Functional Characterization of Nitric Oxide and YC-1 Activation of Soluble Guanylyl Cyclase:  Structural Implication for the YC-1 Binding Site?

Lamothe, Maria; Chang, Fu‐Jung; Balashova, Nataliya V.; Shirokov, Roman; Beuve, Annie

doi:10.1021/bi0360051

Cited by 81 publications

(108 citation statements)

References 40 publications

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“…Interestingly, C243 was proposed to be part of a regulatory binding site for BAY 41-2772 (NOindependent activator) (30) but this observation was not supported by site-directed mutagenesis (31). As part of a mutational analysis of conserved Cys residues, Cys-78 and Cys-214 in the ␤1-subunit were mutated but the lost of NO-stimulated activity of the corresponding mutants was caused by heme depletion (32).…”

Section: Discussionmentioning

confidence: 99%

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Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation

Sayed

Baskaran

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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199

129

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The molecular mechanism of desensitization of soluble guanylyl cyclase (sGC), the NO receptor, has long remained unresolved. Posttranslational modification and redox state have been postulated to affect sGC sensitivity to NO but evidence has been lacking. We now show that sGC can be S-nitrosylated in primary aortic smooth muscle cells by S-nitrosocysteine (CSNO), an S-nitrosylating agent, in human umbilical vein endothelial cells after vascular endothelial growth factor treatment and in isolated aorta after sustained exposure to acetylcholine. Importantly, we show that S-nitrosylation of sGC results in decreased responsiveness to NO characterized by loss of NO-stimulated sGC activity. Desensitization of sGC is concentration-and time-dependent on exposure to CSNO, and sensitivity of sGC to NO can be restored and its S-nitrosylation prevented with cellular increase of thiols. We confirm in vitro with semipurified sGC that S-nitrosylation directly causes desensitization, suggesting that other cellular factors are not required. Two potential S-nitrosylated cysteines in the ␣-and ␤-subunits of sGC were identified by MS. Replacement of these cysteines, C243 in ␣ and C122 in ␤, created mutants that were mostly resistant to desensitization. Structural analysis of the region near ␤-C122 in the homologous Nostoc H-NOX crystal structure indicates that this residue is in the vicinity of the heme and its S-nitrosylation could dampen NO activation by affecting the positions of key residues interacting with the heme. This study suggests that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells and could be a mechanism of NO tolerance.cGMP ͉ tolerance ͉ redox ͉ S-nitrosothiols I n the cardiovascular system, nitric oxide (NO) is critical for regulation of vascular tone and homeostasis (1, 2). In mammals, the main sensor of NO is the soluble guanylyl cyclase (sGC), a heme-containing heterodimer formed by an ␣-and a ␤-subunit. When NO binds to the heme, catalytic activity of sGC increases several hundredfold to produce the second messenger cGMP (3). Despite the importance of the NO-cGMP pathway in the biology and pathology of the cardiovascular and neuronal systems, modulation of sGC is poorly understood (4, 5). In particular, the mechanism of desensitization of sGC has remained unresolved despite 30 years of effort. Desensitization is the transition to a state in which sGC's response to a new NO stimulation is reduced or abolished. This direct effect on sGC differs from the desensitization of the NO-cGMP pathway caused by decrease in cGMP levels [e.g., because of increased phosphodiesterase activity (6)] or decrease in NO availability (7). Desensitization of sGC itself has been reported in various cell types and tissues after exposure to NO (8-11) but its mechanism is unknown. Ser/Thr phosphorylation was proposed to be involved but evidence is still lacking (12), and Tyr phosphorylation was ruled out recently (13). Elucidating the mechanism of sGC desensitization is crucial considering i...

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Section: Discussionmentioning

confidence: 99%

“…sGC activity was determined by formation of [␣ 32 P]cGMP from [␣ 32 P]GTP as described (31). Reactions were performed for 5 min at 33°C in a final volume of 100 l, in 50 mM Hepes, pH 8.0, reaction buffer containing 500 M GTP, 1 mM DTT, and 5 mM MgCl 2 .…”

mentioning

confidence: 99%

Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation

Sayed

Baskaran

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

199

129

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“…The ␣ 1 /␤ 1 -His tag sGC was expressed in a Sf21/baculovirus system and purified using Talon cobalt resin (Clontech) followed by MonoQ 5/5 FPLC (Amersham Biosciences), as previously described (12). Fractions with the highest sGC activity were pooled in 10% glycerol and 5 mM DTT and snap frozen.…”

Section: Sgc Expression and Purificationmentioning

confidence: 99%

“…sGC Activity Assay GC activity was determined by formation of [␣- 32 P]cGMP from [␣- 32 P]GTP, as previously described (12). Reactions were performed for 10 min at 30°C in a final volume of 100 l, in a 50 mM HEPES, pH 8.0, reaction buffer containing 500 M GTP, 1 mM DTT, and 5 mM MgCl 2 .…”

Section: Sgc Expression and Purificationmentioning

confidence: 99%

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Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase

Balashova

Chang

Lamothe

et al. 2005

Journal of Biological Chemistry

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Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.

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Inhibition of soluble guanylyl cyclase by small molecules targeting the catalytic domain

et al. 2016

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Soluble guanylyl cyclase (sGC) plays a crucial role in cyclic nucleotide signaling that regulates numerous important physiological processes. To identify new sGC inhibitors that may prevent the formation of the active catalytic domain conformation, we carried out an in silico docking screen targeting a ‘backside pocket’ of the inactive sGC catalytic domain structure. Compounds 1 and 2 were discovered to inhibit sGC even at high/saturating nitric oxide concentrations. Both compounds also inhibit the BAY 58-2667-activated sGC as well as BAY 41-2272-stimulated sGC activity. Additional biochemical analyses showed that compound 2 also inhibits the isolated catalytic domain, thus demonstrating functional binding to this domain. Both compounds have micromolar affinity for sGC and are potential leads to develop more potent sGC inhibitors.

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Functional Characterization of Nitric Oxide and YC-1 Activation of Soluble Guanylyl Cyclase: Structural Implication for the YC-1 Binding Site?

Cited by 81 publications

References 40 publications

Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation

Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation

Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase

Inhibition of soluble guanylyl cyclase by small molecules targeting the catalytic domain

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