Abstract-Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the ␣ heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both ␣ and  subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC. O ne major risk factor for the development of cardiovascular diseases, such as coronary heart disease, stroke, and myocardial infarction, is an imbalance of the production and elimination of reactive oxygen species, also termed as oxidative stress. [1][2][3] As a consequence, the nitric oxide (NO)/ cGMP signaling cascade is impaired, eg, through the excessive production of superoxide, which reacts with NO in a diffusion-limited reaction, yielding peroxynitrite. 4 The biological impact of NO scavenging is further aggravated by the progressive inhibition and downregulation of the NO receptor soluble guanylate cyclase (sGC). [5][6][7][8][9] Circumstantial evidence has implicated proteasomal pathways in this downregulation of sGC. 10 -12 Conversely, a novel class of sGC activators, represented by BAY 58-2667, are potentiated under oxidative stress conditions and represent thus an entirely new diseasespecific vasodilator class. 13 sGC is a heterodimer consisting of an ␣ and a Fe 2ϩ /hemecontaining  subunit that complexes NO with high affinity and specificity. 14 Binding of NO to the Fe 2ϩ /heme results in allosteric activation of the enzyme and enhanced conversion of GTP into the vasorelaxant and antiproliferative second messenger cGMP. 14,15 In vitro experiments have demonstrated that oxidation of sGC heme to its ferric (Fe 3ϩ ) form by the sGC inhibitor 1H-[1,2,4]-oxadiazolo [3,4-a]quinoxalin-1-one (ODQ) attenuates NO-mediated cGMP production, suggesting that the ferro (Fe 2ϩ ) form of sGC is crucial for activation by NO. 16 -18 In addition, studies with primary endothelial and smooth muscle cells have revealed that, within 24 hours, ODQ causes a dramatic decrease in sGC protein levels. 13 Similar results were obtained with other oxidizing compounds such as methylene blue or the peroxynitrite donor 1,3-morpholino-sydnonimine hydrochloride (SIN-1), indicating that oxidative stress triggers downregulation of sGC protein levels. 13 At present, the molecular mechanisms under...
Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.
Oxidative stress attenuates the NO-cGMP pathway, e.g. in the vascular system, through scavenging of free NO radicals by superoxide O 2•-, by inactivation of soluble guanylyl cyclase (sGC) via oxidation of its central Fe 2+ ion, and by down-regulation of sGC protein levels. While the former pathways are well established, the molecular mechanisms underlying the latter are still obscure. Using oxidative sGC inhibitor ODQ we demonstrate rapid down-regulation of sGC protein in mammalian cells. Coincubation with proteasomal inhibitor MG132 results in accumulation of ubiquitinated sGC whereas sGC activator BAY 58-2667 prevents ubiquitination. ODQ-induced down-regulation of sGC is mediated through selective ubiquitination of its b subunit, and BAY 58-2667 abrogates this effect. Ubiquitination of sGC-b is dramatically enhanced by E3 ligase CHIP. Our data indicate that oxidative stress promotes ubiquitination of sGC b subunit through E3 ligase CHIP, and that sGC activator 58-2667 reverts this effect, most likely through stabilization of the heme-free b subunit. Thus the deleterious effects of oxidative stress can be counter-balanced by an activator of a key enzyme of vascular homeostasis.
NO-sensitive guanylyl cyclases (sGCs) are cytosolic receptors for nitric oxide (NO) catalyzing the conversion of GTP to cGMP. sGCs are obligate heterodimers composed of one α and β subunit each. The allosteric mechanism of sGC activation via NO is well understood, however, our knowledge about alternative mechanisms such as proteinprotein interactions regulating activity, availability, translocation and expression of sGC is rather limited. In a search by the yeast two-hybrid system using the catalytic domain of the α 1 subunit as the bait, we have identified two structurally related proteins AGAP1 [1] and MRIP2 as novel sGC interacting proteins. MRIP2 is a multi-domain protein of 75 kDa comprising a single PH and ArfGAP domain each and two ankyrin repeats. Co-immunoprecipitation experiments using COS1 cells overexpressing both proteins demonstrated the interaction of MRIP2 with both subunits of the sGC α 1 β 1 . Confocal microscopical analysis showed a prominent plasma membrane staining of MRIP2. This membrane association is mediated through an N-terminal myristoylation site and through binding of its PH domain to phospholipids such as phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ). We hypothesize that MRIP2 may represent an acceptor protein for sGC that mediates recruitment of cytosolic sGC to the plasma membrane or other subcellular compartments. References 1.Meurer S, Pioch S, Wagner K, Müller-Esterl W, Gross S: AGAP1 -a novel binding partner of NO-sensitive guanylyl cyclase.
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