Association of human neuroglobin with cystatin C, a cysteine proteinase inhibitor

Neuroglobin (Ngb) is a newly discovered globin that is expressed in vertebrate brain. It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that Ngb protects neurons from hypoxia. However, the mechanism of this neuroprotection remains unclear. In the present study, we identified human cystatin C, a cysteine proteinase inhibitor, as an Ngb-binding protein by using a yeast two-hybrid system. Surface plasmon resonance experiments verified that Ngb binds to cystatin C dimers, not to the monomers. Because both intracellular cystatin C and the amyloidogenic variant of cystatin C form dimers, Ngb may modulate the intracellular transport (or secretion) of cystatin C to protect against neuronal death under conditions of oxidative stress and/or it may have a role in the development of neurodegenerative diseases.     

Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta

Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures. Ngb expression is strongly up-regulated by hypoxia and post-anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H₂O₂ release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H₂O₂ release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.     

Neuroglobin Involvement in the Course of Arsenic Toxicity in Rat Cerebellar Granule Neurons

Exposure to arsenic in drinking water results in a widespread environmental problem in the world, and the brain is a major target. Neuroglobin is a vertebrate heme protein regarded as playing neuroprotective role in hypoxia or oxidative stress. In this study, we investigated the toxic effects of sodium arsenite (NaAsO₂) on primary cultured rat cerebellar granule neurons (CGNs) and detected neuroglobin (Ngb) expression in rat CGNs exposed to NaAsO₂. Our results show that apoptosis was obviously induced by NaAsO₂ treatment in rat CGNs by annexin V-fluorescein isothiocyanate assay. Intracellular reactive oxygen species generation increased significantly in the cells exposed to NaAsO₂, and the apoptotic effects could be partially reversed by antioxidant N-acetyl-l-cysteine. Ngb protein and mRNA expression were significantly downregulated in rat CGNs shortly after NaAsO₂ exposure and then upregulated after a longer time of exposure. Furthermore, mRNA expression changed more than protein expression and the toxic effect of NaAsO₂ on Ngb expression is dose dependent. Higher Ngb expression was also detected in rat cerebellum, but not in other parts (cerebrum, hippocampus, and midbrain) of the brain exposed to NaAsO₂ for 16 weeks. Taken together, cytotoxic effects of NaAsO₂ on rat CGNs is induced at least partly by oxidative stress and Ngb may influence the course of arsenic toxicity in rat CGNs and rat cerebellum.     

Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor

Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in response to oxygen deprivation and that it protects neurons from hypoxia in vitro and in vivo. However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid autoxidation, binds exclusively to the GDP-bound form of the alpha subunit of heterotrimeric G protein (Galphai). In GDP dissociation assays or guanosine 5'-O-(3-thio)triphosphate binding assays, ferric Ngb behaved as a guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP. The interaction of GDP-bound Galphai with ferric Ngb will liberate Gbetagamma, leading to protection against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI activities. Taken together, we propose that human Ngb may be a novel oxidative stress-responsive sensor for signal transduction in the brain.     

脑红蛋白和细胞红蛋白：携氧蛋白质家族2个新成员

脑红蛋白(neuroglobin, Ngb)和细胞红蛋白(cytoglobin, Cygb)是新发现的2个携氧蛋白家族的成员.脑红蛋白主要存在于脑中,而细胞红蛋白在全身各个组织都含有,它们和另外2个携氧蛋白——血红蛋白和肌红蛋白的同源性<25%,但它们在种属之间的同源性很高(>95%).脊椎动物脑红蛋白基因定位于14q24,细胞红蛋白基因定位于17q25,都含有4个外显子和3个内含子.2种蛋白在生理条件下含有6个配位键,不同于血红蛋白和肌红蛋白的5个配位键结构.这2种新蛋白和氧都具有很高的亲和力,在缺氧条件下其基因及蛋白表达都有明显的提升,对细胞的存活有一定保护作用.对于脑红蛋白和细胞红蛋白的功能研究,有助于更好地了解机体氧代谢和氧利用过程,并为临床在缺氧损伤时的治疗提供新的观点和途径.     

Neuroglobin and friends

In the year 2000, the third member of the globin family was discovered in human and mouse brain and named neuroglobin (Ngb). Neuroglobin overexpression significantly protects both heart and brain from hypoxic/ischemic and oxidative stress‐related insults, whereas decreased Ngb levels lead to an exacerbation of tissue injuries. Moreover, Ngb overexpression protects neurons from mitochondrial dysfunctions and neurodegenerative disorders such as Alzheimer disease; however, it facilitates the survival of cancer cells. Neuroglobin, representing a switch point for cell death and survival, has been reported to recognize a number of proteins involved in several metabolic pathways including ionic homeostasis maintenance, energy metabolism, mitochondrial function, and cell signaling. Here, the recognition properties of Ngb are reviewed to highlight its roles in health and disease.     

A neuroglobin-overexpressing transgenic mouse

Neuroglobin (Ngb) is a recently discovered vertebrate globin expressed primarily in neurons. Ngb expression is induced by hypoxia and ischemia, and Ngb protects neurons from these insults. However, its normal physiological role and the mechanism underlying its neuroprotective action are uncertain. We report production of a transgenic mouse in which Ngb is overexpressed under the control of the chicken beta-actin promoter. This mouse should prove helpful for studying Ngb-mediated effects in vitro and in vivo.     

