Binding of neuronal nitric-oxide synthase (nNOS) to carboxyl-terminal-binding protein (CtBP) changes the localization of CtBP from the nucleus to the cytosol: a novel function for targeting by the PDZ domain of nNOS

Recent work suggests a role for PDZ domains in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, we performed affinity chromatography of brain extract with the nNOS PDZ domain. We identified the carboxyl-terminal-binding protein (CtBP), a phosphoprotein first identified as a binding partner to adenovirus E1A, as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, our data suggest a new function for nNOS as a regulator of CtBP nuclear localization.     

Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885-22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with N(G)-nitro-L-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca(2+)-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination.     

Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation

We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca²⁺/calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser⁷⁴¹ to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser⁷⁴¹ by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser⁷⁴¹ in transfected cells, resulting in 60–70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser⁷⁴¹ in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser⁷⁴¹ on nNOS.     

Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i)

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.     

Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac β-adrenergic signaling.     

B-MYB positively regulates serine-threonine kinase receptor-associated protein (STRAP) activity through direct interaction

Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-β and p53 signaling. However, the regulatory mechanism of STRAP activity is not understood. In this study, we report that B-MYB is a new STRAP-interacting protein, and that an amino-terminal DNA-binding domain and an area (amino acids 373-468) between the acidic and conserved regions of B-MYB mediate the B-MYB·STRAP interaction. Functionally, B-MYB enhances STRAP-mediated inhibition of TGF-β signaling pathways, such as apoptosis and growth inhibition, by modulating complex formation between the TGF-β receptor and SMAD3 or SMAD7. Furthermore, coexpression of B-MYB results in a dose-dependent increase in STRAP-mediated stimulation of p53-induced apoptosis and cell cycle arrest via direct interaction. Confocal microscopy showed that B-MYB prevents the normal translocation of SMAD3 in response to TGF-β1 and stimulates p53 nuclear translocation. These results suggest that B-MYB acts as a positive regulator of STRAP.     

A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase: implications for catalysis

Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP(+) reductase (FNR)-like module that contains NADPH- and FAD-binding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaM-dependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPH-dependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS.     

Modulation of hypothalamic neuronal activity through a novel G-protein-coupled estrogen membrane receptor

Estrogens are involved in the hypothalamic control of multiple homeostatic functions including reproduction, stress responses, energy metabolism, sleep cycles, temperature regulation and motivated behaviors. The actions of 17beta-estradiol (E(2)) in the brain have been attributed to the activation of estrogen receptors alpha and beta, as well as G-protein-coupled or other membrane-associated estrogen receptors. Recently, we have identified a putative membrane-associated estrogen receptor that is coupled to desensitization of GABA(B) receptors in guinea pig and mouse hypothalamic neurons including proopiomelanocortin (POMC) neurons. We have synthesized a new nonsteroidal compound, STX, which selectively targets the Galphaq-coupled phospholipase C-protein kinase C-protein kinase A pathway, and have established that STX is more potent than E(2) in mediating this desensitization in an ICI 182,780-sensitive manner in both guinea pig and mouse neurons. Both E(2) and STX are fully efficacious in estrogen receptor alpha, beta knock-out mice. Finally, we observed that the putative membrane-associated estrogen receptor is different from GPR30 in arcuate neurons using whole-cell patch recording in hypothalamic slices from GPR30 knock-out mice. Collectively, these findings suggest that the mER is distinct from ERalpha, ERbeta or GPR30.     

SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

Characterization of mouse nNOS2, a natural variant of neuronal nitric-oxide synthase produced in the central nervous system by selective alternative splicing.

Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no L-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either L-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction.     

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.     

Neuronal nitric oxide synthase (nNOS) modulates the JNK1 activity through redox mechanism: a cGMP independent pathway

Nitric oxide (NO) is a small, uncharged molecule, which is primarily generated by the nitric oxide synthase (NOS) family of proteins, including neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO has been implicated in diverse roles in biological systems, such as the regulation of cell death and survival signaling pathways of a variety of cell types, including neuronal cells. In this study, we determined that the NO generated from l-arginine by ectopically overexpressed nNOS in HEK293 cells exerted an inhibitory effect against the activity of c-Jun N-terminal kinase (JNK), an important modulator of neuronal cell death and survival signaling pathways. NO repressed the activation of JNK, but exerted no significant effects on the activities of SEK1/MKK4 and MEKK1, which are the upstream MAPKK and MAPKKK of JNK1, respectively. This NO-mediated inhibition of JNK1 was not affected by the addition of ODQ, a guanylyl cyclase inhibitor, indicating that the effect is independent of the level of cyclic GMP. In an in vitro kinase assay, SNAP, a NO donor, was shown to directly suppress JNK1 activity, thereby indicating that NO is a direct modulator of JNK1. Moreover, the NO-mediated suppression of JNK1 was demonstrated to be redox-sensitive and dependent on the cysteine-116 in JNK1. Finally, according to the results of an immunohistochemical study using rat striatal neurons, we were able to determine that nNOS-expressing neurons evidenced significantly reduced JNK1 activation. Collectively, these data suggest that JNK1 is regulated by nNOS-mediated NO production in neurons, via a thiol-redox-sensitive mechanism.     

