Nitric Oxide Binding Research Articles

Nitric Oxide Binding Geometry in Heme-Proteins: Relevance for Signal Transduction.

Nitric oxide (NO) synthesis, signaling, and scavenging is associated to relevant physiological and pathological events. In all tissues and organs, NO levels and related functions are regulated at different levels, with heme proteins playing pivotal roles. Here, we focus on the structural changes related to the different binding modes of NO to heme-Fe(II), as well as the modulatory effects of this diatomic messenger on heme-protein functions. Specifically, the ability of heme proteins to bind NO at either the distal or proximal side of the heme and the transient interchanging of the binding site is reported. This sheds light on the regulation of O2 supply to tissues with high metabolic activity, such as the retina, where a precise regulation of blood flow is necessary to meet the demand of nutrients.

Systemic treatment with ubiquitin carboxy terminal hydrolase L1 TAT protein ameliorates axonal injury and reduces functional deficits after traumatic brain injury in mice

Traumatic brain injury (TBI) is often associated with axonal injury that leads to significant motor and cognitive deficits. Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in neurons and loss of its activity plays an important role in the pathogenesis of TBI. Fusion protein was constructed containing wild type (WT) UCHL1 and the HIV trans-activator of transcription capsid protein transduction domain (TAT-UCHL1) that facilitates transport of the protein into neurons after systemic administration. Additional mutant proteins bearing cysteine to alanine UCHL1 mutations at cysteine 152 (C152A TAT-UCHL1) that prevents nitric oxide and reactive lipid binding of C152, and at cysteine 220 (C220A TAT-UCHL1) that inhibits farnesylation of the C220 site were also constructed. WT, C152A, and C220A TAT-UCHL1 proteins administered to mice systemically after controlled cortical impact (CCI) were detectable in brain at 1 h, 4 h and 24 h after CCI by immunoblot. Mice treated with C152A or WT TAT-UCHL1 decreased axonal injury detected by NF200 immunohistochemistry 24 h after CCI, but C220A TAT-UCHL1 treatment had no significant effect. Further study indicated that WT TAT-UCHL1 treatment administered 24 h after CCI alleviated axonal injury as detected by SMI32 immunoreactivity 7 d after CCI, improved motor and cognitive deficits, reduced accumulation of total and K48-linked poly-Ub proteins, and attenuated the increase of the autophagy marker Beclin-1. These results suggest that UCHL1 activity contributes to the pathogenesis of white matter injury, and that restoration of UCHL1 activity by systemic treatment with WT TAT-UCHL1 after CCI may improve motor and cognitive deficits. These results also suggest that farnesylation of the C220 site may be required for the protective effects of UCHL1.

Anemia Outside the Box: Nonimmune Intramedullary Hemolytic Anemia Driven By Vitamin B12 Deficiency

Introduction: Vitamin B12 and folate deficiencies are common etiologies of macrocytic anemia, mostly encountered with chronic alcohol use, and uncommon for healthy individuals with unremarkable medical history. The pathogenesis of the resulting anemia is described by two possible mechanisms: either ineffective erythropoiesis, driven by vitamin B12 deficiency, or more rarely, via nonimmune intramedullary hemolysis. We present a very rare case of severe vitamin B12 deficiency leading to nonimmune intramedullary hemolysis and highlight the diagnostic evaluation and tailored management. Case Presentation: A 49-year-old female with history of depression, chronic alcohol use and chronic nitrous oxide inhalation, presented with lethargy, hypotension and fevers with visual hallucinations, and was subsequently admitted for acute encephalopathy. Findings on physical examination included diminished proprioception and diminished sensation to fine touch, pinprick and vibration of the lower extremities with distal hyperreflexia of the patellar and Achilles tendon reflexes bilaterally. Lab work was notable for leukopenia and macrocytic anemia with a significant drop in hemoglobin to 9.6 from a baseline of 13.2 within 5 months and MCV of 103. Extensive investigation of encephalopathy was undertaken, of which we note infectious workup, prompted by the septic presentation, which showed Staphylococcus aureus bacteremia. On the other hand, the patient's anemia was concurrently investigated. Hemolysis workup was positive (LDH 394 U/L; direct bilirubin 1.6 mg/dL; haptoglobin<8 mg/dL; reticulocyte count 6.1%); however, schistocytes were low on peripheral smear (Figures 1 and 2). As such, nonimmune intramedullary hemolysis was suspected. Further anemia workup revealed severe vitamin B12 deficiency (75 ng/L), consistent with the physical exam findings, but normal folate levels. Of note, methylmalonic acid levels and homocysteine were also elevated (respectively 4.39 nmol/mL, and 142 umol/L). The constellation of the clinical presentation and all the laboratory findings confirmed a nonimmune intramedullary hemolytic process, elicited by vitamin B12 deficiency. This deficiency was thought to be multifactorial, mainly caused by chronic alcohol abuse, in addition to persistent nitrous oxide use. It was also suggested that hemolysis could be a consequence of drug-induced carboxyhemoglobinemia (level was 9.4%), whereby carboxyhemoglobin can form during the binding and release of nitric oxide from hemoglobin. Treatment was focused on replacing vitamin B12 with 1000 mcg daily intramuscular injections for 7 days, then weekly thereafter for 4 weeks, followed by monthly injections. Improvement of hemoglobin to 11.1 with improvement of neurologic exam findings were noted prior to discharge. Conclusion: Nonimmune hemolytic anemia must be included in the differential diagnosis when evaluating patients with anemia concurrent with vitamin B12 deficiency, mostly encountered with chronic alcohol use. Vitamin B12 supplementation remains the key treatment to correct this rare, but serious outcome of vitamin B12 deficiency.

