| Nitric oxide synthase (NOS), a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our lab has found that certain drugs, such as guanabenz, inactivate neuronal NOS (nNOS) and lead to the ubiquitination of the nNOS. To better understand the molecular trigger for nNOS ubiquitination, we characterized the proteins that are involved in nNOS ubiquitination, examined a mutant nNOS that can serve as a model for inactivated nNOS, and identified the sites of ubiquitin attachment to nNOS. Using an in vitro model for ubiquitination containing Fraction II, the DE52-retained fraction of reticulocyte lysates that contains all ubiquitinating enzymes and the proteasome, we found that CHIP (C-terminus of Hsp70-interacting protein) and Hsp7() play a major role in promoting the ubiquitination of nNOS, whereas Hsp90, in concert with calmodulin. protects nNOS from ubiquitination. We found a C331A nNOS mutant that is highly susceptible to CHIP-dependent ubiquitination in cells and in vitro systems. Substrates and other ligands stabilize the C331A nNOS against ubiquitination, suggesting that subtle alterations to the active site cleft are sufficient for triggering the ubiquitination. The C33IA nNOS is ubiquitinated in an IIsp70/CHIP-dependent manner, similar to the wildtype enzyme. With the use of an in vitro system containing purified proteins, including E1 ubiquitin-activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase CHIP, we recapitulated the ubiquitination of nNOS seen in cells. We identified the sites of ubiquitination on nNOS through LC-MS/MS analysis of the ubiquitinated nNOS obtained from the in vitro purified system. Of the twelve sites that were identified, nine are located in the oxygenase domain, two in the calmodulin-binding region, and one in the reductase domain of the enzyme. These data are consistent with studies showing that the oxygenase domain and the calmodulin-binding domain play a major role in regulating the ubiquitination of nNOS. Thus, alterations to the heme active site structure, whether by drug-mediated inactivation or the destabilizing C331A mutation. leads to ubiquitination by I Isp7O and CHIP in the calmodulin-binding region and/or the oxygenase domain of nNOS. |
