Redox-active Proteins Research Articles

Attenuation of indomethacin-induced gastric mucosal injury by prophylactic administration of sake yeast-derived thioredoxin

BackgroundIndomethacin is one of the group of nonsteroidal anti-inflammatory drugs, which often cause gastric mucosal injury as a side effect. Infiltration and activation of inflammatory cells, production of proinflammatory cytokines and chemokines, generation of reactive oxygen species, and activation of apoptotic signaling are involved in the pathogenesis of indomethacin-induced gastric injury. We examined whether sake yeast-derived thioredoxin (a small redox-active protein with anti-oxidative activity and various redox-regulating functions) reduced indomethacin-induced gastric injury.MethodsGastric injury was produced by the intraperitoneal administration of indomethacin (40 mg/kg body weight) to C57BL/6 mice. Prior to the administration of indomethacin, the mice were offered food pellets containing non-genetically modified sake yeast-derived thioredoxin (thioredoxin 200 μg/g) for 3 days. Histological examinations, assessment of myeloperoxidase activity, and analysis of the gene expressions of proinflammatory cytokines and a chemokine (interleukin [IL]-1β, IL-6, and CXCL1) were statistically evaluated. Indomethacin cytotoxicity was determined by lactate dehydrogenase release from murine gastric epithelial GSM06 cells induced by 24-h treatment with 200 and 400 μM indomethacin after 1-h preincubation with 100 μg/ml sake yeast-derived thioredoxin.ResultsMacroscopic (edema, hemorrhage, and ulcers) and histological (necrosis, submucosal edema, neutrophil infiltration) findings induced by indomethacin were significantly reduced by pretreatment with food pellets containing thioredoxin. Gastric myeloperoxidase activity and the gene expressions of proinflammatory cytokines (IL-1β and IL-6) were also significantly reduced by this pretreatment compared with findings in the mice not pretreated with thioredoxin-containing food pellets. The administration of sake yeast-derived thioredoxin significantly reduced indomethacin-induced cytotoxicity in GSM06 cells.ConclusionsWe conclude that oral administration of sake yeast-derived thioredoxin reduces indomethacin-induced gastric injury. Sake yeast-derived thioredoxin may have therapeutic potential against indomethacin-induced gastric injury.

Surface multiheme c -type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

Identifying proteins that can form tyrosine-cysteine crosslinks

Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.

Orientation-Dependent Electron Transport in a Single Redox Protein

The redox-active protein cytochrome b(562) has been engineered to introduce pairs of thiol groups in the form of cysteine residues at specified sites. Successful STM imaging of the molecules adsorbed on a gold surface indicated that one thiol group controls the orientation of the molecule and that the protein maintains its native form under the experimental conditions. Stable protein-gold STM tip electrical contact was directly observed to form via the second free thiol group in current-voltage and current-distance measurements. Proteins with thiol contacts positioned across the protein's short axis displayed a conductance of (3.48 ± 0.05) × 10(-5)G(0). However proteins with thiol groups placed along the long axis reproducibly yielded two distinct values of (1.95 ± 0.03) × 10(-5)G(0) and (3.57 ± 0.11) × 10(-5)G(0), suggesting that the placement of the asymmetrically located haem within the protein influences electron transfer. In contrast, the unengineered wild-type cytochrome b(562) had conductance values at least 1 order of magnitude less. Here we show that an electron transfer protein engineered to bind gold surfaces can be controllably oriented and electrically contacted to metallic electrodes, a prerequisite for potential integration into electronic circuits.

Functional Regulation of an Immobilized Redox Protein on an Oriented Metal Coordinated Peptide Monolayer as an Electron Mediator

