title: 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.
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.
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213. Electronic address: tja12@pitt.edu.
authors.forename: Thottala
authors.initials: T
authors.lastname: Jayaraman
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Jesús
authors.initials: J
authors.lastname: Tejero
authors.affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Bill B
authors.initials: BB
authors.lastname: Chen
authors.affiliation: Division of Neonatology, Department of Pediatrics, School of Medicine, Loma Linda University, Loma Linda, California 92354.
authors.forename: Arlin B
authors.initials: AB
authors.lastname: Blood
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Sheila
authors.initials: S
authors.lastname: Frizzell
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Calli
authors.initials: C
authors.lastname: Shapiro
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Mauro
authors.initials: M
authors.lastname: Tiso
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Hood
authors.affiliation: NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Xunde
authors.initials: X
authors.lastname: Wang
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; Medical Specialty Service Line, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 15240.
authors.forename: Rama K
authors.initials: RK
authors.lastname: Mallampalli
authors.affiliation: Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213. Electronic address: gladwinmt@upmc.edu.
authors.forename: Mark T
authors.initials: MT
authors.lastname: Gladwin
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D005914:Globins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006801:Humans; D000860:Hypoxia; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D000078787:Neuroglobin; D009569:Nitric Oxide / Q000737:chemistry*; D009573:Nitrites / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000378:metabolism; D012756:Sheep
chemical_list: D048948:14-3-3 Proteins; D008024:Ligands; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D009573:Nitrites; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D006418:Heme; D005914:Globins