Emil Martin

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation

Hydrogen sulfide (H2S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H2S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H2S and NO exert their effects via separate pathways, the results of the current study show that H2S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H2S increases intracellular cyclic guanosine 5′-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H2S-stimulated angiogenic response, and attenuated H2S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H2S signaling. Conversely, silencing of the H2S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H2S in the vascular activity of NO. The actions of H2S and NO converge at cGMP; though H2S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H2S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H2S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H2S are mutually required for the physiological control of vascular function.

Histone H1.2 is a substrate for denitrase, an activity that reduces nitrotyrosine immunoreactivity in proteins

Several reports have described an activity that modifies nitrotyrosine-containing proteins and their immunoreactivity to nitrotyrosine Abs. Without knowing the product of the reaction, this new activity has been called a “denitrase.” In those studies, some nonspecific proteins, which have multiple tyrosine residues, e.g., albumin, were used as a substrate. Therefore, the studies were based on an unknown mechanism of reaction and potentially a high background. To solve these problems, one of the most important things is to find a more suitable substrate for assay of the enzyme. We developed an assay strategy for determining the substrate for denitrase combining 2D-gel electrophoresis and an on-blot enzyme assay. The resulting substrate from RAW 264.7 cells was Histone H1.2, an isoform protein of linker histone. Histone H1.2 has only one tyrosine residue in the entire molecule, which ensures the exact position of the substrate to be involved. It has been reported that Histones are the most prominent nitrated proteins in cancer tissues. It was also demonstrated that tyrosine nitration of Histone H1 occurs in vivo. These findings lead us to the idea that Histone H1.2 might be an intrinsic substrate for denitrase. We nitrated recombinant and purified Histone H1.2 chemically and subjected it to an on-blot enzyme assay to characterize the activity. Denitrase activity behaved as an enzymatic activity because the reaction was time dependent and was destroyed by heat or trypsin treatment. The activity was shown to be specific for Histone H1.2, to differ from proteasome activity, and to require no additional cofactors.

Ligand Selectivity of Soluble Guanylyl Cyclase EFFECT OF THE HYDROGEN-BONDING TYROSINE IN THE DISTAL HEME POCKET ON BINDING OF OXYGEN, NITRIC OXIDE, AND CARBON MONOXIDE

Although soluble guanylyl cyclase (sGC) functions in an environment in which O2, NO, and CO are potential ligands for its heme moiety, the enzyme displays a high affinity for only its physiological ligand, NO, but has a limited affinity for CO and no affinity for O2. Recent studies of a truncated version of the sGC β1-subunit containing the heme-binding domain (Boon, E. M., Huang, S H., and Marletta, M. A. (2005) Nat. Chem. Biol., 1, 53–59) showed that introduction of the hydrogen-bonding tyrosine into the distal heme pocket changes the ligand specificity of the heme moiety and results in an oxygen-binding sGC. The hypothesis that the absence of hydrogen-bonding residues in the distal heme pocket is sufficient to provide oxygen discrimination by sGC was put forward. We tested this hypothesis in a context of a complete sGC heterodimer containing both the intact α1- and β1-subunits. We found that the I145Y substitution in the full-length β-subunit of the sGC heterodimer did not produce an oxygen-binding enzyme. However, this substitution impeded the association of NO and destabilized the NO·heme complex. The tyrosine in the distal heme pocket also impeded both the binding and dissociation of the CO ligand. We propose that the mechanism of oxygen exclusion by sGC not only involves the lack of hydrogen bonding in the distal heme pocket, but also depends on structural elements from other domains of sGC.

YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components

YC-1 [3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.

Kinetic and Cellular Characterization of Novel Inhibitors of S-Nitrosoglutathione Reductase

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of β-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-κB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.

A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation

Heterodimeric αβ soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the β subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type αβ enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant αβCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the β subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer.

Soluble guanylyl cyclase: the nitric oxide receptor.

Soluble guanylyl cyclase is recognized as the most sensitive physiologic receptor for nitric oxide. Binding of nitric oxide to the heme moiety of the cyclase induces its capacity to synthesize the second messenger cGMP. Although the changes in the state of the heme moiety upon exposure of enzyme to NO and its correlation to the stimulation of sGC catalytic activity are well documented, the exact mechanism of such coupling is not understood. Structure-functional studies are required to elucidate this process. In this chapter, we describe the method of expression and purification of recombinant human alpha1/beta1 isoform of sGC in insect cells, which can be a useful tool for such studies. Several approaches that enable characterization of the binding of NO to sGC heme moiety are also described.

