A Futile Redox Cycle Involving Neuroglobin Observed at Physiological Temperature.

Previous studies identifying the potential anti-apoptotic role of neuroglobin raise the question as to how cells might employ neuroglobin to avoid the apoptotic impact of acute hypoxia whilst also avoiding chronic enhancement of tumour formation. We show that under likely physiological conditions neuroglobin can take part in a futile redox cycle. Determination of the rate constants for each of the steps in the cycle allows us to mathematically model the steady state concentration of the active anti-apoptotic ferrous form of neuroglobin under various conditions. Under likely normal physiological conditions neuroglobin is shown to be present in the ferrous state at approximately 30% of its total cellular concentration. Under hypoxic conditions this rapidly rises to approximately 80%. Temporal analysis of this model indicates that the transition from low concentrations to high concentration of ferrous neuroglobin occurs on the seconds time scale. These findings indicate a potential control model for the anti-apoptotic activity of neuroglobin, under likely physiological conditions, whereby, in normoxic conditions, the anti-apoptotic activity of neuroglobin is maintained at a low level, whilst immediately a transition occurs to a hypoxic situation, as might arise during stroke, the anti-apoptotic activity is drastically increased. In this way the cell avoids unwanted increased oncogenic potential under normal conditions, but the rapid activation of neuroglobin provides anti-apoptotic protection in times of acute hypoxia.

An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: application to neuroserpin reveals regions that differentially regulate inhibitory activity.

The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution-matrix-based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface-exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site-directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins.

Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant-tuning receptor-dependent activation.

Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca(2+) levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.

Novel haemoglobin mutation (α127Lys→Glu) increases oxygen affinity and has a minor effect on haptoglobin binding.

OBJECTIVES: To determine if a new haemoglobin (Hb) variant was the underlying cause of erythrocytosis in a subject with a high apparent HbA(1)c. DESIGN AND METHOD: Haemolysate was analysed by ESI MS, and individual components purified by ion exchange and reverse phase chromatography. Peptide mapping was used to pinpoint the substitution and DNA sequencing to confirm the precise mutation. Oxygen affinity was measured and relative haptoglobin (Hp) binding estimated. RESULTS: Intact protein analysis and peptide mapping suggested a mutation in peptide α13 and DNA sequencing confirmed a novel α127Lys→Glu substitution in the α 2 gene. The abnormal Hb had a significantly higher O(2) affinity (5.8 mmHg) than HbA (12.4 mmHg). In addition the mutation caused a small but significant decrease in Hp binding. CONCLUSION: Molecular models show that the side chain of α127Lys stabilises the T structure of deoxy Hb and that mutation to Glu would favour conversion to the high affinity R state. Notwithstanding this and the demonstrated high affinity, there was only a small increase in RBCs, Hb concentration and PCV in other female carriers of the mutation. The absence of a significant phenotype of erythrocytosis is most probably due to the low level (19%) of the variant.

Extended survival of SH-SY5Y cells following overexpression of Lys67Glu neuroglobin is associated with stabilization of ΔψM.

Overwhelming evidence indicates that a high level of expression of the protein neuroglobin protects neurons in vitro, in animal models, and in humans, against cell death associated with hypoxic and amyloid insult. We have previously showed that neuroglobin protects neuronal cells from the mitochondrial pathway of apoptosis induced by the BH3 mimetic, by preventing cytochrome c-triggered activation of caspase 9. Here, using cell and molecular biology approaches, we generated a particular neuroglobin mutant, Lys67Glu, overexpression of which confers a significant protection from the BH3 mimetic (TW-37)-induced apoptosis in human neuroblastoma SH-SY5Y cells. The cumulative inhibition of caspase 9 activation is significantly enhanced in Lys67Glu neuroglobin-expressing cells, as compared to wild-type neuroglobin expressing cells. A multiparameter flow cytometry analysis of TW-37-treated cells revealed that inhibition of caspase 9 activity by Lys67Glu neuroglobin is associated with the preservation of the mitochondrial transmembrane potential (Δψ(M) ), as well as a decreased rate of cytochrome crelease from the mitochondria.

Does a redox cycle provide a mechanism for setting the capacity of neuroglobin to protect cells from apoptosis?