The sarcolemmal calcium pump, alpha-1 syntrophin, and neuronal nitric-oxide synthase are parts of a macromolecular protein complex

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of signal transduction pathways. This paper shows the physical interaction between PMCA (isoforms 1 and 4) and alpha-1 syntrophin and proposes a ternary complex of interaction between endogenous PMCA, alpha-1 syntrophin, and NOS-1 in cardiac cells. We have identified that the linker region between the pleckstrin homology 2 (PH2) and the syntrophin unique (SU) domains, corresponding to amino acids 399-447 of alpha-1 syntrophin, is crucial for interaction with PMCA1 and -4. The PH2 and the SU domains alone failed to interact with PMCA. The functionality of the interaction was demonstrated by investigating the inhibition of neuronal nitric-oxide synthase-1 (NOS-1); PMCA is a negative regulator of NOS-1-dependent NO production, and overexpression of alpha-1 syntrophin and PMCA4 resulted in strongly increased inhibition of NO production. Analysis of the expression levels of alpha-1 syntrophin protein in the heart, skeletal muscle, brain, uterus, kidney, or liver of PMCA4-/- mice, did not reveal any differences when compared with those found in the same tissues of wild-type mice. These results suggest that PMCA4 is tethered to the syntrophin complex as a regulator of NOS-1, but its absence does not cause collapse of the complex, contrary to what has been reported for other proteins within the complex, such as dystrophin. In conclusion, the present data demonstrate for the first time the localization of PMCA1b and -4b to the syntrophin.dystrophin complex in the heart and provide a specific molecular mechanism of interaction as well as functionality.     

Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability

Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O₂ responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²⁺ transients, at least in some neurons: in maco-1 mutants the O₂-sensing neuron PQR is unable to generate a Ca²⁺ response to a rise in O₂. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O₂, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²⁺ channels, also fails to disrupt Ca²⁺ responses in the PQR cell body to O₂ stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²⁺ channel α1 subunit, recapitulate the Ca²⁺ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators.     

Crucial role of Lys(423) in the electron transfer of neuronal nitric-oxide synthase.

Nitric-oxide synthase (NOS) is composed of an oxygenase domain having cytochrome P450-type heme active site and a reductase domain having FAD- and FMN-binding sites. To investigate the route of electron transfer from the reductase domain to the heme, we generated mutants at Lys(423) in the heme proximal site of neuronal NOS and examined the catalytic activities, electron transfer rates, and NADPH oxidation rates. A K423E mutant showed no NO formation activity (<0.1 nmol/min/nmol heme), in contrast with that (72 nmol/min/nmol heme) of the wild type enzyme. The electron transfer rate (0.01 min(-1)) of the K423E on addition of excess NADPH was much slower than that (>10 min(-1)) of the wild type enzyme. From the crystal structure of the oxygenase domain of endothelial NOS, Lys(423) of neuronal NOS is likely to interact with Trp(409) which lies in contact with the heme plane and with Cys(415), the axial ligand. It is also exposed to solvent and lies in the region where the heme is closest to the protein surface. Thus, it seems likely that ionic interactions between Lys(423) and the reductase domain may help to form a flavin to heme electron transfer pathway.     

p21-Activated kinase 3 (PAK3) protein regulates synaptic transmission through its interaction with the Nck2/Grb4 protein adaptor

Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.     

Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.     

Differential effect of 2-hydroxyoleic acid enantiomers on protein (sphingomyelin synthase) and lipid (membrane) targets

The complex dual mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent anti-tumor compound used in membrane lipid therapy (MLT), has yet to be fully elucidated. It has been demonstrated that 2OHOA increases the sphingomyelin (SM) cell content via SM synthase (SGMS) activation. Its presence in membranes provokes changes in the membrane lipid structure that induce the translocation of PKC to the membrane and the subsequent overexpression of CDK inhibitor proteins (e.g., p21^Cip1). In addition, 2OHOA also induces the translocation of Ras to the cytoplasm, provoking the silencing of MAPK and its related pathways. These two differential modes of action are triggered by the interactions of 2OHOA with either lipids or proteins. To investigate the molecular basis of the different interactions of 2OHOA with membrane lipids and proteins, we synthesized the R and S enantiomers of this compound. A molecular dynamics study indicated that both enantiomers interact similarly with lipid bilayers, which was further confirmed by X-ray diffraction studies. By contrast, only the S enantiomer was able to activate SMS in human glioma U118 cells. Moreover, the anti-tumor efficacy of the S enantiomer was greater than that of the R enantiomer, as the former can act through both MLT mechanisms. The present study provides additional information on this novel therapeutic approach and on the magnitude of the therapeutic effects of type-1 and type-2 MLT approaches. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Galpha(i2,3)

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K(d) = 48.7 pm) and saturable (B(max) = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPgammaS binding by 157%. DHEA stimulated [35S]GTPgammaS binding to Galpha(i2) and Galpha(i3), but not to Galpha(i1) or Galpha(o). Pretreatment of plasma membranes with antibody to Galpha(i2) or Galpha(i3), but not to Galpha(i1), inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Galpha(i2) and Galpha(i3). These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.