Abstract
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.
Highlights
Reactions have been proposed on the basis of observations obtained mainly with immunoprecipitation experiments and using recombinant proteins [11, 14, 15]
It is well known that endothelial form of nitric-oxide synthase (eNOS) is a sensitive calpain substrate (16 –21) and that the elevation of [Ca2ϩ]i required for the initiation of the eNOS activation cycle induces the activation of this protease through its translocation to the membranes [22,23,24,25]
Coimmunolocalization experiments revealed that eNOS is mostly associated with caveolin-1 protein, present in both plasma membranes and the Golgi apparatus (Fig. 1A)
Summary
Reactions have been proposed on the basis of observations obtained mainly with immunoprecipitation experiments and using recombinant proteins [11, 14, 15]. Following the addition of the HSP90 inhibitor geldanamycin [37,38,39,40,41,42,43,44] to Ca2ϩ-loaded cells, the fluorescence of eNOS associated to the membranes is largely decreased, and only a small amount of eNOS becomes detectable in the cytosolic fraction (Fig. 2A).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.