We fabricated a vertically and unidirectionally oriented metal coordinated α-helical peptide monolayer, Leu(2)Ala(Pyri)(Co(II))Leu(6)Ala(4-Pyri)(Co(II))Leu(6), by stepwise polymerization on a mixed self-assembled monolayer consisting of amino-alkanethiol, dialkyl disulfide, and ferrocenyl alkanethiol acted as a photoresponsive electron donor. Redox-active protein, nitrate reductase (NR), was fixed on the surface of the peptide monolayer. By contrast, we fixed NR on the mixed self-assembled monolayer directly. Upon photoirradiation, electron flow occurred from the excited ferrocenyl group on the substrate to the electron acceptor, NR, on the surface of the molecular layers. The activated NR on the molecular layers reduced the nitrate to nitrite. The amount of the bioelectrocatalytic product, nitrite, generated by the immobilized NR on the peptide monolayer was larger than that produced by the immobilized NR on the mixed self-assembled monolayer directly. That is to say, the NR on the peptide monolayer has been more activated rather than that on the peptide absent monolayer by photoirradiation. The effective activation of the NR on the peptide monolayer can be explained in terms of enhancement of the vectorial electron flow along the macro-dipole moment of the α-helical peptide that arranged unidirectionally. It suggested that the ordered metal coordinated α-helical peptide monolayer acted as an efficient electron mediator to achieve a communication between the electron donor and the redox-active moiety. Such a hybrid molecular system looks promising for novel nanodevices, such as nano-photoreactors.

Methionine Ligand Lability in Bacterial Monoheme Cytochromes c: An Electrochemical Study

The direct electrochemical analysis of adsorbed redox active proteins has proven to be a powerful technique in biophysical chemistry, frequently making use of the electrode material pyrolytic "edge-plane" graphite. However, many heme-bearing proteins such as cytochromes c have been also examined systematically at alkanethiol-modified gold surfaces, and previously we reported the characterization of the redox properties of a series of bacterial cytochromes c in a side-by-side comparison of carbon and gold electrode materials. In our prior findings, we reported an unanticipated, low potential (E(m) ∼ -100 mV vs SHE) redox couple that could be analogously observed when a variety of monoheme cytochromes c are adsorbed onto carbon-based electrodes. Here we demonstrate that our prior phenomological data can be understood quantitatively in the loss of the methionine ligand of the heme iron, using the cytochrome c from Hydrogenbacter thermophilum as a model system. Through the comparison of wild-type protein with M61H and M61A mutants, in direct electrochemical analyses conducted as a function of temperature and exogenous ligand concentration, we are able to show that Met-ligated cytochromes c have a propensity to lose their Met ligand at graphite surfaces, and that energetics of this process (6.3 ± 0.2 kJ/mol) is similar to the energies associated with "foldons" of known protein folding pathways.

Measuring EGSH and H 2O 2 with roGFP2-based redox probes

Redox biochemistry plays an important role in a wide range of cellular events. However, investigation of cellular redox processes is complicated by the large number of cellular redox couples, which are often not in equilibrium with one another and can vary significantly between subcellular compartments and cell types. Further, it is becoming increasingly clear that different redox systems convey different biological information; thus it makes little sense to talk of an overall “cellular redox state”. To gain a more differentiated understanding of cellular redox biology, quantitative, redox couple-specific, in vivo measurements are necessary. Unfortunately our ability to investigate specific redox couples or redox-reactive molecules with the necessary degree of spatiotemporal resolution is very limited. The development of genetically encoded redox biosensors offers a promising new way to investigate redox biology. Recently developed redox-sensitive green fluorescent proteins (roGFPs), genetically fused to redox-active proteins, allow rapid equilibration of the roGFP moiety with a specific redox couple. Two probes based on this principle are now available: Grx1-roGFP2 for the measurement of glutathione redox potential ( E GSH) and roGFP2-Orp1 for measuring changes in H 2O 2 concentration. Here we provide a detailed protocol for the use of these probes in both yeast and mammalian systems using either plate-reader- or microscopy-based measurements.

