Guanylyl cyclase sensitivity to nitric oxide is protected by a thiol oxidation-driven interaction with thioredoxin-1

Abstract

Nitric oxide (NO) modulates many physiological events through production of cGMP from its receptor, the NO-sensitive guanylyl cyclase (GC1). NO also appears to function in a cGMP-independent manner, via S-nitrosation (SNO), a redox-based modification of cysteine thiols. Previously, we have shown that S-nitrosated GC1 (SNO-GC1) is desensitized to NO stimulation following prolonged NO exposure or under oxidative/nitrosative stress. In animal models of nitrate tolerance and angiotensin II-induced hypertension, decreased vasodilation in response to NO correlates with GC1 thiol oxidation, but the physiological mechanism that resensitizes GC1 to NO and restores basal activity is unknown. Because GC1 interacts with the oxidoreductase protein-disulfide isomerase, we hypothesized that thioredoxin-1 (Trx1), a cytosolic oxidoreductase, could be involved in restoring GC1 basal activity and NO sensitivity because the Trx/thioredoxin reductase (TrxR) system maintains thiol redox homeostasis. Here, by manipulating activity and levels of the Trx1/TrxR system and by using a Trx1-Trap assay, we demonstrate that Trx1 modulates cGMP synthesis through an association between Trx1 and GC1 via a mixed disulfide. A proximity ligation assay confirmed the endogenous Trx1-GC1 complex in cells. Mutational analysis suggested that Cys⁶⁰⁹ in GC1 is involved in the Trx1-GC1 association and modulation of GC1 activity. Functionally, we established that Trx1 protects GC1 from S-nitrosocysteine-induced desensitization. A computational model of Trx1-GC1 interaction illustrates a possible mechanism for Trx1 to maintain basal GC1 activity and prevent/rescue GC1 desensitization to NO. The etiology of some oxidative vascular diseases may very well be explained by the dysfunction of the Trx1-GC1 association.

Keywords: S-nitrosylation; cyclic GMP (cGMP); guanylate cyclase (guanylyl cyclase); nitric oxide; oxidation-reduction (redox); thioredoxin.

Figure 1.

Trx1 enhances GC1 activity. COS-7 cells were transfected with plasmids overexpressing GC1-α and -β subunits with or without FLAG-Trx1^WT. The cytosolic fractions of COS-7 cells were assayed for GC1 activity under basal and NO-stimulated conditions. SNAP was used as an NO donor at 100 μm and added to the reaction mix. The NO-stimulated GC1 activity in the presence of Trx1^WT overexpression was compared with the GC1 activity without Trx1^WT expression. #, p < 0.05, by unpaired Student's t test; *, p < 0.05, two-way ANOVA with Tukey's post hoc test; n = 5. Each data point corresponds to two separate transfected wells, pulled together for lysis and with their activity measured in duplicate. Right, cell lysates were analyzed by immunoblots to assess the expression of FLAG-Trx1^WT, GC1, and β-actin, a loading control. Each sample contained 15 μg of total protein.

Figure 2.

Trx1-mediated regulation of GC activity is redox-dependent. A, NO-stimulated and basal (inset) GC1 activities were assayed in the lysate of A7r5 cells infected with GC1-α- and GC1-β-expressing adenoviruses, pretreated with CSNO (100 μm, 30 min) or buffer, and then treated or not with DNCB (50 μm, 30 min). Diethylamine (DEA)-NO was added to the reaction mixture and used at 10 μm. #, p = 0.07; *, p < 0.05; ***, p < 0.001 versus untreated GC1. n ≥ 4. One-way ANOVA with Tukey's post hoc test was used. Each data point corresponds to activity measurements done in duplicate of two infected dishes pulled together for lysis. B, SNO content of GC1-α and -β subunits were resolved by Western blotting following the biotin switch assay. Lysates prepared from cells were subjected to the same CSNO treatment with or without DNCB as in A. Band intensity indicated the extent of protein S-nitrosation. α-Tubulin was used for a loading control. The input blots indicate that a similar amount of protein was used for analyses. Ethanol and phosphate buffer are the solvent controls for DNCB and CSNO treatment, respectively. Error bars, S.D.

Figure 3.