Expression and function of Four AAV-based constructs for dystrophin restoration in the mdx mouse model of duchenne muscular dystrophy.

Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared efficacy of the following four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e., reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.

Nitric oxide binding to ferrous nitrobindins: A computer simulation investigation

Nitrobindins (Nbs) represent an evolutionary conserved all-β-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum–classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed.

Low Fouling Marine Coatings Based on Nitric Oxide-Releasing Polysaccharide-Based Hybrid Materials

Diethylenetriamine-modified heparin and alginates as well as unmodified polysaccharides were combined using sol–gel chemistry to construct hybrid material thin-film coatings. Due to the high density of amine groups, the hybrid material coatings are capable of binding nitric oxide when exposed to NO at elevated pressures, which were characterized by UV–Vis and ATR-FTIR spectroscopy. When exposed to aqueous environments, the hybrid material coatings showed nitrogen oxide release rates between 17 and 30 pmol·cm–2·s–1. Marine antifouling properties were tested against the marine bacterium Cobetia marina and the diatom Navicula perminuta. The diatom assays revealed a lower settlement on the nitric oxide-loaded hybrid materials compared to hybrid materials without nitrogen oxide release. All coatings readily suppressed attachment of the marine bacterium C. marina. The advantages of hybrid materials, composed purely of polysaccharides and siloxanes, are that their building blocks are environmentally friendly, biodegradable, and biocompatible. This makes them interesting candidates for environmentally benign marine protective coating applications. In particular, the binding of nitric oxide and release of nitrogen oxide species as naturally occurring signaling molecules were found to be promising mechanisms to add additional fouling inhibiting functionality to deter marine organisms from settlement.

Dental Pulp Inflammation Initiates the Occurrence of Mast Cells Expressing the α1 and β1 Subunits of Soluble Guanylyl Cyclase.

The binding of nitric oxide (NO) to heme in the β1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1β1 and α2β1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and β1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and β1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the β1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations.

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity > 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.

Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects.

SUMMARYNuclear protein high-mobility group box 1 (HMGB1) can be actively secreted by activated immune cells and functions as a proinflammatory cytokine. Regulation of HMGB1 secretion is critical for treatment of HMGB1-mediated inflammation and related diseases. This study demonstrates that S-nitrosylation (SNO; the covalent binding of nitric oxide [NO] to cysteine thiols) by inducible nitric oxide synthase (iNOS)-derived NO at Cys106 is essential and sufficient for inflammation-elicited HMGB1 secretion. iNOS deletion or inhibition or Cys106Ser mutation prevents lipopolysaccharide (LPS)- and/or poly(I:C)-elicited HMGB1 secretion. NO donors induce SNO of HMGB1 and reproduce inflammogen-triggered HMGB1 secretion. SNO of HMGB1 promotes its proinflammatory and neurodegenerative effects. Intranigral HMGB1 injection induces chronic microglial activation, dopaminergic neurodegeneration, and locomotor deficits, the key features of Parkinson’s disease (PD), in wild-type, but not Mac1 (CD11b/CD18)-deficient, mice. This study indicates pivotal roles for SNO modification in HMGB1 secretion and HMGB1-Mac1 interaction for inflammatory neurodegeneration, identifying a mechanistic basis for PD development.

Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyzes formation of the β-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O2)-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O2 to the IPNS–Fe(II)–ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed 19F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O2 analog nitric oxide, using the less mobile α6 for comparison. 19F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron–electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.

A Short Review of New Drugs for Heart Failure: Omecamtiv Mecarbil and Vericiguat

Heart failure is associated with increased morbidity and mortality. Although developments of therapeutic agents have shown benefit and have improved the patients’ condition, there is only a slight decreased of mortality rate throughout the years. This condition becomes a reason to develop efficient drugs which can further improve the condition and reducing the mortality significantly. New therapeutic agents have been proposed and studied. Among them is cardiac myosin activator, such as omecamtiv mecarbil. This drug will increase the binding of myosin head to the actin and therefore form a stronger contraction. Omecamtiv mecarbil is beneficial due to its mechanism which not related to higher oxygen consumption and less adverse events. Another promising drug is vericiguat, a soluble guanylate cyclase (sGC) stimulator. This drug enhances the binding of nitric oxide to sGC and therefore increasing cyclic guanosine monophosphate (cGMP) production. cGMP important in regulating physiological processed in the cardiovascular system. In this review, we will discuss about the current evidence about these novel agents.
 Keywords: heart failure, novel agent, omecamtiv mecarbil, vericiguat, management

Experimental and Computational Insight into the Mechanism of NO Binding to Ferric Microperoxidase. The Likely Role of Tautomerization to Account for the pH Dependence.

According to the current paradigm, the metal-hydroxo bond in a six-coordinate porphyrin complex is believed to be significantly less reactive in ligand substitution than the analogous metal-aqua bond, due to a much higher strength of the former bond. Here, we report kinetic studies for nitric oxide (NO) binding to a heme-protein model, acetylated microperoxidase-11 (AcMP-11), that challenge this paradigm. In the studied pH range 7.4-12.6, ferric AcMP-11 exists in three acid-base forms, assigned in the literature as [(AcMP-11)FeIII(H2O)(HisH)] (1), [(AcMP-11)FeIII(OH)(HisH)] (2), and [(AcMP-11)FeIII(OH)(His-)] (3). From the pH dependence of the second-order rate constant for NO binding (kon), we determined individual rate constants characterizing forms 1-3, revealing only a ca. 10-fold decrease in the NO binding rate on going from 1 (kon(1) = 3.8 × 106 M-1 s-1) to 2 (kon(2) = 4.0 × 105 M-1 s-1) and the inertness of 3. These findings lead to the abandonment of the dissociatively activated mechanism, in which the reaction rate can be directly correlated with the Fe-OH bond energy, as the mechanistic explanation for the process with regard to 2. The reactivity of 2 is accounted for through the existence of a tautomeric equilibrium between the major [(AcMP-11)FeIII(OH)(HisH)] (2a) and minor [(AcMP-11)FeIII(H2O)(His-)] (2b) species, of which the second one is assigned as the NO binding target due to its labile Fe-OH2 bond. The proposed mechanism is further substantiated by quantum-chemical calculations, which confirmed both the significant labilization of the Fe-OH2 bond in the [(AcMP-11)FeIII(H2O)(His-)] tautomer and the feasibility of the tautomer formation, especially after introducing empirical corrections to the computed relative acidities of the H2O and HisH ligands based on the experimental pKa values. It is shown that the "effective lability" of the axial ligand (OH-/H2O) in 2 may be comparable to the lability of the H2O ligand in 1.

Resonance Raman studies of gas sensing heme proteins

AbstractHeme sensor proteins are present in organisms ranging from bacteria to mammals, typically consisting of a catalytic domain and a heme‐containing sensor domain, which can bind diatomic gas molecules such as O2, CO, and NO. Such ligand binding event can induce active site conformational changes that are subsequently propagated to the functional domain, ultimately activating/deactivating a series of biological events, such as aerotaxis, gene expression, changes the metabolic pathway, and so forth. Such regulatory events are crucial for the cell to fulfill the physiological functions by regulating enzyme activity in response to the external stimuli. Resonance Raman (rR) spectroscopy is an effective method to investigate the sensing mechanism of these structurally different associated sensor proteins that exhibit diverse inherent physiological functions. This review summarizes the results of rR studies on a diverse collection of these heme sensor proteins, including FixL, Escherichia coli direct oxygen sensor (EcDOS), HemAT from Bacillus subtilis (HemAT‐Bs), CooA, soluble guanylyl cyclase (sGC), heme nitric oxide and/or oxygen binding domain (H‐NOX), and nitric oxide sensing proteins (NosP).

