sGC Subunits Research Articles

Nitric Oxide Receptor Soluble Guanylyl Cyclase Undergoes Splicing Regulation in Differentiating Human Embryonic Cells

Nitric oxide (NO), an important mediator molecule in mammalian physiology, initiates a number of signaling mechanisms by activating the enzyme soluble guanylyl cyclase (sGC). Recently, a new role for NO/cyclic guanosine monophosphate signaling in embryonic development and cell differentiation has emerged. The changes in expression of NO synthase isoforms and various sGC subunits has been demonstrated during human and mouse embryonic stem (ES) cells differentiation. Previously, our laboratory demonstrated that nascent α1 sGC transcript undergoes alternative splicing and that expression of α1 sGC splice forms directly affects sGC activity. Expression of sGC splice variants in the process of human ES (hES) cells differentiation has not been investigated. In this report, we demonstrate that α1 sGC undergoes alternative splicing during random hES differentiation for the first time. Our results indicate that C-α1 sGC splice form is expressed at high levels in differentiating cells and its intracellular distribution varies from canonical α1 sGC subunit. Together, our data suggest that alternative splicing of sGC subunits is associated with differentiation of hES cells.

Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.

Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

BackgroundTo examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC β1 and α subunits.Methodology/Principal FindingsCyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged α subunits with the carboxy-terminally tagged β1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the α subunits is close to the carboxy-terminus of the β1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO.Conclusions/SignificanceBased on the ability of an amino-terminal fragment of the β1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (αcatβcat), Winger and Marletta (Biochemistry 2005, 44:4083–90) have proposed a direct interaction of the amino-terminal region of β1 with the catalytic domains. In support of such a concept of “trans” regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed “fluorescent-conjoined” sGC's by fusion of the α amino-terminus to the β1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of alpha1beta1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain beta1(1-385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (beta1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat beta1 distal heme pocket (Ile-145 --> Tyr and Ile-149 --> Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe(II)-O(2) complexes and is likely the first observation of a Fe(II)-O(2) complex in the full-length alpha1beta1 protein. Additionally, these data suggest that atypical sGCs stabilize O(2) binding by a hydrogen bonding network involving tyrosine and glutamine.

Crystal structure of the signaling helix coiled-coil domain of the β1 subunit of the soluble guanylyl cyclase

BackgroundThe soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner.ResultsTo investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGCβ1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 Å resolution.ConclusionsThe CC structure of sGCβ1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

Nitric oxide sensitive guanylyl cyclase activity decreases during cerebral postnatal development because of a reduction in heterodimerization

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co-precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.

Role of soluble guanylyl cyclase–cyclic GMP signaling in tumor cell proliferation

Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO–cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO-receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGC α 1, robust levels of sGC β 1 were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1 H-pyrazolo[3,4- b]pyridine); sGC activator), NOC-18 + BAY41-2272, IBMX (3-isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC-18 + BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO–sGC–cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms.

Atypical guanylyl cyclase from the pond snail Lymnaea stagnalis: cloning, sequence analysis and characterization of expression

Soluble guanylyl cyclases (sGCs) are traditionally recognized as the main molecular receptor for nitric oxide (NO), a gaseous transmitter involved in many functions of the nervous system. Some sGCs are however insensitive to NO and therefore are known as atypical. Although atypical sGCs have been shown to exist in both vertebrate and invertebrate nervous systems, our understanding of their functional role is incomplete. Here we report on the cloning, sequencing and localization of an atypical sGC named Lym-sGCβ3 from the snail Lymnaea stagnalis. We found that Lym-sGCβ3 shares a number of structural characteristics with some previously characterized atypical sGCs including the presence of Tyr140 in the regulatory domain. This residue is thought to be of a critical importance in determining sensitivity of atypical sGCs to oxygen. These findings raise the possibility that Lym-sGCβ3 is an oxygen receptor. The results of our in situ hybridization and RT-PCR experiments support this idea further by showing that Lym-sGCβ3 is expressed in the osphradium, a peripheral sense organ in which oxygen-sensing neurons are located. Also of interest are our observations that many neurons in Lymnaea CNS co-express conventional and atypical sGC subunits. These data are consistent with a possible dominant negative regulatory role of atypical sGC subunits through the formation of heterodimers exhibiting low enzymatic activity.