Interaction of GC1 with Trx1 in situ. A, Duolink in situ PLA using anti-GC1-α, anti-GC1-β, and anti-Trx1 antibodies in rat NCM (which are amenable to PLA and express detectable levels of endogenous GC1). Interaction between molecules is indicated by a red positive reaction. No reaction was detected in the negative control, in which no anti-GC1-α or anti-GC1-β antibodies were added. Nuclei were stained with DAPI in blue. Differential interference contrast (DIC) was used to visualize cell shape. Magnification was a ×40 objective using a Zeiss 200 microscope. B, quantification of PLA reactions from fluorescent microscopy of GC1-α/Trx1 and GC1-β/Trx1 indicates that Trx1 strongly interacts with GC1-α subunit. CSNO treatment significantly enhanced the interaction between GC1-α and Trx1. ***, p < 0.001; two-way ANOVA with Tukey's post hoc test was used; n = 8–10. Error bars, S.D.

Figure 4.

GC1 interacts with the active site of Trx1 (³²CXXC³⁵). A, COS-7 cells were transfected with GC1 and Trx1^WT, Trx1^C35S (Trap mutant), or Trx1^DM (Trx1^C32S/C35S). The interaction between GC1 and Trx1 was resolved by electrophoresis under reducing conditions and by Western blotting (IB) following immunoprecipitation with anti-FLAG. Input, starting material; β-actin is a loading control. This blot is representative of three independent experiments. Fifteen micrograms of total protein was used for input of each sample. M.W., molecular weight. B, quantification of immunoblot densitometry is shown. **, p < 0.01; one-way ANOVA with Bonferroni's post hoc test was used. n = 3. The β1 signal is not detectable in the IP elute unless overexposed and as such was not included in the statistical analysis. C, cytosolic fractions of COS-7 cells, co-transfected with plasmids encoding GC1-α, GC1-β, FLAG-Trx1, or active site mutants, were assayed for GC1 activity in the presence of SNAP (10 μm). GC1 activity was measured as in Fig. 2A. *, p < 0.05; ***, p < 0.001; one-way ANOVA with Bonferroni's post hoc test was used; n = 5. Values are mean ± S.D. (error bars).

Figure 5.

αCys⁶⁰⁹ of GC1 is involved in Trx1-mediated modulation and interaction. A, GC1 activity under basal and NO-stimulated conditions was measured in COS-7 cells co-transfected with GC1-α^C609Sβ with or without Trx1, as described under “Experimental procedures” (10 μm SNAP was added to the reaction mixture for NO-stimulated conditions). Two-way ANOVA with Tukey's post hoc test was used; n = 4. Values are expressed as mean ± S.D. (error bars). N.S., not significant. B, COS-7 cells were transfected with GC1 or GC1-α^C609S and Trx1^WT, Trx1^C35S, or Trx1^DM. The cytosol was prepared and immunoprecipitated with anti-FLAG antibodies as described under “Experimental Procedures.” A Western blot probed with anti-FLAG antibodies under non-reducing conditions shows that GC1–Trx1^C35S was pulled down with anti-FLAG as a slightly more than 150-kDa complex (as expected), whereas a GC1-α^C609S–Trx1^C35S complex could not be detected. No or little complex could be detected with Trx1^WT or Trx1^C32S/C35S. Input of each sample contained 15 μg of total protein. M.W., molecular weight. A Western blot under reducing conditions is shown in supplemental Fig. S3.

Figure 6.

Trx1 protects GC1 from CSNO-induced desensitization. COS-7 cells were co-transfected with plasmids encoding GC1 or GC1-α^C609S and with or without FLAG-Trx1. Cells were incubated with 200 μm CSNO for 1 h in the dark prior to lysis. NO-stimulated activity (10 μm SNAP) of lysates containing GC1 or GC1-α^C609S (inset) was measured as above. **, p < 0.01; two-way ANOVA with Bonferroni's post hoc test was used. Values are mean ± S.D. (error bars); n = 3–4.

Figure 7.

Proposed docking models between Trx1 and the catalytic domain of GC1. The GC1-α subunit is shown in red, and the GC1-β subunit is in cyan. Predicted interaction regions (as proposed in Ref. 22), residues 573–601 and 610–654, are depicted in magenta, and 602–609 is in pink. Trx1 is shown in dark gray. A, Cys³² and Cys³⁵ of Trx1 active site are indicated, as are Cys⁶⁰⁹ and Cys⁶⁵³ in the α subunit. B, surface rendering of the model. Of note, the docking model predicts a distance of 9.8 Å between αCys⁶⁵³ and αCys⁶⁰⁹ of GC1, offering an opportunity of disulfide bonding upon conformational changes.