Nanosheet-based titania microspheres with hollow core-shell structure encapsulating horseradish peroxidase for a mediator-free biosensor

Nanosheet-based titania (TiO2) microspheres with a hollow core-shell structure have been synthesized and employed to immobilize horseradish peroxidase (HRP) in order to fabricate a mediator-free biosensor. The morphology and structure of the TiO2 microspheres were characterized by X-ray diffraction, scanning electron microscopy and transmission electronic microscopy. A possible growth mechanism has been proposed. Spectroscopic and electrochemical measurements revealed that the TiO2 microspheres are an immobilization support with biocompatibility for enzymes, affording good enzyme stability and bioactivity. Due to the nanosheet-based hollow core-shell structure of the TiO2 microspheres, the direct electron transfer of HRP is facilitated and the resulting biosensor displayed good performance for the detection of H2O2, with both a low detection limit of 0.05 μM and a wide linear range of 0.4–140 μM, as well as a fast response and excellent long-term stability. The nanosheet-based TiO2 microspheres with hollow core-shell structure, can be used for the efficient entrapment of other redox-active proteins and have wide potential applications in biosensors, biocatalysis, biomedical devices and bioelectronics.

Redox biochemistry of mammalian metallothioneins

Metallothionein (MT) is a generic name for certain families of structurally rather variable metal-binding proteins. While purely chemical or biological approaches failed to establish a single physiologic function for MTs in any species, a combination of chemical and biological approaches and recent progress in defining the low but significant concentrations of cytosolic free zinc(II) ions have demonstrated that mammalian MTs function in cellular zinc metabolism in specific ways that differ from conventional knowledge about any other metalloprotein. Their thiolate coordination environments make MTs redox-active zinc proteins that exist in different molecular states depending on the availability of cellular zinc and the redox poise. The zinc affinities of MTs cover a range of physiologic zinc(II) ion concentrations and are modulated. Oxidative conditions make more zinc available, while reductive conditions make less zinc available. MTs move from the cytosol to cellular compartments, are secreted from cells, and are taken up by cells. They provide cellular zinc ions in a chemically available form and participate in cellular metal muffling: the combination of physiologic buffering in the steady state and the cellular redistribution and compartmentalization of transiently elevated zinc(II) ion concentrations in the pre-steady state. Cumulative evidence indicates that MTs primarily have a redox-dependent function in zinc metabolism, rather than a zinc-dependent function in redox metabolism.

Human serum albumin–thioredoxin fusion protein with long blood retention property is effective in suppressing lung injury

Thioredoxin (Trx) is a redox-active protein with anti-inflammatory effects but with a short half life of 1 h. Genetic fusion of Trx to human serum albumin (HSA) extended its half life without causing significant loss of its biological activities. HSA–Trx caused a decrease in the number of cells in brochoalveolar lavage fluid, the wet/dry ratio and the inflammation at the respiratory tract of the ovalbumin (OVA) induced lung injury model mouse. Three intraperitoneal doses of Trx alone produced the same extent of suppression of those three detrimental effects of OVA as one intravenous dose of HSA–Trx. Inhibition experiments confirmed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) involved in the progression of the injury. HSA–Trx inhibited the production of ROS as confirmed in the EPR experiment, but lung tissue staining suggested that induced nitrogen oxide synthase (iNOS) was not suppressed by the fusion protein. Instead, the production of nitrotyrosine, 8-nitro-cGMP, and 8-hydroxy-2′-deoxyguanosine downstream to the iNOS has been inhibited. This suggested that HSA–Trx produced lung protection effect via different mechanisms from Trx alone. HSA–Trx retains the biological properties of Trx thus has great potential in treating oxidative stress related diseases.

Thioredoxin and lactoferrin differentially regulate Langerhans’ cell migration (102.4)

Abstract Epidermal LC play a critical role in the initiation and regulation of skin immune and allergic responses. LC migration, including that stimulated by allergen, is dependent upon cytokine signals, provided by tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. We have examined the ability of two human recombinant proteins with immunomodulatory properties to influence LC migration : thioredoxin (Trx), a (12kDa) redox-active protein and lactoferrin (LF), a (79.5kDa) iron-binding protein, produced in E. coli and rice, respectively. LC migration in response to chemical contact allergen (oxazolone; Ox) or to murine TNF-α or IL-1β was assessed in BALB/c mice. Mice were treated by simple topical exposure to TRX or LF 2 h prior to treatment with the migratory stimuli. LC densities were assessed in epidermal sheets by staining for MHC class II or Langerin. Topical application of Trx inhibited Ox- and TNF-α-induced migration whereas LF prevented LC mobilisation stimulated by Ox and IL-1β only. Inhibition of LC migration by TRX was selective for IL-1β-dependent stimuli (Ox and TNF-α) whereas TNF-α-dependent migration (that provoked by Ox and IL-1β) was inhibited by LF. These data suggest that TRX modulates IL-1β production/signaling whereas LF inhibits the activity of TNF-α. The bioactivity of these molecules following topical exposure shows that these proteins can access the viable epidermis and may represent an opportunity for the development of novel anti-inflammatory reagents.