Reproductive toxicity of acrylamide-treated male rats

Acrylamide content is elevated in fried, baked and heat-processed starchy foods. The present experiment was conducted to investigate the reproductive toxicity of oral acrylamide in male rats. Thirty weaned SD male rats of 21-day-old were randomly allotted to three groups, and acrylamide was administered to each group at doses of 0, 5 and 10 mg/kg-d for 8 consecutive weeks. The results indicated that the growth of rats treated with acrylamide was retarded (P<0.05), but relative weights of testes and epididymides compared to body weight were not significantly different (P>0.05). Our results also indicate that the epididymal sperm reserves decreased significantly (P<0.05), suggesting partial depletion of germ cells. In addition, histopathologic lesions were also present in the testes of treated rats. Furthermore, distinct expression patterns of sGC heterodimers were observed in this animal model. This may suggest different physiologic roles for sGC subunits in spermiogenesis and steroidogenesis.

Regulation of soluble guanylyl cyclase by phosphorylation

Results In vitro kinase assays revealed that the α1, but not the β1, subunit of sGC is a PKG substrate and that the phosphorylation site is located within the first 360 residues of the α1. A constitutively active form of PKG stimulated incorporation of 32P into the α1 subunit in vivo. In addition, PKG could be detected in sGC immunoprecipitates, suggesting that the two proteins interact in cells. Serine to alanine mutation of putative PKG sites revealed that Ser64 is the main phosphorylation site for PKG. After generating a phospho-specific antibody for Ser64, we could demonstrate that phosphorylation of endogenous sGC on Ser64 increases in cells and tissues exposed to NO. Phosphorylation of sGC in cells following SNP stimulation was blocked by the peptide inhibitor of PKG, DT-3. Wild-type (wt) sGC when co-expressed in COS cells with a constitutively active form of PKG exhibited lower basal and NOstimulated cGMP accumulation. In contrast, the S64A α1/ β1 sGC was resistant to the PKG-induced reduction in activity. Moreover, co-cultures of endothelial cells with smooth muscle expressing a phosphorylation-resistant form of sGC, exhibited higher levels of cGMP (both basally and following stimulation with bradykinin) compared to co-cultures expressing wild-type sGC. Phosphorylation of the α1 subunit did not alter its ability to form heterodimers with β1. Using purified sGC and mutants, we observed that the S64D α1 phosphomimetic/β1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D α1/β1 compared to wt sGC. Expression of a phosphorylation deficient form of sGC showed reduced desensitization to acute NO exposure and allowed for greater VASP phosphorylation.

Direct evidence for close proximity of catalytic and regulatory domains of heterodimeric sGC based on fluorescence resonance energy transfer

Methods The FRET donor, CFP, and FRET acceptor, YFP, were fused to aminoand carboxy-terminal ends of sGC subunits. After generation of recombinant baculovirus strains fluorescent tagged sGC subunits were co-expressed in Sf9cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. In addition, fluorescent tagged sGC subunits were analyzed in vivo using confocal laser scanning microscopy and fluorescence lifetime imaging (FLIM) on an inverted microscope.

Lack of correlation between sGC subunit expression and sGC activity in cerebral development is due to non-heterodimerizing subunits in vivo

Background A number of studies have shown that the nitric oxide (NO)/cGMP signalling pathway plays a major role in neuronal cell differentiation and in the central nervous system during development, but much less is known about the expression and regulation of the different subunits of NO sensitive guanylyl cyclase (sGC) in the developing brain. In the present study, we have analysed the regulation and expression of sGC in brain of rats during postnatal development using biochemical methods.

I036 Activation of soluble guanylate cyclase by BAY 58-2667 decreases systolic blood pressure and proteinuria in spontaneous hypertensive stroke-prone rats (SHR-SP)

