Irina G. Gazaryan

Zinc Is a Potent Inhibitor of Thiol Oxidoreductase Activity and Stimulates Reactive Oxygen Species Production by Lipoamide Dehydrogenase

Submicromolar zinc inhibits α-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the α-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A.  J.  L. (2000) J. Biol. Chem. 275, 13441–13447). Lipoamide dehydrogenase, a component of the α-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD+. This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD+. The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn2+(K i ∼0.15 μm) in both directions. Steady-state kinetic studies revealed that Zn2+ competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn2+ with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn2+ accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 ± 0.02 μm. Activation is a consequence of Zn2+binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn2+ on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn2+ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Zinc Irreversibly Damages Major Enzymes of Energy Production and Antioxidant Defense Prior to Mitochondrial Permeability Transition

Recent observations point to the role played by Zn2+ as an inducer of neuronal death. Two Zn2+ targets have been identified that result in inhibition of mitochondrial respiration: the bc1 center and, more recently, α-ketoglutarate dehydrogenase. Zn2+ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn2+ is imported through the Ca2+ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2+ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2+ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2+ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 μm Zn2+. An additional, unidentified import route functions at higher Zn2+ concentrations. (g) No enzyme inactivation is observed for Ca2+-induced MPT. These observations strongly suggest that, unlike Ca2+, exogenous Zn2+ interferes with mitochondrial NADH production and directly alters redox protection in the matrix, contributing to mitochondrial dysfunction. Inactivation of these enzymes by Zn2+ is irreversible, and thus only their de novo synthesis can restore function, which may underlie persistent loss of oxidative carbohydrate metabolism following transient ischemia.

Targeting Nrf2-Mediated Gene Transcription by Extremely Potent Synthetic Triterpenoids Attenuate Dopaminergic Neurotoxicity in the MPTP Mouse Model of Parkinson's Disease

UNLABELLED Although the etiology of Parkinsons disease (PD) remains unclear, ample empirical evidence suggests that oxidative stress is a major player in the development of PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that upregulates a battery of antioxidant response element (ARE)-driven antioxidative and cytoprotective genes that defend against oxidative stress. AIMS We evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with small molecule synthetic triterpenoids (TP) attenuate MPTP-induced PD in mice. RESULTS We show that synthetic TP are thus far the most potent and direct activators of the Nrf2 pathway using a novel Neh2-luciferase reporter. They upregulate several cytoprotective genes, including those involved in glutathione biosynthesis in vitro. Oral administration of TP that were structurally modified to penetrate the brain-induced messenger RNA and protein levels for a battery of Nrf2-dependent cytoprotective genes reduced MPTP-induced oxidative stress and inflammation, and ameliorated dopaminergic neurotoxicity in mice. The neuroprotective effect of these TP against MPTP neurotoxicity was dependent on Nrf2, since treatment with TP in Nrf2 knockout mice failed to block against MPTP neurotoxicity and induce Nrf2-dependent cytoprotective genes. INNOVATION Extremely potent synthetic TP that are direct activators of the Nrf2 pathway block dopaminergic neurodegeneration in the MPTP mouse model of PD. CONCLUSION Our results indicate that activation of Nrf2/antioxidant response element (ARE) signaling by synthetic TP is directly associated with their neuroprotective effects against MPTP neurotoxicity and suggest that targeting the Nrf2/ARE pathway is a promising approach for therapeutic intervention in PD.

Neurotoxic lupus autoantibodies alter brain function through two distinct mechanisms

Damaging interactions between antibodies and brain antigenic targets may be responsible for an expanding range of neurological disorders. In the case of systemic lupus erythematosus (SLE), patients generate autoantibodies (AAbs) that frequently bind dsDNA. Although some symptoms of SLE may arise from direct reactivity to dsDNA, much of the AAb-mediated damage originates from cross-reactivity with other antigens. We have studied lupus AAbs that bind dsDNA and cross-react with the NR2A and NR2B subunits of the NMDA receptor (NMDAR). In adult mouse models, when the blood–brain barrier is compromised, these NMDAR-reactive AAbs access the brain and elicit neuronal death with ensuing cognitive dysfunction and emotional disturbance. The cellular mechanisms that underlie these deleterious effects remain incompletely understood. Here, we show that, at low concentration, the NMDAR-reactive AAbs are positive modulators of receptor function that increase the size of NMDAR-mediated excitatory postsynaptic potentials, whereas at high concentration, the AAbs promote excitotoxicity through enhanced mitochondrial permeability transition. Other synaptic receptors are completely unaffected by the AAbs. NMDAR activation is required for producing both the synaptic and the mitochondrial effects. Our study thus reveals the mechanisms by which NMDAR-reactive AAbs trigger graded cellular alterations, which are likely to be responsible for the transient and permanent neuropsychiatric symptoms observed in patients with SLE. Our study also provides a model in which local AAb concentration determines the exact nature of the cellular response.

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway.

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H2O2) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H2O2 in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H2O2 in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H2O2 production in astrocytes cocultured with neurons, we heterologously expressed the H2O2-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H2O2. Seven hours of low-level H2O2 production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H2O2, suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H2O2 yet H2O2-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H2O2-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H2O2 concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.

Harnessing hypoxic adaptation to prevent, treat, and repair stroke

The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics.

Direct electron transfer catalysed by recombinant forms of horseradish peroxidase: insight into the mechanism

Abstract The paper presents the first results on recombinant horseradish peroxidase (HRP) electrochemistry obtained on graphite with a rotating disk electrode system. Recombinant HRP demonstrates a higher percentage of properly oriented molecules than the native enzyme. The first important conclusion based on the recombinant HRP electrochemistry is that glycosylation hinders direct electron transfer (ET). The single-point mutants with limited activity toward phenolic substrates, viz. Asn70Val and Asn70Asp showed no changes in the registered current upon the addition of p -cresol, catechol, p -aminophenol and guaiacol and, thus, in this particular case mediated ET was not more advantageous than direct ET. The rate constants for direct ET were comparable for all mutants tested in this study demonstrating that direct ET does not depend on the enzymes ability or inability to oxidise phenolic substrates. The results obtained in this study demonstrate the true mediatorless nature of enzyme-catalysed direct ET.

Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found--nordihydroguaiaretic acid, fisetin, and gedunin--induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

Comparison of rotating disk and wall-jet electrode systems for studying the kinetics of direct and mediated electron transfer for horseradish peroxidase on a graphite electrode

Abstract This paper presents a comparison between the results obtained for the kinetics of direct and mediated electroreduction of hydrogen peroxide at horseradish peroxidase (HRP) modified graphite electrodes, used either as a rotating disk electrode (RDE) or as the working electrode in a wall-jet flow through cell. The advantages of using a wall-jet flow system compared with a RDE system for kinetic investigations are that there is no need to account for substrate consumption, especially in the case of desorption of enzyme, and also when studying product-inhibited enzymes. The comparison reveals that identical results can be obtained using either technique. The ratio of HRP molecules on the graphite surface in direct electron transfer contact was determined as 48±4%, and the turnover number of the heterogeneous electron transfer between adsorbed HRP and the graphite electrode was determined as 1.9±0.3 s −1 .