Neuroglobin (Ngb) is a member of the vertebrate globin superfamily involved in cellular oxygen homeostasis. Structural analyses (Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity, 2003) show that human Ngb displays the typical globin fold, made of 151 amino acids (molecular mass= 17kDa), with only 20-25% of sequence identity with mioglobins and hemoglobins. Differently from the heterotetrameric hemoglobins, Ngb is a monomer that reversibly binds oxygen with an affinity higher than that of hemoglobin.
PDB NEUROGLOBIN STRUCTURE
Like Mb, Ngb is a monomeric protein that features the typical 3/3 α-helical globin fold. The main structural difference between Mb and Ngb is the coordination of the iron atom in the deoxy state. In Mb and Hb the central iron atom is pentacoordinated in the deoxy state and external ligands can bind to the free sixth coordination site. In Ngb, the sixth coordination site is occupied by the distal histidine of the E helix (HisE7), indicating a His-Fe(2+)-His binding scheme. Thus, this internal ligand has to be displaced before any external ligand (O2 or CO) can bind to the iron atom. The ferric (Fe(3+)) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k(on)) and a slow dissociation rate (k(off)) for both O(2) and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O(2) and CO binding is suggested to be slow in vivo.
The 3D structure of human neuroglobin was determined in 2003, and the next year, murine neuroglobin was determined at a higher resolution (The structure of murine neuroglobin: Novel pathways for ligand migration and binding, 2004).
Ngb is an intracellular hemoprotein expressed in the central and peripheral nervous system, cerebrospinal fluid, retina and endocrine tissues. It increases oxygen availability to brain tissue and provides protection under hypoxic or ischemic conditions, potentially limiting brain damage. Recent research on Neuroglobin presence confirmed that Human neuroglobin protein in cerebrospinal fluid (CSF) (Human neuroglobin protein in cerebrospinal fluid, 2005).
Italian researchers finally suggest that neuroglobin is more likely to usher in nitric oxide to protect neuron survival and recovery in areas where oxygen supply is reduced.
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NGB AMINO ACIDS PERCENTAGE
Ngb is a particularly highly conserved protein, with mouse and human Ngb differing in only 6% of
the amino acid positions. It has a substitution rate nearly four times lower than that of other vertebrate globins. Ngb shares as little as 25% of the amino acids with vertebrate hemoglobins and myoglobins but resembles some nerve-specific invertebrate globins. This observation is in line with phylogenetic analyses, which suggest that Ngb is a derivative of an ancient branch of the metazoan globins that emerged before the separation of Deuterostomia and Protostomia.
SYNTHESIS AND TURNOVER
mRNA and protein synthesis
NEUROGLOBIN CELLULAR FUNCTIONS
Globins are usually considered either oxygen transport or storage proteins. Thus Ngb may function as a “neuronal myoglobin,” providing oxygen to the respiratory chain.
• Ngb exerts a Mb-like role, enhancing O2 supply to the mitochondria of the metabolically active neurons. The hypothesis that Ngb functions as an O2 supply protein is supported by the observation that Ngb preferentially resides in metabolically active cells and subcellular compartments. A particularly high expression level was observed in the mammalian retina (How does the eye breathe?, 2003). The estimated concentration of Ngb in this tissue is 100 μM, higher than that in total brain and in the same range as the concentration of myoglobin in the skeletal muscle. Within the retina, Ngb protein is accumulated in the plexiform layers and the inner segment of the photoreceptor cells, thus correlating with the high oxygen demands of these regions. On the subcellular level, Ngb is concentrated in regions containing many mitochondria. Thus both cellular and subcellular distributions support the notion that in the eye Ngb expression levels are positively correlated with cellular oxygen consumption rates.
• Ngb scavenges damaging reactive oxygen or nitrogen species (ROS/RNS) (see The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger, 2010).The capacity of Ngb to scavenge the superoxide anion and hydrogen peroxide was even comparable to that of vitamin C.
• Ngb detoxifies harmful excess of nitric oxide (NO) to nitrate (NO3–) at normoxia or produce NO for signalling functions from nitrite (NO2–) at hypoxia for the control of blood pressure (see Human neuroglobin functions as a redox-regulated nitrite reductase, 2011).
• Ngb is involved in a signal transduction pathway, by inhibiting the dissociation of GDP from G protein α, and prevent hypoxia-induced apoptosisis via reduction of cytochrome c.