Re‐evaluation of the function of the F420 dehydrogenase in electron transport of Methanosarcina mazei

Methanosarcina mazei is a methanogenic archaeon that is able to thrive on various substrates and therefore contains a variety of redox-active proteins involved in both cytoplasmic and membrane-bound electron transport. The organism possesses a complex branched respiratory chain that has the ability to utilize different electron donors. In this study, two knockout mutants of the membrane-bound F(420) dehydrogenase (ΔfpoF and ΔfpoA-O) were constructed and analyzed. They exhibited severe growth deficiencies with trimethylamine, but not with acetate, as substrates. In cell lysates of the fpo mutants, the F(420):heterodisulfide oxidoreductase activity was strongly reduced, although soluble F(420) hydrogenase was still present. This led to the conclusion that the predominant part of cellular oxidation of the reduced form of F(420) (F(420)H(2)) in Ms. mazei is performed by F(420) dehydrogenase. Enzyme assays of cytoplasmic fractions revealed that ferredoxin (Fd):F(420) oxidoreductase activity was essentially absent in the ΔfpoF mutant. Subsequently, FpoF was produced in Escherichia coli and purified for further characterization. The purified FpoF protein catalyzed the Fd:F(420) oxidoreductase reaction with high specificity (the K(M) for reduced Fd was 0.5 μM) but with low velocity (V(max) = 225 mU·mg(-1)) and was present in the Ms. mazei cytoplasm in considerable amounts. Consequently, soluble FpoF might participate in electron carrier equilibrium and facilitate survival of the Ms. mazei Δech mutant that lacks the membrane-bound Fd-oxidizing Ech hydrogenase.

Blood Aging, Safety, and Transfusion: Capturing the “Radical” Menace

Throughout their life span, circulating red blood cells (RBCs) transport oxygen (O(2)) primarily from the lungs to tissues and return with carbon dioxide (CO(2)) from respiring tissues for final elimination by lungs. This simplistic view of RBCs as O(2) transporter has changed in recent years as other gases, for example, nitric oxide (NO), and small molecules, such as adenosine triphosphate (ATP), have been shown to either be produced and/or carried by RBCs to perform other signaling and O(2) sensing functions. In spite of the numerous biochemical and metabolic changes occurring within RBCs during storage, prior to, and after transfusion, perturbations of RBC membrane are likely to affect blood flow in the microcirculation. Subsequent hemolysis due to storage conditions and/or hemolytic disorders may have some pathophysiological consequences as a result of the release of Hb. In this review, we show that evolution has provided a multitude of protection and intervention strategies against free Hb from "cradle" to "death"; from early biosynthesis to its final degradation and a lot more in between. Furthermore, some of the same naturally occurring protective mechanisms can potentially be employed to oxidatively inactivate this redox active protein and control its damaging side reactions when released outside of the RBC.

ChemInform Abstract: Synthesis and Functionalization of Chalcogenide Nanotubes

AbstractReview: 184 refs.

Treatments with sodium selenate or doxycycline offset diabetes-induced perturbations of thioredoxin-1 levels and antioxidant capacity

