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PubMed2006

Characterization of human cytoglobin gene promoter region.

Cytoglobin (CYGB) is a member of the vertebrate globin family together with hemoglobin, myoglobin and neuroglobin. Although the physiological function of CYGB is still unclear, spectroscopic studies show that CYGB contains a hexacoordinated heme pocket similar to other pentacoordinated globin proteins. CYGB shares a common phylogenetic ancestry with vertebrate myoglobin from which it diverged by duplication before the appearance of jawed vertebrates. The objective of this study is to identify the regulatory and promoter region of the human cytoglobin gene. 5' unidirectional deletion constructs demonstrated that the proximal promoter elements of human CYGB gene are located between -1113 to -10 relative to the translation start site. Site-directed mutagenesis showed that mutation of a c-Ets-1 motif at -1008 and Sp1 motifs at -400, -230 and -210 remarkably decreased the promoter activity. Gel shift assays confirmed the binding of DNA-nuclear proteins to these motifs. All these results indicate that CYGB gene expression can be up-regulated by c-Ets-1 and Sp1 motifs.

Base Sequence↗

PubMedDate not supplied

Tracing the structure-function relationship of neuroglobin and cytoglobin using resonance Raman and electron paramagnetic resonance spectroscopy.

The physiological role of neuroglobin and cytoglobin, two vertebrate globins discovered in the last 5 years, is not yet clearly understood. In this work, we review the structural information on these globins and its implication on the possible protein function, obtained by electron paramagnetic resonance and resonance Raman spectroscopy. All studies reveal a high flexibility in the heme-pocket region of neuroglobin. Together with the observation that the distal ligand of the heme iron is the endogenous E7-histidine in both the ferric and ferrous form of neuroglobin and cytoglobin, the flexibility of the heme environment in neuroglobin will play a crucial role in the globins' ability to bind and stabilize exogenous ligands.

Animals↗

PubMed2006

Hypoxia differentially regulates the expression of neuroglobin and cytoglobin in rat brain.

Neuroglobin (Ngb) and Cytoglobin (Cygb) are new members of the globin family and display heterotopic expression patterns. To examine the effect of different hypoxia profiles on expression of Ngb and Cygb in rodent brain, rats were exposed to either sustained hypoxia (SH; 10% O(2)) or intermittent hypoxia (IH; 10% and 21% O(2) alternating every 90 s) for 1, 3, 7 and 14 days, and mRNA and protein expression of Ngb and Cygb were assessed in brain cortex. SH increased Ngb mRNA and protein expression throughout the exposure, while IH only elicited slight increases in Ngb expression at day 1. Neither SH nor IH elicited increases in Cygb expression. Thus, hypoxic stimulus presentation is a major determinant of the regulation of hypoxic sensitive genes such as Ngb. Furthermore, disparities between Ngb and Cygb responses to hypoxia further suggest that these two globins may play divergent roles in brain.

Animals↗

PubMed2004

Structural basis of human cytoglobin for ligand binding.

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.

Amino Acid Sequence↗

PubMed2004

Functional adaptation and its molecular basis in vertebrate hemoglobins, neuroglobins and cytoglobins.

Hemoglobin (Hb), the paradigm for allosteric proteins through decades, has gained renaissance in recent years following discovery of globins or their genes in all living organisms and in all tissues of higher animals, and of new members of the globin family, such as neuroglobins, Ngb, found predominantly in neural and nerve tissues and cytoglobins, Cygb, that has unprecedented nuclear location. The recent progresses in this field have been prompted by the development of sophisticated techniques to probe molecular structure and functions, which have revealed novel functions, such as the scavenging and release of vasoactive nitric oxide and the regulation of cellular metabolism. This review deals with the functional adaptations and the underlying molecular mechanisms in globins and presents case examples of molecular adaptations encountered in vertebrates and agnathans.

Acclimatization↗

PubMed2006

Cytoglobin overexpression protects against damage-induced fibrosis.

