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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-009-0436-5) contains supplementary material, which is available to authorized users.
Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b 5 is relatively slow (k = 6 · 10 2 M À1 s À1 at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k = 2 · 10 7 M À1 s À1 ) with an apparent overall equilibrium constant of 1 lM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.
BH3 mimetics have been proposed as new anticancer therapeutics. They target anti-apoptotic Bcl-2 proteins, up-regulation of which has been implicated in the resistance of many cancer cells, particularly leukemia and lymphoma cells, to apoptosis. Using probabilistic computational modeling of the mitochondrial pathway of apoptosis, verified by single-cell experimental observations, we develop a model of Bcl-2 inhibition of apoptosis. Our results clarify how Bcl-2 imparts its anti-apoptotic role by increasing the time-to-death and cell-to-cell variability. We also show that although the commitment to death is highly impacted by differences in protein levels at the time of stimulation, inherent stochastic fluctuations in apoptotic signaling are sufficient to induce cell-to-cell variability and to allow single cells to escape death. This study suggests that intrinsic cell-to-cell stochastic variability in apoptotic signaling is sufficient to cause fractional killing of cancer cells after exposure to BH3 mimetics. This is an unanticipated facet of cancer chemoresistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-010-0515-7) contains supplementary material, which is available to authorized users.
SummaryWe propose a new hypothesis for the molecular mechanism by which neuroglobin exerts its protective effect in hypoxiainduced 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.2008 IUBMB IUBMB Life, 60(6): 398-401, 2008
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