Anoxia or oxygen and glucose deprivation in SH-SY5Y cells: a step closer to the unraveling of neuroglobin and cytoglobin functions

Several studies support the hypothesis that neuroglobin and cytoglobin play a protective role against cell death when cellular oxygen supply is critical. Although the underlying molecular mechanisms are unknown, previous reports suggest that this protection can be realised by the fact that they act as ROS scavengers. In this study, expression of neuroglobin and cytoglobin was evaluated in a human neuroblastoma cell line (SH-SY5Y) under conditions of anoxia or oxygen and glucose deprivation (OGD). The cells could survive prolonged anoxia without significant loss of viability. They became anoxia sensitive when deprived of glucose. OGD induced significant cell death after 16 h resulting in 54% dead cells after 32 h. Necrosis was the main process involved in OGD-induced cell death. After reoxygenation, apoptotic neurons became more abundant. Real-time quantitative PCR and Western blotting revealed that neuroglobin and cytoglobin were upregulated, the former under OGD and the latter under anoxic conditions. Under OGD, cell survival was significantly reduced after inhibiting cytoglobin expression by transfection with antisense ODN. Moreover, cell survival was significantly enhanced by neuroglobin or cytoglobin overexpression. When neuroglobin or cytoglobin protein expression increased or decreased, the H(2)O(2) level was found to be lower or higher, respectively. We conclude that neuroglobin or cytoglobin act as ROS scavengers under ischemic conditions.     

Neuroglobin,an oxygen-binding protein in the mammalian nervous system (localization and putative functions)

The review summarizes current data on neuroglobin, the heme-containing protein discovered in mammalian nerve cells in 2000. It presents general characteristics of neuroglobin as well as data on its evolutionary changes and expression across different taxa. Neuroglobin distribution in specific brain structures and outside the brain is described. The issue of the occurrence of neuroglobin not only in neurons but also in astroglial cells is discussed. Subcellular localization of neuroglobin is characterized with a special focus on its detection in the nucleus of nerve cells, suggesting its involvement in nuclear functions. Current ideas on the probable functional significance of neuroglobin are reported. Neuroglobin is presumed to be involved in metabolism of reactive nitrogen and oxygen species as well as in intracellular signaling pathways. Besides, neuroglobin has neuroprotective and antiapoptotic functions. Since its expression changes during ontogenesis, its neuroprotective role in ageing is specifically highlighted. Changes in expression and localization of neuroglobin are suggested to influence the adaptive potential of an organism.     

Functional properties of neuroglobin and cytoglobin. Insights into the ancestral physiological roles of globins

Neuroglobin and cytoglobin are two recently discovered vertebrate globins, which are expressed at low levels in neuronal tissues and in all tissues investigated so far, respectively. Based on their amino acid sequences, these globins appear to be phylogenetically ancient and to have mutated less during evolution in comparison to the other vertebrate globins, myoglobin and hemoglobin. As with some plant and bacterial globins, neuroglobin and cytoglobin hemes are hexacoordinate in the absence of external ligands, in that the heme iron atom coordinates both a proximal and a distal His residue. While the physiological role of hexacoordinate globins is still largely unclear, neuroglobin appears to participate in the cellular defence against hypoxia. We present the current knowledge on the functional properties of neuroglobin and cytoglobin, and describe a mathematical model to evaluate the role of mammalian retinal neuroglobin in supplying O2 supply to the mitochondria. As shown, the model argues against a significant such role for neuroglobin, that more likely plays a role to scavenge reactive oxygen and nitrogen species that are generated following brain hypoxia. The O2 binding properties of cytoglobin, which is upregulated upon hypoxia, are consistent with a role for this protein in O2-requiring reactions, such as those catalysed by hydroxylases.     

Neuroglobin involvement in visual pathways through the optic nerve

Neuroglobin is a member of the globin superfamily proposed to be only expressed in neurons and involved in neuronal protection from hypoxia or oxidative stress. A significant fraction of the protein localizes within the mitochondria and is directly associated with mitochondrial metabolism and integrity. The retina is the site of the highest concentration for neuroglobin and has been reported to be up to 100-fold higher than in the brain. Since neuroglobin was especially abundant in retinal ganglion cell layer, we investigated its abundance in optic nerves. Remarkably in optic nerves, neuroglobin is observed, as expected, in retinal ganglion cell axon profiles but also astrocyte processes, in physiological conditions, possess high levels of the protein. Neuroglobin mRNA and protein levels are ~ 10-fold higher in optic nerves than in retinas, indicating an important accumulation of neuroglobin in these support cells. Additionally, neuroglobin levels increase in Müller cells during reactive gliosis in response to eye injury. This suggests the pivotal role of neuroglobin in retinal glia involved in neuronal support and/or healing. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Identification of residues critical for the cell-membrane-penetrating activity of zebrafish neuroglobin

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.     

Functional characterization of fish neuroglobin: Zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells

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 conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3 days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Zebrafish neuroglobin is a cell-membrane-penetrating globin

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.     

Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O(2) supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a hexacoordinated heme. O(2) and CO bind to the heme iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold adapted to host the reversible bis-histidyl heme complex and an elongated protein matrix cavity, held to facilitate O(2) diffusion to the heme. The neuroglobin structure suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.     

缺血缺氧疾病中脑红蛋白的表达

脑红蛋白是继血红蛋白和肌红蛋白后由Burmester等[1]于2000年发现的体内第3类携氧蛋白,脑红蛋白在视网膜组织中的含量高达100μmol/L,占视网膜总蛋白量的2%-4%,其浓度约为脑中浓度的100倍,而体内不同组织脑红蛋白表达量的增加均与组织的缺血缺氧密切相关。视网膜作为中枢神经系统的重要组成部分之一,对氧的需求远远大于其他神经组织,临床实践中存在着与视网膜相关的大量的缺血缺氧性疾病,视网膜的这种特征为我们深入研究脑红蛋白与视网膜细胞之间的关系提供了临床基础。本文就脑红蛋白在缺血缺氧疾病中的表达展开论述,为视网膜缺血缺氧损伤中脑红蛋白的表达提供相关依据。