Abstract

Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1-5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

Highlights

  • Neuroglobin protects neurons from hypoxia; the underlying mechanisms for this effect remain poorly understood

  • Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding

  • To further examine neuroglobin phosphorylation during ischemic and hypoxic insults in live neuronal cells, which express minimal neuroglobin in culture, we developed stable cell lines by transducing GFP-tagged wild-type (GFPneuroglobin) and H64L mutant neuroglobin into neuroblastoma SH-SY5Y cells

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Summary

Background

Neuroglobin protects neurons from hypoxia; the underlying mechanisms for this effect remain poorly understood. (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. One example is that oxidation induces a disulfide bridge formation between two surface cysteines (Cys and Cys55) in neuroglobin [25] and decreases the distal histidine binding affinity for heme iron (KHis value has been shown to decrease from ϳ3000 to 280 and is calculated as kon/koff) [25] This is accompanied by a five-coordinate neuroglobin subpopulation increase with enhanced affinity for endogenous ligands such as oxygen (P50 shift from ϳ9 to 1 mm Hg) [26]. We sought to determine whether neuroglobin is a target for these kinase pathways and 14-3-3 binding, how these post-translational modifications are influenced by hypoxia, and what effects they have on the heme pocket coordination and rates of nitrite reduction

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