Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ～150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme‐Nitric Oxide Oxygen (H‐NOX), Per‐ARNT‐Sim (PAS), coiled‐coil and cyclase domains. Here, we present the crystal structure of the α₁ PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β₁ H‐NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.     

Solution structures of the Shewanella woodyi H‐NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP‐051

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.     

Glutathione depletion switches nitric oxide neurotrophic effects to cell death in midbrain cultures: implications for Parkinson's disease

Nitric oxide (NO) exerts neurotrophic and neurotoxic effects on dopamine (DA) function in primary midbrain cultures. We investigate herein the role of glutathione (GSH) homeostasis in the neurotrophic effects of NO. Fetal midbrain cultures were pretreated with GSH synthesis inhibitor, l ‐buthionine‐(S,R)‐sulfoximine (BSO), 24 h before the addition of NO donors (diethylamine/nitric oxide‐complexed sodium and S‐nitroso‐N‐acetylpenicillamine) at doses tested previously as neurotrophic. Under these conditions, the neurotrophic effects of NO disappeared and turned on highly toxic. Reduction of GSH levels to 50% of baseline induced cell death in response to neurotrophic doses of NO. Soluble guanylate cyclase (sGC) and cyclic GMP‐dependent protein kinase (PKG) inhibitors protected from cell death for up to 10 h after NO addition; the antioxidant ascorbic acid also protected from cell death but its efficacy decreased when it was added after NO treatment (40% protection 2 h after NO addition). The pattern of cell death was characterized by an increase in chromatin condensed cells with no DNA fragmentation and with breakdown of plasmatic membrane. The inhibition of RNA and protein synthesis and of caspase activity also protected from cell death. This study shows that alterations in GSH levels change the neurotrophic effects of NO in midbrain cultures into neurotoxic. Under these conditions, NO triggers a programmed cell death with markers of both apoptosis and necrosis characterized by an early step of free radicals production followed by a late requirement for signalling on the sGC/cGMP/PKG pathway.     

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α₁β₁ has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn²⁺ but not Mg²⁺, the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α₁β₁ and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn²⁺ is a physiological sGC cofactor.     

The dimerization of PSGL‐1 is driven by the transmembrane domain via a leucine zipper motif

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is a homodimeric mucin ligand that is important to mediate the earliest adhesive event during an inflammatory response by rapidly forming and dissociating the selectin‐ligand adhesive bonds. Recent research indicates that the noncovalent associations between the PSGL‐1 transmembrane domains (TMDs) can substitute for the C320‐dependent covalent bond to mediate the dimerization of PSGL‐1. In this article, we combined TOXCAT assays and molecular dynamics (MD) simulations to probe the mechanism of PSGL‐1 dimerization. The results of TOXCAT assays and Martini coarse‐grained molecular dynamics (CG MD) simulations demonstrated that PSGL‐1 TMDs strongly dimerized in a natural membrane and a leucine zipper motif was responsible for the noncovalent dimerization of PSGL‐1 TMD since mutations of the residues that occupied a or d positions in an (abcdefg)n leucine heptad repeat motif significantly reduced the dimer activity. Furthermore, we studied the effects of the disulfide bond on the PSGL‐1 dimer using MD simulations. The disulfide bond was critical to form the leucine zipper structure, by which the disulfide bond further improved the stability of the PSGL‐1 dimer. These findings provide insights to understand the transmembrane association of PSGL‐1 that is an important structural basis for PSGL‐1 preferentially binding to P‐selectin to achieve its biochemical and biophysical functions.     

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone–rod dystrophy but not for stationary night blindness

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber''s congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca²⁺ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca²⁺ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser⁸³⁸-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.     

TNF‐α‐mediated signal transduction pathway is a major determinant of apoptosis in dilated cardiomyopathy

Although J2N-k strain of cardiomyopathic hamsters is an excellent model of dilated cardiomyopathy, the presence and mechanisms of apoptosis in the hearts of these genetically modified animals have not been investigated. This study examined the hypothesis that cardiac dysfunction and apoptosis in the cardiomyopathic hamsters were associated with tumour necrosis factor-alpha (TNF-α)-mediated signalling pathway involving the activation of some pro-apoptotic proteins and/or deactivation of some antiapoptotic proteins. Echocardiographic assessment of 31-week-old hamsters indicated an increase in the internal dimension of the left ventricle as well as decreases in the ejection fraction, fractional shortening and cardiac output without any evidence of cardiac hypertrophy. Increased level of TNF-α and apoptosis in cardiomyopathic hearts were accompanied by increased protein content for protein kinase C (PKC) -α and -ɛ isozymes as well as caspases 3 and 9. Phosphorylated protein content for p38 MAPK and NFκB was increased whereas that for Erk1/2, BAD and Bcl-2 was decreased in cardiomyopathic hearts. These results support the view that TNF-α and PKC isozymes may promote apoptosis due to the activation of p38 MAPK and deactivation of Erk1/2 pathways, and these changes may contribute toward the development of cardiac dysfunction in dilated cardiomyopathy.     