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INCREASED EXPRESSION OF NGB GENE INDUCED BY HYPOXIA
The oxygen-binding property and neuron specific expression of Ngb are strong indications of Ngb’s neuroprotection role against hypoxic/ischemic neuron injury.The first report in this category (Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury, 2001) showed that antisense-mediated knock-down of Ngb rendered cortical neurons more vulnerable to hypoxia, whereas Ngb overexpression conferred protection of cultured neurons against hypoxia. In the Ngb-overexpressing transgenic
(Ngb-Tg) mice, Dr. Greenberg’s group found that the cerebral infarct area was reduced by approximately 30% compared to wild type (Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia, 2006).
- Hypoxia-inducible factor-1 (HIF-1) enhances the expression of a large number of hypoxia-inducible genes, including erythropoietin, VEGF and glycolytic enzymes. Transcription factors that are induced by HIF-1 and for which binding sites are present in the Ngb promoter region probably increase Ngb expression. Binding sites for Sp1, NF-1, AP-1 and NFκB have been identified within the Ngb promoter (see Hypoxia-inducible factor-1 and neuroglobin expression, 2012; Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression, 2012).
Finally, Neuroglobin regulates hypoxic response of neuronal cells through Hif-1α- and Nrf2-mediated mechanism, 2012 shows that the expression of both Hif-1α and Nrf2 is decreased in neuroglobin-silenced hypoxic cells, whereas upregulation of both Hif-1α and Nrf2 is observed in neuroglobin-overexpressed hypoxic cells.
- Other mechanisms for the induction of Ngb include that activated by hemin (Hemin induces neuroglobin expression in neural cells, 2002). However, the relevance of this pathway to that activated by hypoxia is uncertain.
MOLECULAR MECHANISMS OF NGB NEUROPROTECTION
ROS and RNS scavenging
Soon after the discovery of Ngb there was speculation that – in analogy to Mb – it protects neurons from noxious ROS or RNS, such as nitrogen monoxide, peroxynitrite, and hydrogen peroxide, which are generated at high levels in the brain during hypoxia. In the presence of excess of NO, Fe(III) (metNGB) is converted into NGBFe(II)NO by reductive nitrosylation, in analogy to the reactions of NO with metmyoglobin and methemoglobin. The Fe(II)NO form of neuroglobin is oxidized to metNGB by peroxynitrite (NO3-) and dioxygen(O2). In contrast to myoglobin and hemoglobin, metNGB unexpectedly does not generate the cytotoxic ferryl form of the protein after addition of either peroxynitrite or hydrogen peroxide (H2O2). As we can see in many studies (Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger?, 2007), human neuroglobin may be an efficient SCAVENGER of reactive oxidizing species and thus may play a role in the cellular defense against oxidative stress.
In vitro, oxygenated Ngb (NGBFe(II)O2) may also react with NO, producing NGBFe(III)+ and NO3–. This pathway may protect cellular respiration damaged by the inhibitory effect of NO on cytochrome c oxidase activity. Ngb was proposed to have a similar role to that of Mb, acting as a NO-dioxygenase when PO2 is low (for example after an ischemic insult) and NO levels are increased. Under low-oxygen conditions deoxygenated Ngb may react with NO2–, resulting in the formation of NO. Thus, depending on the oxygen partial pressures, Ngb may either decompose or produce NO, which may be instrumental in the control of vasoconstriction or relaxation, and the level of mitochondrial respiration.
Protection from cell death
- In addition to the possible O2 sensing and ROS scavenging functions described above, Ngb has also been hypothesized to act as a signal transducer. It has been found that ferric human Ngb (met-Ngb) binds to the GDP-bound state of G protein α subunit (Gα), and acts as guanine nucleotide dissociation inhibitor (GDI). Ferric Ngb inhibits the exchange of GDP for GTP, thus prevents the Gα subunit from binding to the Gβγ complex and activates the signal transduction pathway, which is protective against oxidative stress (Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity, 2008)
Ngb binds two members of the Rho GTPase family , Rac1 and Rho A, as well as the Pak1 kinase, a key regulator of actin assembly and Rho-GDI-GTPase signaling complex
activity under hypoxia (Regulation of hypoxic neuronal death signaling by neuroglobin, 2008).