In SHR-SP, oxidative stress participates to altered relaxation in response to acetylcholine (ACh) and sodium-nitroprusside (SNP). The aim of this study was to evaluate the effects of the activator of soluble guanylate cyclase (sGC) BAY 58-2667 on SHR-SP in comparison to control Wistar-Kyoto rats (WKY). Aortas from 15 week-old SHRSP and WKY were removed and isometric tension as well as cGMP generation were determined in response to ACh, SNP and BAY 58-2667. After contraction of aortic rings by phenylephrine (10-6 M), the relaxations of SHR-SP rings were smaller in response to ACh (60 % vs. 90 % maximal relaxation, n=15), to SNP (EC50 : 6.4 vs. 0.9 nM, n=20) and to BAY 58-2667 (EC50 : 95 vs. 9 nM, n=6). In contrast, cGMP generation (in the presence of the PDE inhibitor IBMX, 2 mM) caused by 10-8M BAY 58-2667 was not significantly altered in SHR-SP vs. WKY (34¡Ó11 vs. 64¡Ó23 pmol/mg protein, ns t-test, n=7) whereas that caused by 10-5M ACh and 10-6M SNP were significantly smaller (−90 % and −60 % vs. WKY, respectively, p,,T0.05, t-test, n=9). Moreover, the amount of (fÑf¡fyfÒf¡)-sGC subunits, quantified by western-blot, was decreased by 35 % in SHR-SP aorta (n=12). Interestingly, an oral chronic treatment of rats with 1 mg/kg/day of BAY 58-2667 for 5 days (mini-pump Alzet, IP, n=11-13) decreased SHR-SP systolic blood pressure by 15¡Ó4 mmHg, proteinuria by 50¡Ó5 % and was associated with a aortic and urinary cGMP increase (20¡Ó2 vs. 9¡Ó1.3 pmol/mg proteins and 11¡Ó1 vs. 7.6¡Ó1.4 nmol/24h, respectively, t-test). These results demonstrate that BAY 58-2667 activates sGC in SHRSP aorta, even if the enzyme amount is decreased and suggest the presence of oxidized and/or heme free form of sGC unresponsive to NO in SHR-SP. In conclusion, activators of sGC may constitute an alternative treatment of resistant hypertension associated to vascular complications.

Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands

17beta-estradiol (E2) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing alpha1- and decreasing beta1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E2 is the main effector of these changes. Since the sGC alpha-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC alpha-subunit are expressed. Here we show for the first time that sGCalpha2 and sGCalpha2 inhibitory (alpha2i) isoforms are expressed in this gland, but only sGCalpha2i mRNA increased after E2 acute treatment. Finally, to test whether E2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGCalpha1, -beta1, and -alpha2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland.

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni 2+-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α ( M r ∼77,000) and β ( M r ∼70,000) sGC subunits. It showed an A 430/A 280 = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k cat/ K m = 1.7 × 10 −3 s −1 μM −1 that increased to 5.8 × 10 −1 s −1 μM −1 upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.

Characterization of Soluble Guanylate Cyclase in NO-Induced Increases in Aqueous Humor Outflow Facility and in the Trabecular Meshwork

Nitric oxide (NO) increases the rate at which aqueous humor exits the eye; however, the involvement of soluble guanylate cyclase (sGC) is unknown. This study investigated the role of sGC in mediating the NO-induced increases in outflow facility. Outflow facility was measured in porcine eyes using the anterior segment organ culture perfusion system. sGC activity was assessed by cGMP production in low-passage porcine and human trabecular meshwork (TM) cells and transformed human TM cells, as measured by enzyme immunoassay. sGCalpha and sGCbeta isoform expression were determined using Western blot analysis. Activation of sGC is necessary for the NO-induced increases in outflow facility (0.3215 microL/min per mm Hg [baseline outflow facility]+/-0.0837 [SEM]). NO resulted in increased sGC activity that was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ). Western blot analysis of total protein demonstrated an equivalent ratio of sGCalpha and sGCbeta subunit expression. In transformed cell fractions, however, the level of cytoplasmic sGCalpha subunit expression was decreased compared with low-passage human TM cells. Activation of sGC is involved in the NO-induced increases in outflow facility. The expression of alpha and beta sGC subunits in an equivalent ratio would suggest a functional sGC heterodimer because DETA-NO increased cGMP levels in low-passage human and porcine TM cells. However, the inability of DETA-NO to cause increases in cGMP levels in transformed TM cells suggests that though the sGC heterodimer is necessary, it is not sufficient and may require other factors not present in transformed cells.

High yield purification of soluble guanylate cyclase from bovine lung

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), is a cytosolic, heme-containing, heterodimeric enzyme that catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3,5′-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) in the presence of Mg 2+. Cyclic GMP is then involved in transmitting the NO activating signals to a variety of downstream effectors such as cyclic-nucleotide-gated channels, protein kinases, and phosphodiesterases. In this work, sGC has been purified from bovine lung. The lungs were subjected to grinding and extraction with buffer at physiological pH followed by centrifugation. The resulting solution was subjected to successive column chromatography on DEAE- and Q-Sepharose, Ceramic Hydroxyapatite, Resource Q, and GTP–agarose. The purified enzyme migrated as a two-band protein on SDS–PAGE corresponding to sGC subunits α ( M r = 77,532) and β ( M r = 70,500) and had an A 280nm/ A 430nm of ∼1 indicating one heme per heterodimer. The yield of enzyme was 8–10 mg from 4 to 5 kg bovine lungs. V max and K m of non-stimulated sGC were 22 nmol/mg/min and 180 μM, respectively. Upon stimulation with the NO donor 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, the V max increased to 1330 nmol/mg/min while the K m dropped to 43 μM. The quality and quantity of enzyme make it suitable for studies to probe the structure and catalytic mechanism of this enzyme and for research related to drug discovery.