Role of PKCζ‐NADPH oxidase signaling axis in PKCα‐mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C‐ζ (PKC‐ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC‐α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ, NADPH oxidase, and PKC‐α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC‐ζ and NADPH oxidase attenuate AngII induced increase in PKC‐α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ and NADPH oxidase activities. Overall, PKCζ‐NADPH oxidase‐PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII‐mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.     

GDF‐15 is a better complimentary marker for risk stratification of arrhythmic death in non‐ischaemic,dilated cardiomyopathy than soluble ST2

Growth differentiation factor (GDF)‐15 and soluble ST2 (sST2) are established prognostic markers in acute and chronic heart failure. Assessment of these biomarkers might improve arrhythmic risk stratification of patients with non‐ischaemic, dilated cardiomyopathy (DCM) based on left ventricular ejection fraction (LVEF). We studied the prognostic value of GDF‐15 and sST2 for prediction of arrhythmic death (AD) and all‐cause mortality in patients with DCM. We prospectively enrolled 52 patients with DCM and LVEF ≤ 50%. Primary end‐points were time to AD or resuscitated cardiac arrest (RCA), and secondary end‐point was all‐cause mortality. The median follow‐up time was 7 years. A cardiac death was observed in 20 patients, where 10 patients had an AD and 2 patients had a RCA. One patient died a non‐cardiac death. GDF‐15, but not sST2, was associated with increased risk of the AD/RCA with a hazard ratio (HR) of 2.1 (95% CI = 1.1‐4.3; P = .031). GDF‐15 remained an independent predictor of AD/RCA after adjustment for LVEF with adjusted HR of 2.2 (95% CI = 1.1‐4.5; P = .028). Both GDF‐15 and sST2 were independent predictors of all‐cause mortality (adjusted HR = 2.4; 95% CI = 1.4‐4.2; P = .003 vs HR = 1.6; 95% CI = 1.05‐2.7; P = .030). In a model including GDF‐15, sST2, LVEF and NYHA functional class, only GDF‐15 was significantly associated with the secondary end‐point (adjusted HR = 2.2; 95% CI = 1.05‐5.2; P = .038). GDF‐15 is superior to sST2 in prediction of fatal arrhythmic events and all‐cause mortality in DCM. Assessment of GDF‐15 could provide additional information on top of LVEF and help identifying patients at risk of arrhythmic death.     

IGF‐1 deficiency impairs neurovascular coupling in mice: implications for cerebromicrovascular aging

Aging is associated with marked deficiency in circulating IGF‐1, which has been shown to contribute to age‐related cognitive decline. Impairment of moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of age‐related cognitive impairment. To establish the link between IGF‐1 deficiency and cerebromicrovascular impairment, neurovascular coupling mechanisms were studied in a novel mouse model of IGF‐1 deficiency (Igf1^f/f‐TBG‐Cre‐AAV8) and accelerated vascular aging. We found that IGF‐1‐deficient mice exhibit neurovascular uncoupling and show a deficit in hippocampal‐dependent spatial memory test, mimicking the aging phenotype. IGF‐1 deficiency significantly impaired cerebromicrovascular endothelial function decreasing NO mediation of neurovascular coupling. IGF‐1 deficiency also impaired glutamate‐mediated CBF responses, likely due to dysregulation of astrocytic expression of metabotropic glutamate receptors and impairing mediation of CBF responses by eicosanoid gliotransmitters. Collectively, we demonstrate that IGF‐1 deficiency promotes cerebromicrovascular dysfunction and neurovascular uncoupling mimicking the aging phenotype, which are likely to contribute to cognitive impairment.     

The GLT‐1 (EAAT2; slc1a2) glutamate transporter is essential for glutamate homeostasis in the neocortex of the mouse

Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with [1‐¹³C]glucose and [1,2‐¹³C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and ¹³C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and ¹³C labeling of metabolites, glutamate level was decreased but labeling from [1‐¹³C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2‐¹³C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex.

     

Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells

Agrin released from motor nerve terminals directs differentiation of the vertebrate neuromuscular junction (NMJ). Activity of nitric oxide synthase (NOS), guanylate cyclase (GC), and cyclic GMP-dependent protein kinase (PKG) contributes to agrin signaling in embryonic frog and chick muscle cells. Stimulation of the NO/cyclic GMP (cGMP) pathway in embryos potentiates agrin's ability to aggregate acetylcholine receptors (AChRs) at NMJs. Here we investigated the timing and mechanism of NO and cGMP action. Agrin increased NO levels in mouse C2C12 myotubes. NO donors potentiated agrin-induced AChR aggregation during the first 20 min of agrin treatment, but overnight treatment with NO donors inhibited agrin activity. Adenoviruses encoding siRNAs against each of three NOS isoforms reduced agrin activity, indicating that these isoforms all contribute to agrin signaling. Inhibitors of NOS, GC, or PKG reduced agrin-induced AChR aggregation in mouse muscle cells by ∼ 50%. However, increased activation of the GTPase Rac1, an early step in agrin signaling, was dependent on NOS activity and was mimicked by NO donors and a cGMP analog. Our results indicate that stimulation of the NO/cGMP pathway is important during the first few minutes of agrin signaling and is required for agrin-induced Rac1 activation, a key step leading to reorganization of the actin cytoskeleton and subsequent aggregation of AChRs on the surface of skeletal muscle cells.