In neurons, after the exposure to neurotoxic stimuli (hypoxia, Aβ, NMDA), Rho GTPase–GDI cycle is activated, thus leading to actin polymerization and microdomain aggregation. Cultured cortical neurons respond rapidly to oxygen deprivation, demonstrating pronounced changes in morphology (bleb formation, process retraction, change in shape of the soma from pyramidal to round) within minutes. Disruption of cytoskeletal architecture during hypoxia in epithelial cells is mediated in large part by Rho proteins. Rho GTPases, such as Rac1, act as molecular switches to regulate biological responses. To perform this function, they must cycle between GDP-bound inactive states and GTP-bound active states. GDP dissociation inhibitors (GDIs: RhoGDI) sequester the inactive GTPase, preventing the dissociation of GDP and interactions with regulatory and effector molecules. In response to cell stimulation, Rac1 is induced to dissociate from GDI through Pak1 kinase-mediated phosphorylation of GDI. The GDI-free Rac1 is converted to the active GTP-bound form, which is then able to bind to effectors, promote actin polymerization and associate with membranes. The interaction of Rac1 with the membrane is terminated by the conversion of Rac1 to the GDP form and re-association with GDI.
The study mentioned above suggests that Ngb over-expression promotes Rac1 association with GDI maintaining Rac1 in an inactive state and retarding actin polymerization and subsequent cytoskeletal and microdomain aggregation, early events that drive neurons to death.
- Furthermore, Neuroglobin is involved in the regulation of mitochondrial pathway of apoptosis, generating a complex signalling network that limit stress signals, protecting neurons from excessive death. Ngb occurs in the intrinsic pathway of apoptosis. Pre-mitochondrial events that regulate mitochondrial outer membrane permeabilisation (MOMP) in neurons include increase in Ca2+ and reactive oxygen species (ROS) levels. MOMP is regulated by the multi-protein Bcl-2 family, which consists of both anti- and pro-apoptotic members. Cytochrome c is a key protein: it is released into cytosol consequently to MOMP. Following its release from the mitochondria, cytochrome c binds to Apaf-1, and promotes oligomerisation of Apaf-1 into the caspase 9 activating platform called the apoptosome. Down-stream of mitochondria, the pathway is regulated by the probability event of apoptosome assembly, and inhibitor of apoptosis proteins such as XIAP. Neuroglobin binding to and reduction of cytochrome c interferes with the mitochondrial pathway of apoptosis: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death, 2010.
In the absence of direct structural determinations of the complex formed between cytochrome c and neuroglobin, computational methods have been used: a close examination of the lowest energy complex (The binding of cytochrome c to neuroglobin: A docking and Surface Plasmon Resonance study, 2008) has allowed the identification of amino acids probably involved. In particular, Lys25 and Lys72 appear to have a significant role in the interface between neuroglobin and cytochrome c. It is interesting to note that these two amino acids have previously been identified as key residues in the interaction of cytochrome c with Apaf-1.
The reaction of neuroglobin with cytochrome c have multiple effects on apoptosis:
- Directly, the reaction of ferrous neuroglobin with (pro-apoptotic) ferric cytochrome c, released from the mitochondria, would produce (non-apoptotic) ferrous cytochrome c;
- Through the sequestration of cytochrome c, Ngb suppresses the normal suppression of auto-inhibition calcium release from IP3 receptors in the ER, thus reducing calcium levels and cyt c release. In vivo, support for this function of neuroglobin can be found in the work of Duong (Multiple protective activities of neuroglobin in cultred neuronal cells exposed to hypoxia re-oxygenation injury, 2009) and Liu et al. (Effects of neuroglobin overexpression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons, 2009).
- Finally, ferric neuroglobin has been identified as a potent inhibitor of GPCR (through mechanisms described above, about the role of Ngb as GDI) and therefore IP3 production.
NGB AS A TARGET FOR DEVELOPMENT OF THERAPEUTICS AGAINST STOKE AND NEURODEGENERATIVE DISORDERS
Accumulating evidence has proved that Ngb is an endogenous neuroprotective molecule against hypoxic/ischemic insults. Ngb gene expression inversely correlates with the severity of histological and functional deficits after ischemic stroke (Effects of neuroglobin overexpression on acute brain injury and long-term outcomes after focal cerebral ischemia, 2008).
But Ngb overexpression is also protective against beta-amyloid induced neurotoxicity and transgenic Alzheimer (Neuroglobin attenuates beta-amyloid neurotoxicity in vitro and transgenic Alzheimer phenotype in vivo, 2007).