Stimulation of Inducible Nitric Oxide by Hepatitis B Virus Transactivator Protein HBx Requires MTA1 Coregulator

Nitric oxide has been implicated in the pathogenesis of inflammatory disorders, including hepatitis B virus-associated hepatocellular carcinoma. Transactivator protein HBx, a major regulator of cellular responses of hepatitis B virus, is known to induce the expression of MTA1 (metastasis-associated protein 1) coregulator via NF-κB signaling in hepatic cells. However, the underlying mechanism of HBx regulation of the inducible nitric-oxide synthase (iNOS) pathway remains unknown. Here we provide evidence that MTA1 is a positive regulator of iNOS transcription and plays a mechanistic role in HBx stimulation of iNOS expression and activity. We found that the HBx-MTA1 complex is recruited onto the human iNOS promoter in an NF-κB-dependent manner. Pharmacological inhibition of the NF-κB signaling prevented the ability of HBx to stimulate the transcription, the expression, and the activity of iNOS; nevertheless, these effects could be substantially rescued by MTA1 dysregulation. We further discovered that HBx-mediated stimulation of MTA1 is paralleled by the suppression of miR-661, a member of the small noncoding RNAs, recently shown to target MTA1. We observed that miR-661 controls of MTA1 expression contributed to the expression and activity of iNOS in HBx-expressing HepG2 cells. Accordingly, depletion of MTA1 by either miR-661 or siRNA in HBx-expressing cells severely impaired the ability of HBx to modulate the endogenous levels of iNOS and nitrite production. Together, these findings reveal an inherent role of MTA1 in HBx regulation of iNOS expression and consequently its function in the liver cancer cells.

Dynamic ligand exchange in soluble guanylyl cyclase (sGC): implications for sGC regulation and desensitization.

Background: The enzyme soluble guanylyl cyclase (sGC) converts the NO signal into cGMP. Results: Stoichiometric NO forms an unstable sGC-NO complex, which is stabilized by extra NO, GTP, or substitution of βHis-107. Conclusion: Exchange of the proximal heme ligand contributes to sGC activation and desensitization. Significance: Understanding the dynamics of sGC/NO interaction and its functional consequences is necessary to understand the process of NO/cGMP signaling. Accumulating evidence indicates that the functional properties of soluble guanylyl cyclase (sGC) are affected not only by the binding of NO but also by the NO:sGC ratio and a number of cellular factors, including GTP. In this study, we monitored the time-resolved transformations of sGC and sGC-NO complexes generated with stoichiometric or excess NO in the presence and absence of GTP. We demonstrate that the initial five-coordinate sGC-NO complex is highly activated by stoichiometric NO but is unstable and transforms into a five-coordinate sGC-2 state. This sGC-2 rebinds NO to form a low activity sGC-NO complex. The stability of the initial complex is greatly enhanced by GTP binding, binding of an additional NO molecule, or substitution of βHis-107. We propose that the transient nature of the sGC-NO complex, the formation of a desensitized sGC-2 state, and its transformation into a low activity sGC-NO adduct require βHis-107. We conclude that conformational changes leading to sGC desensitization may be prevented by GTP binding to the catalytic site or by binding of an additional NO molecule to the proximal side of the heme. The implications of these observations for cellular NO/cGMP signaling and the process of rapid desensitization of sGC are discussed in the context of the proposed model of sGC/NO interactions and dynamic transformations.

Cellular signaling with nitric oxide and cyclic guanosine monophosphate

The understanding of the formation and biological actions of nitric oxide (NO) has grown extensively during the past two decades. With the discoveries of the biological effects of NO and nitrovasodilators on cyclic guanosine monophosphate, with the elucidation of the biochemical mechanisms of NO synthesis, and with the growing knowledge of regulation of NO synthases, the complexities of this signal transduction cascade and its participation in numerous cell signaling processes continues. NO can be recognized as an intracellular second messenger, a local substance for regulation of neighboring cells, a neurotransmitter, and probably a hormone acting at distant sites.