TY - JOUR
T1 - 14-3-3 Binding and phosphorylation of neuroglobin during hypoxia modulate six-to-five heme pocket coordination and rate of nitrite reduction to nitric oxide
AU - Jayaraman, Thottala
AU - Tejero, Jesús
AU - Chen, Bill B.
AU - Blood, Arlin B.
AU - Frizzell, Sheila
AU - Shapiro, Calli
AU - Tiso, Mauro
AU - Hood, Brian L.
AU - Wang, Xunde
AU - Zhao, Xuejun
AU - Conrads, Thomas P.
AU - Mallampalli, Rama K.
AU - Gladwin, Mark T.
PY - 2011/12/9
Y1 - 2011/12/9
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=82755167730&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.271973
DO - 10.1074/jbc.M111.271973
M3 - Article
C2 - 21965683
AN - SCOPUS:82755167730
SN - 0021-9258
VL - 286
SP - 42679
EP - 42689
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -