Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis.

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and .NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker beta-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the .NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization.

Induction of nitric oxide synthase-2 proceeds with the concomitant downregulation of the endogenous caveolin levels.

Several cell types express inducible nitric oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide (LPS) or proinflammatory cytokines. For instance, muscular cells treated with LPS and interferon gamma (IFN-gamma) respond by increasing the mRNA and protein levels of NOS2, and synthesize large amounts of nitric oxide. We show here that transcriptional induction of NOS2 in muscular cells proceeds with a concomitant decrease in the levels of caveolin-1, -2 and -3. Addition of *NO-releasing compounds to C2C12 muscle cells reveals that this downregulation of the caveolin (cav) levels is due to the presence of *NO itself in the case of caveolin-3 and to the action of the LPS/IFN-gamma in the case of cav-1 and cav-2. Likewise, muscle cells obtained from NOS2(-/-) knockout mice challenged with LPS/IFN-gamma could downregulate their levels of cav-1 but not of cav-3, unlike wild-type animals, in which both cav-1 and cav-3 levels diminished in the presence of the proinflammatory insult. Laser confocal immunofluorescence analysis proves that *NO exerts autocrine and paracrine actions, hence diminishing the cav-3 levels. When the induced NOS2 was purified using an affinity resin or immunoprecipitated from muscular tissues, it appears strongly bound not only to calmodulin but also to cav-1, and marginally to cav-2 and cav-3. When the cav levels where reduced using antisense oligonucleotides, an increase in the NOS2-derived.NO levels could be measured, demonstrating the inhibitory role of the three cav isoforms. Our results show that cells expressing NOS2 diminish their cav levels when the synthesis of *NO is required.