PAS-mediated Dimerization of Soluble Guanylyl Cyclase Revealed by Signal Transduction Histidine Kinase Domain Crystal Structure

Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) because it is often associated with the heme-nitric oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 A resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCbeta1 H-NOXA homodimerization, heterodimerization of full-length heterodimeric sGC was mostly unaffected, likely due to the additional dimerization contacts of sGC in the coiled-coil and catalytic domains. Exceptions are mutations sGCalpha1 F285A and sGCbeta1 F217A, which each caused a drastic drop in NO stimulated activity, and mutations sGCalpha1 Q368A and sGCbeta1 Q309A, which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture.

Heat shock protein 90 regulates stabilization rather than activation of soluble guanylate cyclase

Endothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminished possibly involving the sGC binding chaperone, heat shock protein 90 (hsp90). Here we show that prolonged hsp90 inhibition in different cell types reduces protein levels of both sGC subunits by about half, an effect that was prevented by the proteasome inhibitor MG132. Conversely, acute hsp90 inhibition affected neither basal nor NO-stimulated sGC activity. Thus, hsp90 is a molecular stabilizer for sGC tonically preventing proteasomal degradation rather than having a role in short-term activity regulation.

The Cyclic GMP-Dependent Pathway Is Involved in the Mechanisms of Action of Hydroxyurea.

Hydroxyurea (HU) is the standard therapeutic agent for patients with sickle cell disease (SCD). Administration of HU reduces the frequency of pain crisis, increases total hemoglobin levels, and decreases the numbers of reticulocytes, leukocytes, and platelets. However, the mechanism underlying these ameliorating effects of HU on the clinical expression of SCD patients still remains to be clarified. Several lines of evidence that were obtained from our laboratory and others suggest that HU exerts its molecular effects through the cyclic GMP (cGMP)-dependent pathway. To examine whether the cGMP-dependent pathway is involved in the molecular actions of HU, we generated mice that overexpress soluble guanylate cyclase (sGC), a key enzymes of the cGMP-dependent pathway and have an activated cGMP-dependent pathway in peripheral blood cells. Adult mice expressed the transgenes encoding sGC subunits in bone marrow cells, spleen cells and peripheral leukocytes. In these cells of transgenic sGC mice, intracellular cGMP levels were elevated and the phosphorylation levels of vasodilator-stimulated phosphoprotein, which is a substrate of protein kinase G of the cGMP-dependent pathway, were increased. These results demonstrated activation of the cGMP-dependent pathway in the sGC mice. Interestingly, high-level expression of sGC subunit transgenes resulted in down-regulation of endogenous mouse sGC mRNA expression, suggesting that mRNAs encoding sGC subunits are unstable in cells which have high cGMP levels. More interestingly, adult sGC mice had elevated levels of total hemoglobin, and decreased numbers of white blood cell counts, which is consistent with a clinical picture of SCD patients who are administered with HU. In vitro colony assays demonstrated that cGMP has positive and negative effects on the differentiation of erythroid- and myeloid-lineage cells, respectively. Furthermore, in sickle cell patients treated with HU, intracellular cGMP levels of red blood cells positively correlated with total hemoglobin levels, while leukocyte counts were inversely correlated with the cGMP levels of leukocytes. These results indicate the roles of the cGMP-dependent pathway at least in part in regulating blood cell counts in sickle cell patients. We previously reported that expression of the human gamma-globin gene is induced by activating the cGMP-dependent pathway (PNAS 98: 1847, 2001). Next, we examined whether expression of human gamma-globin, which is dormant in adult erythroid cells, is induced in sGC mice. We bred sGC mice with a YAC mouse carrying the entire human beta-globin gene locus. Although human gamma-globin was not detectable by HPLC in sGC/YAC mice, low but appreciable levels of human gamma-globin expression was induced following the activation of the cGMP-dependent pathway with protoporphyrin IX, which is an activator of sGC. This demonstrated a role of the pathway in inducing gamma-globin expression in adult erythroid cells. Collectively, these results shown in this study demonstrate that sGC mice exhibit hematologic characteristics that are similar to those of SCD patients on HU therapy, suggesting that the cGMP-dependent pathway is involved at least in part in the mechanisms of action of HU.