Diabetes is associated with increased oxidative stress and impaired antioxidant defenses. Thioredoxin-1 (TRX-1) is a cytosolic thiol antioxidant and redox-active protein which plays a vital role in the maintenance of reduced intracellular redox state. In this study, the authors examined whether 4-week treatments with sodium selenate and doxycycline--a metalloproteinase-2 inhibitor which also has antioxidant-like effects--offset perturbations in oxidative stress and antioxidant protection in rat liver and skeletal muscle in streptozotocin-induced diabetes (SID) model. Experimental diabetes decreased TRX-1 levels in skeletal muscle and liver. On the other hand, SID increased oxidative stress marker protein carbonyl levels and decreased oxygen radical absorbance capacity (ORAC), an indicator of antioxidant capacity, in liver. A 4-week treatment of sodium selenate to diabetic rats decreased blood glucose levels moderately, while doxycycline treatment caused a reduction in weight loss of diabetic rats. Both doxycycline and sodium selenate prevented diabetes-induced decrease of TRX-1 levels in skeletal muscle, whereas only doxyxycline was effectively preventing diabetes-induced decrease of TRX-1 in liver. Furthermore, both treatments prevented diabetes-induced altered levels of protein carbonyls and ORAC in liver, and restored free and total protein thiol levels in both skeletal muscle and liver. In conclusion, the data of this study provides further evidence that sodium selenate and doxycycline treatments may control oxidative stress and improve antioxidant defense in diabetes.

Strategies for “wiring” redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.

How the xap Locus Put Electrical “Zap” in Geobacter sulfurreducens Biofilms

Investigation of microbial mineral respiration remains an experimental challenge. In this issue of Journal of Bacteriology, Rollefson et al. (11) present a foundational study on the functionality of the biofilm matrix in Geobacter sulfurreducens, a model dissimilatory metal respiring bacterium (DMRB). In this study, the investigators identify an extracellular polysaccharide scaffold or network that entraps redox-active proteins, thus positioning these proteins for optimal electron transfer from the membrane-bound respiratory supercomplexes to a mineral phase electron acceptor. The distinguishing feature of this study is the perspective, in that the team examined specifically exopolysaccharide formation and how it enables entrapment and tethering of redox proteins in the vicinity of the cell. Previous studies on Geobacter (10) and Shewanella (4) have focused primarily on the presence and functionality of conductive pili and nanowires, proteinaceous structures that also enable and enhance extracellular electron transfer. Rollefson et al. remind investigators in this field that many microbial systems have redundancy in essential functions, and in the case of DMRB, it is clearly critical that more than one mechanism exists to ensure vectoral electron transport to mineral phase electron acceptors. The major findings of Rollefson et al. (11) were (i) identification of a biofilm locus in G. sulfurreducens that harbors exopolysaccharide synthesis and export genes; (ii) detection of c-type cytochromes in exopolysaccharide materials; (iii) genetic mutation of a gene (xapD [GSU1501], an ATP-dependent ABC transporter) in the locus which results in reduced functionality of the biofilm matrix, i.e., reduced agglutination and attachment, and less matrix-bound cytochrome; and (iv) microscopic confirmation of exopolysaccharide materials in the biofilm matrix. The research employed a combination of traditional microbiological techniques, genetic manipulation and mutation, and fluorescence and electron microscopy to obtain an integrated view of biofilm matrix composition and functionality in G. sulfurreducens. The elegance of the study is in its relative simplicity. For example, while agglutination has been examined in bacteria for decades, basic assessment of this phenomenon has not been made in many DMRB models, thus showing that classical microbiology still can reveal a great deal about DMRB biofilms. For example, Fig. 2 and 3 of the Rollefson et al. paper clearly reveal both macroscopic and microscopic differences in culture and biofilm morphology. Differential extraction procedures purify pili and exopolysaccharide and demonstrate binding of redox proteins and positioning of an extracellular biomolecular conductive network, and although one can argue the efficacy and resolution of these methods, the results seem to suggest that protein-exopolysaccharide complexes can be successfully isolated from biofilm cultures, and these approaches can be reliably used on other model DMRB systems. Unique experiments using polysaccharide binding stains coupled with scanning electron microscopy (SEM) reveal that exopolysaccharide is a key component for attachment and colonization of mineral and electrode surfaces. Indeed, the exopolysaccharide appears as a network of strands wiring cells together and to the substrate. Blocking techniques with safranin O work to strengthen the interpretation of the SEM images by better visualization of the extracellular matrix material.

