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ABSTRACT: Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. The mechanism underlying this function is only partly understood. Here, we report that human Ngb exists in lipid rafts only during oxidative stress and that lipid rafts are crucial for neuroprotection by Ngb. The ferrous oxygen-bound form of Ngb, which exists under normoxia, is converted to the ferric bis-His conformation during oxidative stress, inducing large tertiary structural changes. We clarified that ferric bis-His Ngb, but not ferrous ligand-bound Ngb, specifically binds to flotillin-1, a lipid raft microdomain-associated protein, as well as to α-subunits of heterotrimeric G proteins (Gα(i/o)). Moreover, we found that human ferric bis-His Ngb acts as a guanine nucleotide dissociation inhibitor for Gα(i/o) that has been modified by oxidative stress. In addition, our data shows that Ngb inhibits the decrease in cAMP concentration that occurs under oxidative stress, leading to protection against cell death. Furthermore, by using a mutated Ngb protein that cannot form the bis-His conformation, we demonstrate that the oxidative stress-induced structural changes of human Ngb are essential for its neuroprotective activity.
Journal of Biological Chemistry 07/2012; 287(36):30128-38. · 4.77 Impact Factor
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ABSTRACT: Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Human Ngb is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. We previously demonstrated that, on the one hand, human ferric Ngb binds to the α-subunit of heterotrimeric G proteins (Gα(i)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Gα(i). On the other hand, zebrafish Ngb does not exhibit GDI activity. By using wild-type and Ngb mutants, we demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. The crucial residues for both GDI and neuroprotective activity, corresponding to Glu53, Arg97, Glu118, and Glu151 of human Ngb, are conserved among boreotheria of mammalia. Recently, we found that zebrafish, but not human, Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. By performing site-directed mutagenesis, we showed that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for protein transduction activity. Because these residues are conserved among fishes, but not among mammals, birds, reptilians, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins. Moreover, we clarified that zebrafish Ngb interacts with negatively charged cell-surface glycosaminoglycan. Taken together, these results suggest that the function of Ngb proteins has been changing dynamically throughout the evolution of life.
Marine Genomics 09/2011; 4(3):137-42. · 1.55 Impact Factor
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ABSTRACT: Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins.
PLoS ONE 01/2011; 6(2):e16808. · 4.09 Impact Factor
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ABSTRACT: Neuroglobin (Ngb) is a globin found in the vertebrate brain. Recently, we found that zebrafish Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. In the present study, we used site-directed mutagenesis to identify residues of module M1 that are important for protein transduction. We show that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for its activity. Since these residues are conserved among fishes, but not among mammals, birds, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins.
FEBS letters 06/2010; 584(11):2467-72. · 3.54 Impact Factor
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ABSTRACT: Mammalian neuroglobin (Ngb) is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. However, the neuroprotective mechanism remains unclear. We previously demonstrated that human ferric Ngb binds to the alpha-subunits of heterotrimeric G proteins (Galpha(i/o)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Galpha(i/o). In the present study, we used a protein delivery reagent, Chariot, to investigate whether the GDI activity of human Ngb plays an important role in its neuroprotective activity under oxidative stress conditions. We showed that human Ngb mutants, which retained GDI activities, rescued pheochromocytoma PC12 cell death caused by hypoxia/reoxygenation as did human wild-type Ngb. In contrast, zebrafish Ngb and human Ngb mutants, which did not function as GDI proteins, did not rescue cell death. These results clearly show that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity.
Biochemical and Biophysical Research Communications 06/2008; 369(2):695-700. · 2.48 Impact Factor
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ABSTRACT: Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under oxidative stress conditions, such as ischemia and reperfusion. We previously demonstrated that human ferric Ngb binds to the alpha subunit of heterotrimeric G proteins (Galphai) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Galphai. Recently, we used a protein delivery reagent, Chariot, and demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. In the present study, we found that chimeric ZHHH Ngb, in which module M1 of human Ngb is replaced by that of zebrafish Ngb, protects PC12 cells against oxidative stress-induced cell death even in the absence of Chariot. Using fluorescein isothiocyanate (FITC)-labeled Ngb proteins, we demonstrated that both zebrafish and chimeric ZHHH Ngb can penetrate cell membranes in the absence of Chariot, suggesting that module M1 of zebrafish Ngb can translocate into cells. This is the first report of a native cell-membrane-penetrating globin.
Biochemistry 06/2008; 47(19):5266-70. · 3.42 Impact Factor
