Centre of Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV, 1-16132 Genoa, Italy.
Stifanese
Roberto
R
De Tullio
Roberta
R
Passalacqua
Mario
M
Salamino
Franca
F
Pontremoli
Sandro
S
Melloni
Edon
E
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry; D002154:Calpain / Q000737:chemistry*; D002417:Cattle; D051242:Caveolin 1 / Q000378:metabolism; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D018613:Microscopy, Confocal; D008954:Models, Biological; D009569:Nitric Oxide / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D017510:Protein Folding
D016227:Benzoquinones; D051242:Caveolin 1; D015853:Cysteine Proteinase Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D002154:Calpain; D002118:Calcium; C001277:geldanamycin