Redox Regulation of Soluble Epoxide Hydrolase by 15-Deoxy-Δ-Prostaglandin J 2 Controls Coronary Hypoxic Vasodilation

15-Deoxy-Δ-prostaglandin (15d-PG)J(2) is an electrophilic oxidant that dilates the coronary vasculature. This lipid can adduct to redox active protein thiols to induce oxidative posttranslational modifications that modulate protein and tissue function. To investigate the role of oxidative protein modifications in 15d-PGJ(2)-mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. Proteomic screening with biotinylated 15d-PGJ(2) identified novel vascular targets to which it adducts, most notably soluble epoxide hydrolase (sEH). 15d-PGJ(2) inhibited sEH by specifically adducting to a highly conserved thiol (Cys521) adjacent to the catalytic center of the hydrolase. Indeed a Cys521Ser sEH "redox-dead" mutant was resistant to 15d-PGJ(2)-induced hydrolase inhibition. 15d-PGJ(2) dilated coronary vessels and a role for hydrolase inhibition was supported by 2 structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ(2) and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids consistent with their vasodilatory actions. Indeed 14,15-EET alone induced relaxation and 15d-PGJ(2)-mediated vasodilation was blocked by the EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Additionally, the coronary vasculature of sEH-null mice was basally dilated compared to wild-type controls and failed to vasodilate in response to 15d-PGJ(2). Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ(2) adduction to and inhibition of sEH. Consistent with the importance of hydrolase inhibition, sEH-null mice failed to vasodilate during hypoxia. This represents a new paradigm for the regulation of sEH by an endogenous lipid, which is integral to the fundamental physiological response of coronary hypoxic vasodilation.

The Synucleins Are a Family of Redox-Active Copper Binding Proteins

Thermodynamic studies in conjunction with EPR confirm that α-synuclein, β-synuclein, and γ-synuclein bind copper(II) in a high affinity 1:1 stoichiometry. γ-Synuclein demonstrates the highest affinity, in the picomolar range, while α-synuclein and β-synuclein both bind copper(II) with nanomolar affinity. The copper center on all three proteins demonstrates reversible or partly reversible redox cycling. Various mutations show that the primary coordinating ligand for copper(II) is located within the N-terminal regions between residues 2-9. There is also a contribution from the C-terminus in conjunction with the histidine at position 50 in α-synuclein and position 65 in β-synuclein, although these regions appear to have little effect on overall coordination stability. These histidines and the C-terminus, however, appear to be critical to the redox engine of the proteins.

Intracellular free zinc during cardiac excitation–contraction cycle: calcium and redox dependencies

Zinc exists in biological systems as bound and histochemically reactive free Zn(2+). It is an essential structural constituent of many proteins, including enzymes from cellular signalling pathways, in which it functions as a signalling molecule. In cardiomyocytes at rest, Zn(2+) concentration is in the nanomolar range. Very little is known about precise mechanisms controlling the intracellular distribution of Zn(2+) and its variations during cardiac function. Live-cell detection of intracellular Zn(2+) has become feasible through the recent development of Zn(2+)-sensitive and -selective fluorophores able to distinguish Zn(2+) from Ca(2+). Here, in freshly isolated rat cardiomyocytes, we investigated the rapid changes in Zn(2+) homeostasis using the Zn(2+)-specific fluorescent dye, FluoZin-3, in comparison to Ca(2+)-dependent fluo-3 fluorescence. Zn(2+) sparks and Zn(2+) transients, in quiescent and electrically stimulated cardiomyocytes, respectively, were visualized in a similar manner to known rapid Ca(2+) changes. Both Zn(2+) sparks and Zn(2+) transients required Ca(2+) entry. Inhibiting the sarcoplasmic reticulum Ca(2+) release or increasing the Ca(2+) load in a low-Na(+) solution suppressed or increased Zn(2+) movements, respectively. Mitochondrial inhibitors slightly reduced both Zn(2+) sparks and Zn(2+) transients. Oxidation by H₂O₂ facilitated and acidic pH inhibited the Ca(2+)-dependent Zn(2+) release. It is proposed that Zn(2+) release during the cardiac cycle results mostly from intracellular free Ca(2+) increase, triggering production of reactive oxygen species that induce changes in metal-binding properties of metallothioneins and other redox-active proteins, aside from ionic exchange on these proteins.