As a potential molecular mechanism involved in this effect, it was found that Ngb overexpression attenuates tau hyperphosphorylation, a characterized pathological hallmark of AD brains, probably through activating the Akt signaling pathway, as we can infer from Neuroglobin attenuates Alzheimer-like tau hyperphosphorylation by activating Akt signaling, 2012. The antiapoptotic protein Akt (involved in survival pathway Akt/PI3K), phosphorylated by Ngb, inhibits GSK-3β, that is probably involved in hyperphosphorylation and consequent accumulation of Tau protein. Furthermore, Akt phosphorylation activates cell survival involving CREB. In addition, the recent publication of another article Ibuprofen and Lipoic Acid Conjugate Neuroprotective Activity Is Mediated by Ngb/Akt Intracellular Signaling Pathway in Alzheimer's Disease Rat Model, 2013 suggests that IBU-LA administration has the capability to maintain a high Ngb level, allowing Ngb to perform a neuroprotective and antiapoptotic role, representing a valid tool in the therapeutic strategy of AD progression.
In addition, this study (Neuroglobin and Alzheimer's dementia: genetic association and gene expression changes, 2010) reveals a strong correlation between AD and single nucleotide polymorphisms (SNPs) in five functional genes. Surprisingly, Ngb gene looks like to be one of these candidate genes.
These findings strongly suggest that pharmacological strategies that can up-regulate endogenous Ngb expression may be developed into a new therapeutic approach. A couple of groups have recently reported that Ngb can be up-regulated by a few chemical compounds, including valproic acid, cinnamic acid and 17β-estradiol, which is a good start to develop Ngb-targeted therapeutics against stroke and neurodegenerative disorders. Here we can see these articles: Pharmacological induction of neuroglobin expression, 2011 and 17β-estradiol—A new modulator of neuroglobin levels in neurons: Role in neuroprotection against H2O2-induced toxicity, 2010. In the last publication mentioned, researchers evince that the E₂-induced Ngb increase requires estrogen receptor (ER) β, but not ERα. E₂ exerted a protective effect against H₂O₂-induced neuroblastoma cell death which was completely prevented in Ngb-silenced cells. In this article, published on February 2013, Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H(2)O(2)-induced apoptosis of neuroblastoma cells, 2013, the role of Ngb in E₂ pathway is better explained, underlining that E2 treatment strongly increases NGB-cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of neuroblastoma cells. These data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the main mechanisms underlying E2-dependent NGB neuroprotection against H2O2 toxicity.
Ngb can be regarded as a key mediator of the different protective effects of E(2) in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the birth of several neurodegenerative diseases. The E(2) -mediated anti-inflammatory effect in primary cortical astrocytes can be controlled by reading 17β-Oestradiol Anti-Inflammatory Effects in Primary Astrocytes Require Oestrogen Receptor β-Mediated Neuroglobin Up-regulation, 2013.
Neuroglobin in Tumours: Dr Jekyll or Mr Hyde?
The strategy of blocking apoptosis operates also in cancer cells. If neuroglobin-mediated protection from cell death is of physiological relevance, we could expect that cancer cells will also use this mechanism to help their survival. In agreement with this hypothesis, a particular study has shown that neuroglobin is up-regulated in hypoxic microregions of glioblastoma tumour (Expression and hypoxic up-regulation of neuroglobin in human glioblastoma cells, 2009).
Brain tumours are usually resistant to conventional chemotherapeutics that induce apoptosis up-stream of mitochondria, but remain sensitive to post-mitochondrial induction of apoptosis. This indicates that despite over-expression of anti-apoptotic proteins that regulate MOMP, apoptotic signalling down-stream of mitochondria remains functional in these cancer cells. An increased expression of pro apoptotic Apaf-1 has been noticed in astrocytomas, medulloblastomas, and gliomas as compared to adjacent normal neural tissue (Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues, 2007). It can be hypothesized that not only increased Apaf-1 expression, but also decreased expression of neuroglobin, could contribute to the high sensitivity of brain tumours to postmitochondrial induction of apoptosis. This has not yet been investigated. Undoubtedly further studies are required to understand the role of neuroglobin in tumorigenesis and sensitivity to chemotherapy, and to identify potential of interfering in neuroglobin-cytochrome c interaction in cancer cells for therapeutic purposes.
In conclusion, this recent study Neuroglobin, a Novel Intracellular Hexa-coordinated Globin, Functions as a Tumor Suppressor in Hepatocellular Carcinoma Via Raf/MAPK/Erk, 2013 inquires the possibility of Ngb to function as a tumor suppressor in hepatocelluar carcinoma (HCC): overexpression of Ngb suppressed Raf/MEK/Erk while knock-down of Ngb enhanced Raf/MEK/Erk activation in HepG2 cells in vitro and in vivo. These data suggest that neuroglobin could be considered as a potential new target for cancer therapy.