We hypothesize that the various, previously reported, reactivities of neuroglobin with redox partners and oxygen provide for the establishment of a redox cycle within cells, such as neurons and retinal rod cells. Using native cell lysates, from cultured human cells of neuronal origin, we have estimated the rate of reduction of the oxidized form of neuroglobin in vivo. Furthermore we provide evidence that the cytosol of these cells contains factors (presumably enzymes) capable of employing either glutathione or NADH as re-reductants of ferric neuroglobin. Taken in conjunction with previous rate data, for the various redox reactions of neuroglobin, this information allows us to set up a computer model to estimate the steady state cellular level of the antiapoptotic ferrous form of neuroglobin. This model indicates that the steady state level of antiapoptotic neuroglobin is very sensitive to the cellular oxygen tension and moderately sensitive to the redox status of the cell. Further analysis indicates that such a system would be capable of significant modification, on the seconds time scale, following hypoxic transition, as is likely in stroke. We hypothesize that this mechanism might provide a moderately rapid mechanism for adjusting the antiapoptotic status of a cell, whilst the reaction of neuroglobin with mitochondrial cytochrome c provides a very rapid, but limited, capacity to intervene in the apoptotic pathway.

The anti-apoptotic role of neuroglobin.

The small heme-protein neuroglobin is expressed at high concentrations in certain brain neurons and in the rod cells of the retina. This paper reviews the many studies which have recently identified a protective role for neuroglobin, in a wide range of situations involving apoptotic cell death. The origins of this protective mechanism are discussed in terms of both experimental results and computational modeling of the intrinsic pathway of apoptosis, which shows that neuroglobin can intervene in this process by a reaction with released mitochondrial cytochrome c. An integrated model, based on the various molecular actions of both neuroglobin and cytochrome c, is developed, which accounts for the cellular distribution of neuroglobin.

Nonlinear regulation of commitment to apoptosis by simultaneous inhibition of Bcl-2 and XIAP in leukemia and lymphoma cells.

Apoptosis is a complex pathway regulated by the concerted action of multiple pro- and anti-apoptotic molecules. The intrinsic (mitochondrial) pathway of apoptosis is governed up-stream of mitochondria, by the family of Bcl-2 proteins, and down-stream of mitochondria, by low-probability events, such as apoptosome formation, and by feedback circuits involving caspases and inhibitor of apoptosis proteins (IAPs), such as XIAP. All these regulatory mechanisms ensure that cells only commit to death once a threshold of damage has been reached and the anti-apoptotic reserve of the cell is overcome. As cancer cells are invariably exposed to strong intracellular and extracellular stress stimuli, they are particularly reliant on the expression of anti-apoptotic proteins. Hence, many cancer cells undergo apoptosis when exposed to agents that inhibit anti-apoptotic Bcl-2 molecules, such as BH3 mimetics, while normal cells remain relatively insensitive to single agent treatments with the same class of molecules. Targeting different proteins within the apoptotic network with combinatorial treatment approaches often achieves even greater specificity. This led us to investigate the sensitivity of leukemia and lymphoma cells to a pro-apoptotic action of a BH3 mimetic combined with a small molecule inhibitor of XIAP. Using the computational probabilistic model of the apoptotic pathway, verified by experimental results from human leukemia and lymphoma cell lines, we show that inhibition of XIAP has a non-linear effect on sensitization towards apoptosis induced by the BH3 mimetic HA14-1. This study justifies further ex vivo and animal studies on the potential of the treatment of leukemia and lymphoma with a combination of BH3 mimetics and XIAP inhibitors.

A role for human neuroglobin in apoptosis.

Over the past decade, following the discovery of the human heme protein neuroglobin, many studies have searched for evidence for this protein's mechanism of action. Much data has accrued showing that high levels of neuroglobin will protect cells from apoptotic cell death, following a wide range of challenges. Various explanations of its actions, based on measured reactivity with oxygen, nitric oxide, or free radicals, have been proposed, but none have, as yet, been substantiated in vivo. Following preliminary experiments, it was previously hypothesised that "the central role of neuroglobin in highly metabolically active cells and retinal and brain neurons is to reset the trigger level of mitochondrial cytochrome c release necessary to commit the cells to apoptosis" (I.U.M.B.M. Life (2008) 60, 398). In this article, we review the evidence, which has accumulated to support this hypothesised mechanism of action of neuroglobin and integrate this data, with other reported intracellular functions of neuroglobin, to suggest a plausible central role for neuroglobin in the control of apoptosis.

The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of ß-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes.

Misfolding of the islet ß-cell peptide hA (human amylin) into ß-sheet-containing oligomers is linked to ß-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent ß-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA, but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective ß-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

An antiapoptotic neuroprotective role for neuroglobin.