The Clinical-Stage sGC Stimulator IW-1701 Prevents Increase of Plasma Biomarkers of Intravascular Inflammation and Suppresses Leukocyte-Endothelial Interactions in Tnfα-Treated Mice

Soluble guanylate cyclase (sGC) is a heme containing signaling enzyme that catalyzes the formation of cyclic guanosine 3‘,5‘-monophosphate (cGMP) from GTP in response to nitric oxide (NO) binding to heme. The second messenger cGMP modulates many physiological processes including inflammation. Treatment with NO donors or inhibitors of cGMP-selective phosphodiesterase 9 (PDE9) significantly attenuates TNFα-induced intravascular inflammation in C57BL/6 and sickle cell mice. The anti-inflammatory effect of PDE9 inhibitors was augmented by co-administration of hydroxyurea (HU), which is the current standard of care for sickle cell disease. It has been proposed that HU may exert its anti-inflammatory effect by acting as an NO donor. sGC stimulators are small molecules that bind to sGC and enhance NO-dependent catalytic activity of the enzyme. Here we evaluated the effect of the sGC stimulator IW-1701 in the absence and presence of HU on biomarkers of intravascular inflammation, leukocyte-endothelial cell interactions and neutrophil trafficking in C57BL/6 mice.Treatment of C57BL/6 mice with TNFα (50 ng/mouse, i.p.) increased the plasma levels of biomarkers of endothelial cell activation (sP-selectin, sE-selectin, sICAM-1) and leukocyte activation (MIP-2, sL-selectin). Pretreatment of mice with IW-1701 (10 mg/kg, p.o.) reduced the increase in plasma concentrations of MIP-2 and sL-selectin following the TNFα challenge by 81% and 58%, respectively. The TNFα-induced increase in plasma levels of sP-selectin, sE-selectin and sICAM-1 were also inhibited by 31%, 37% and 34%, respectively in IW-1701-treated mice. Co-treatment with HU augmented the effect of IW-1701 on plasma concentrations of sE-selectin and sICAM-1 in TNFα treated mice.The effects of IW-1701, HU and the combination on leukocyte endothelial cell interactions was assessed using intravital microscopy of post-capillary venules of the mouse cremaster muscle. TNFα treatment of C57BL/6 mice decreased leukocyte velocity from 26.6±3.1 μm/sec to 5.5±0.7 μm/sec and leukocyte rolling flux from 43.3±7.1 cells/min to 16.1±2.7 cells/min as compared to control mice. Pre-treatment of mice with IW-1701 (10 mg/kg) alone increased leukocyte velocity and rolling flux to 10.3±1.1 μm/sec and 24.1±2.3 cells/min, respectively. Similarly, pre-treatment of mice with HU (100 mg/kg, p.o.) alone increased leukocyte velocity to 15.5±1.7 μm/sec and leukocyte rolling flux to 50.2±5.9 cells/min. Co-administration of IW-1701 in combination with HU further increased leukocyte velocity (19.7±1.9 μm/sec) and leukocyte rolling flux (64.5±5.5 cells/min) in post-capillary venules of the mouse cremaster muscle. Treatment with either IW-1701, HU, or the combination did not change the number of adherent leukocytes to the vascular wall following TNFα stimulation. To further evaluate the effect of IW-1701 on neutrophil extravasation and trafficking we used a peritoneal recruitment model. Intraperitoneal injection of TNFα induced accumulation of Gr.1 positive neutrophils in the mouse peritoneal cavity (4±0.4×105 PMNs/peritoneal lavage). Trafficking of PMNs into mouse peritoneum was attenuated by treatment of mice with HU (2.7±0.5×105 PMNs/peritoneal lavage). Co-administration of HU and IW-1701 further decreased the number of neutrophils extravasated into the peritoneum of TNFα-challenged mice (1.4±0.3×105 PMNs/peritoneal lavage).In summary, prophylactic treatment with IW-1701 inhibited the TNFα-induced increase in plasma levels of biomarkers of intravascular inflammation and attenuated the effect of TNFα on transient adhesive interactions between leukocytes and endothelial cells in mice. Co-administration of HU significantly reduced neutrophil trafficking and augmented the effect of IW-1701 on biomarkers of endothelial cell activation and transient adhesive interactions in the mouse cremaster muscle. This study supports further evaluation of the sGC stimulator IW-1701 to reduce complications associated with intravascular inflammation such as vaso-occlusive crisis in patients with sickle cell disease. DisclosuresTchernychev:Ironwood Pharmaceuticals: Employment, Other: own stock options. Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Germano:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Warren:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Lonie:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hadcock:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Graul:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals .

Employing an Unnatural Amino Acid to Examine Local Environments in Multiple Protein Systems

Unnatural amino acids (UAAs) expand the landscape of protein biochemistry, providing new tools to examine and tune protein structure and function. Using the amber codon suppression technology these UAAs can be site-specifically incorporated into proteins. The UAA 4-cyano-L-phenylalanine (pCNF) contains the nitrile functional group whose nitrile stretching frequency is located both in a quiet region of protein IR spectra and is sensitive to local environment. Our work using pCNF site-specifically incorporated at various locations in both the gas-sensing Heme Nitric Oxide and/or Oxygen (H-NOX) binding protein from Caldanaerobacter subterraneus and the super folder Green Fluorescent Protein (sfGFP) will be discussed.

Abstract P093: Cytochrome B5 Reductase 3 And Xanthine Oxidase Coordinately Regulate Soluble Guanylyl Cyclase Physiological Redox State

In cardiovascular disease, oxidative stress can drive soluble guanylyl cyclase (sGC) heme oxidation resulting in the loss of the sGC heme (apo-sGC), the impairment of nitric oxide (NO) binding and cGMP production, and vasoconstriction. Consequently, a new class of therapeutic compounds sGC activators have been developed which target oxidized and apo-sGC to cause irreversible, NO-independent reactivation of cGMP production and vasodilation. While sGC activators have had varied clinical success, surprisingly few studies have defined the impact of NO-independent sGC activation on vascular physiology in healthy conditions. We found mesenteric and pulmonary arteries are two log orders more sensitive to NO-independent sGC activator BAY 58-2667 induced vasodilation than aorta; no difference in NO-dependent sGC vasodilation between vessels was observed. These data indicate the presence of an activatable physiological pool of oxidized and/or apo-sGC in pulmonary and mesenteric arteries. We recently published that smooth muscle cell cytochrome b5 reductase 3 (CYB5R3) acts to reduce oxidized heme sGC back to its NO-sensitive reduced heme state during vascular disease. We found transgenic CYB5R3 overexpression (CYB5R3 OE) mice were more resistant to BAY 58-2667 mesenteric artery vasodilation and blood pressure lowering compared to wild-type controls (n=5-9) under physiologic conditions. Also, healthy CYB5R3 OE pulmonary arteries had a near complete loss of BAY 58-2667 vasodilation suggesting both mesenteric and pulmonary arteries contain a pool of oxidized sGC. We next asked if physiological H 2 O 2 production accounts for changes in BAY 58-2667 responsiveness. We found using mitochondrial-specific catalase overexpression mice, that BAY 58-2667 vasodilation did not differ from controls in any vascular bed (n=4-6). We next tested whether xanthine oxidase (XO), which can produce H 2 O 2 at the endothelial cell surface of vessels, can impact physiological BAY 58-2667 vasodilation. We found that Febuxostat, a XO inhibitor, led to a significant decrease in mesenteric artery BAY 58-2667 induced vasodilation from ~70% to ~30% (n=6). Combined, these data provide evidence for CYB5R3 and XO as regulators of physiological sGC resistance artery vasodilation.

High-throughput single-cell live imaging of photobiomodulation with multispectral near-infrared lasers in cultured T cells.

.Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology.Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light.Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature.Results: A specific combination of wavelengths at low irradiances (250 to for 1064 nm and 55 to for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes.Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.

Structural insights into the mechanism of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is the primary sensor of nitric oxide. It has a central role in nitric oxide signalling and has been implicated in many essential physiological processes and disease conditions. The binding of nitric oxide boosts the enzymatic activity of sGC. However, the mechanism by which nitric oxide activates the enzyme is unclear. Here we report the cryo-electron microscopy structures of the human sGCα1β1 heterodimer in different functional states. These structures revealed that the transducer module bridges the nitric oxide sensor module and the catalytic module. Binding of nitric oxide to the β1 haem-nitric oxide and oxygen binding (H-NOX) domain triggers the structural rearrangement of the sensor module and a conformational switch of the transducer module from bending to straightening. The resulting movement of the N termini of the catalytic domains drives structural changes within the catalytic module, which in turn boost the enzymatic activity of sGC.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic THP-1 cells.

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

Pharmacological Characterization of IW-1973, a Novel Soluble Guanylate Cyclase Stimulator with Extensive Tissue Distribution, Antihypertensive, Anti-Inflammatory, and Antifibrotic Effects in Preclinical Models of Disease

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5'-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1-10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.