Cell death associated with mitochondrial dysfunction is common in acute neurological disorders and in neurodegenerative diseases. Neuronal apoptosis is regulated by multiple proteins, including neuroglobin, a small heme protein of ancient origin. Neuroglobin is found in high concentration in some neurons, and its high expression has been shown to promote survival of neurons in vitro and to protect brain from damage by both stroke and Alzheimer's disease in vivo. Early studies suggested this protective role might arise from the protein's capacity to bind oxygen or react with nitric oxide. Recent data, however, suggests that neither of these functions is likely to be of physiological significance. Other studies have shown that neuroglobin reacts very rapidly with cytochrome c released from mitochondria during cell death, thus interfering with the intrinsic pathway of apoptosis. Systems level computational modelling suggests that the physiological role of neuroglobin is to reset the trigger level for the post-mitochondrial execution of apoptosis. An understanding of the mechanism of action of neuroglobin might thus provide a rational basis for the design of new drug targets for inhibiting excessive neuronal cell death.

Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death.

In the past few years, overwhelming evidence has accrued that a high level of expression of the protein neuroglobin protects neurons in vitro, in animal models, and in humans, against cell death associated with hypoxic and amyloid insult. However, until now, the exact mechanism of neuroglobin's protective action has not been determined. Using cell biology and biochemical approaches we demonstrate that neuroglobin inhibits the intrinsic pathway of apoptosis in vitro and intervenes in activation of pro-caspase 9 by interaction with cytochrome c. Using systems level information of the apoptotic signalling reactions we have developed a quantitative model of neuroglobin inhibition of apoptosis, which simulates neuroglobin blocking of apoptosome formation at a single cell level. Furthermore, this model allows us to explore the effect of neuroglobin in conditions not easily accessible to experimental study. We found that the protection of neurons by neuroglobin is very concentration sensitive. The impact of neuroglobin may arise from both its binding to cytochrome c and its subsequent redox reaction, although the binding alone is sufficient to block pro-caspase 9 activation. These data provides an explanation the action of neuroglobin in the protection of nerve cells from unwanted apoptosis.

Characterization of the hemoglobins of the Australian lungfish Neoceratodus forsteri (Krefft).

We examined for the first time the hemoglobin components of the blood of the Australian lungfish, Neoceratodus forsteri and their functional responses to pH and the allosteric modulators adenosine triphosphate (ATP), guanosine triphosphate (GTP), 2,3-bisphosphoglyceric acid (BPG) and inositol hexaphosphate (IHP) at 25 degrees C. Lysates prepared from stripped, unfractionated hemolysate produced sigmoidal oxygen equilibrium curves with high oxygen affinity (oxygen partial pressure required for 50% hemoglobin saturation, p(50)=5.3 mmHg) and a Hill coefficient of 1.9 at pH 7.5. p(50) was 8.3 and 4.5 mmHg at pH 6 and 8, respectively, which corresponded to a modest Bohr coefficient (Delta log p(50)/Delta pH) of -0.13. GTP increased the pH sensitivity of oxygen binding more than ATP, such that the Bohr coefficient was -0.77 in the presence of 2 mmol L(-1) GTP. GTP was the most potent regulator of hemoglobin affinity, with concentrations of 5 mmol L(-1) causing an increase in p(50) from 5 to 19 mm Hg at pH 7.5, while the order of potency of the other phosphates was IHP>ATP>BPG. Three hemoglobin isoforms were present and each contained both alpha and beta chains with distinct molecular weights. Oxygen affinity and pH-dependence of isoforms I and II were essentially identical, while isoform III had a lower affinity and increased pH-dependence. The functional properties of the hemoglobin system of Neoceratodus appeared consistent with an active aquatic breather adapted for periodic hypoxic episodes.

Intra-molecular electron transfer in proteins.

Intra-molecular electron transfer is a key process, which is of prime importance, in photosynthesis, mitochondrial electron transfer and the action of many multi-centre enzymes. This mini-review considers the possible mechanisms of intra-molecular electron transfer in proteins and reviews the recent developments relating to possible electron tunnelling and electron hopping processes within di-heme cytochrome c peroxidase.

A role for neuroglobin: resetting the trigger level for apoptosis in neuronal and retinal cells.

We propose a new hypothesis for the molecular mechanism by which neuroglobin exerts its protective effect in hypoxia-induced cell death. Our recent observation of a very rapid electron-transfer reaction between ferrous neuroglobin and ferric cytochrome c is central to this hypothesis. In contrast to previously suggested roles for neuroglobin, related to its putative but unlikely oxygen storage/transport properties or its ability to react with nitrogen oxides, we suggest that ferrous neuroglobin exerts its protective effect via modulation of the early events in the intrinsic apoptotic pathway. We suggest this is achieved by the rapid reduction of cytosolic ferric cytochrome c by neuroglobin. The maintenance of cytochrome c in the nonapoptotic ferrous oxidation state and the concomitant generation of ferric neuroglobin in this reaction fit well with known feedback processes in the early events of the intrinsic apoptotic pathway. Our hypothesis also fits well with a number of previously uncorrelated findings, including the localization of neuroglobin in close proximity to mitochondria, the high concentration of neuroglobin in cells whose basal rates of aerobic metabolism are extremely high, and the cell types which are subject to large calcium ion fluxes in their normal physiology.

The interaction of human neuroglobin with hydrogen sulphide.

Neuroglobin has been identified to protect brain neurons from apoptotic stress. Hydrogen sulphide has a role in the brain as a neuromodulator, involving NMDA receptor activation. Here we report on studies of the in vitro interaction of ferric neuroglobin with hydrogen sulphide. Hydrogen sulphide binds very tightly to the heme group of neuroglobin in a biphasic reaction. The faster of the two reaction processes is concentration dependent whilst the slower process is not. The rate of hydrogen sulphide binding is pH sensitive and as the pH is reduced over the physiological range the rate of reaction increases by a factor of approximately 10. This change in reactivity appears to reflect the ionisation of the heme distal His ligand rather than a preference for the binding of H(2)S. We discuss the potential role of neuroglobin in the modulation of hydrogen sulphide sensitivity of neurons in the brain.

Characterization of the hemoglobins of the neonatal brushtailed possum Trichosurus vulpecula (Kerr): evidence for a highly cooperative, aggregated isoform of hemoglobin.

The red blood cells of the neonatal brushtailed possum exhibit unusually strong cooperativity at high levels of oxygen saturation (n=5.4) which appear to arise from a concentration dependent aggregation of one of the neonatal hemoglobin isoforms. Red blood cells from neonatal pouched young exhibit a Bohr factor of -0.36. Stripped hemolysate is sensitive to added 2,3-bisphosphoglycerate (BPG) (apparent binding constant K=35 micromol L(-1)) and ATP (K=180 micromol L(-1)), but is largely insensitive towards chloride ions. Five isoforms of non-adult hemoglobin were identified using isoelectric focusing. Mass spectrometry indicated that two early isoforms contain alpha chains identical to the adult alpha chain. The remaining three isoforms are composed of identical alpha type and beta type gene products, but differ in their isoelectric points due to differential post-translational modification.

Redox-linked structural changes associated with the formation of a catalytically competent form of the diheme cytochrome c peroxidase from Pseudomonas aeruginosa.

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca2+ ions triggers substantial structural rearrangements around the active-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca2+ ions, but a combination of electronic absorption, and EPR spectroscopies suggests that under these circumstances the low potential heme remains six-coordinate, unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that the two mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227-237) correspond to the Ca2+-loaded and -depleted forms of the enzyme.

Characterization of the hemoglobins of the adult brushtailed possum, Trichosurus vulpecula (Kerr) reveals non-genetic heterogeneity.

The hemoglobins contained within the red blood cells of the adult brushtail possum exhibited cooperative (n=2.6) oxygen binding curves with an associated p50 of 38 mm Hg at pH 7.4 and a large Bohr effect (-0.60). Stripped hemolysate showed a Bohr effect of -0.27, and was sensitive to added DPG (K=56 micromol L(-1)), ATP (K=130 micromol L(-1)), and chloride ions. Four isoforms of hemoglobin were identified using isoelectric focusing. Mass spectrometry indicated that all four isoforms most likely represent the same gene products which have differentially undergone post-translational deamidation and glutathionylation. The oxygen binding characteristics of three isolated isohemoglobins have been determined.

Intramolecular electron transfer in the dihaem cytochrome c peroxidase of Pseudomonas aeruginosa.

Mutant forms of the enzyme cytochrome c peroxidase from Pseudomonas aeruginosa, in which the peroxidatic haem ligand (H71) and putative haem-bridging amino acid (W94) have been mutated, were produced in an E. coli expression system as a means of investigating possible mechanisms of intramolecular electron transfer within the enzyme. EPR spectroscopy indicated the presence of a high-spin, presumably five-coordinate, peroxidatic haem site in the H71G and H71G/W94A mutants, whilst the W94A mutant apparently retained the normal six-coordinate haem structures. In turnover experiments, these mutants show 55, 4, and <1% activity, respectively, as compared to the wild-type enzyme. The W94A mutant shows essentially no activity in turnover experiments. Circular dichroism spectroscopy indicates no measurable difference in the secondary structure of the H71G mutant from that of the native enzyme, whilst some small differences are observed for the double mutant. Treatment of the oxidised mutant proteins with hydrogen peroxide, in the absence of preactivation or exogenous reductants, yields products that suggest the formation of a tryptophan radical species in the case of the H71 mutant and the production of a porphyrin radical in the case of the double mutant. These results are discussed in terms of the intramolecular electron transfer in this enzyme.