Cytoglobin (Cygb), a member of the hexacoordinate globin superfamily (hxHb), is expressed in fibroblasts from a broad range of tissues. The physiological functions of hxHb are still unclear, but biochemical studies reveal that they can scavenge toxic species, such as nitric oxide, peroxynitrite, and hydrogen peroxide. We demonstrate that the overexpression of Cygb in rat hepatic stellate cells, both in vitro and in vivo, protects against oxidative stress, inhibiting their differentiation into a myofibroblast-like phenotype. Accordingly, the overexpression of Cygb reduces extracellular matrix deposition in both toxic and cholestatic models of liver injury. The overexpression of Cygb also promotes recovery from previously initiated damage-induced fibrogenesis. By inhibiting free radical-induced activation of hepatic stellate cells, Cygb plays an important role in controlling tissue fibrosis. Therefore, the normal upregulation of Cygb during tissue injury has a homeostatic effect, inhibiting free radical-induced fibroblast activation and tissue fibrosis.

Animals↗

PubMed2003

Characterization of the heme environmental structure of cytoglobin, a fourth globin in humans.

Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E' of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the nu(Fe-CO) and nu(C-O) values in the resonance Raman and infrared spectra of the ferrous-CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The nu(Fe-O2) in the resonance Raman spectra of oxy Cgb can be observed at 572 cm(-1), suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.

Amino Acid Sequence↗

PubMed2005

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin.

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.

Carbon Monoxide↗

PubMed2004

Structural change of the heme pocket due to disulfide bridge formation is significantly larger for neuroglobin than for cytoglobin.

Human neuroglobin (hNgb) and human cytoglobin (hCygb), two recently discovered members of the vertebrate globin family, are known to be able to form an intramolecular disulfide bridge. Using electron paramagnetic resonance (EPR), we show that formation of a disulfide bridge in ferric hNgb causes a considerable change in the heme pocket structure, whereas this is not so clear for ferric hCygb. The structural results can be related nicely to earlier histidine and dioxygen affinity studies of the ferrous proteins.

Cytoglobin↗

PubMed2006

Solution 1H NMR characterization of the axial bonding of the two His in oxidized human cytoglobin.

Solution 1H NMR spectroscopy has been used to determine the relative strengths (covalency) of the two axial His-Fe bonds in paramagnetic, S = 1/2, human met-cytoglobin. The sequence specific assignments of crucial portions of the proximal and distal helices, together with the magnitude of hyperfine shifts and paramagnetic relaxation, establish that His81 and His113, at the canonical positions E7 and F8 in the myoglobin fold, respectively, are ligated to the iron. The characterized complex (approximately 90%) in solution has protohemin oriented as in crystals, with the remaining approximately 10% exhibiting the hemin orientation rotated 180 degrees about the alpha-, gamma-meso axis. No evidence could be obtained for any five-coordinate complex (<1%) in equilibrium with the six-coordinate complexes. Extensive sequence-specific assignments on other dipolar shifted helical fragments and loops, together with available alternate crystal coordinates for the complex, allowed the robust determination of the orientation and anisotropies of the paramagnetic susceptibility tensor. The tilt of the major axis is controlled by the His-Fe-His vector, and the rhombic axes are controlled by the mean of the imidazole orientations for the two His. The anisotropy of the paramagnetic susceptibility tensor allowed the quantitative factoring of the hyperfine shifts for the two axial His to reveal an indistinguishable pattern and magnitudes of the contact shifts or pi spin densities, and hence, indistinguishable Fe-imidazole covalency for both Fe-His bonds.

Anisotropy↗

PubMedDate not supplied

Neuroglobin and cytoglobin: genes, proteins and evolution.

Hemoglobin and myoglobin are oxygen transport and storage proteins of most vertebrates. Neuroglobin (Ngb) and cytoglobin (Cygb)--two recent additions to the vertebrate globin superfamily--have still disputed functions. Combining the data from all available resources, we investigate the evolution of these novel globins. Both Ngb and Cygb show little sequence variation in vertebrate evolution, suggesting conserved structures and functions, and an important role in the animal's metabolism. Exon-intron patterns remained unchanged in Ngb and Cygb, with the exception of the addition of a 3' exon to Cygb early in mammalian evolution. In phylogenetic analyses, Ngb forms a common branch with globin X, another recently identified globin with undefined function in lower vertebrates, and with some invertebrate nerve globins. This shows an early divergence of this branch in animal evolution. Cygb is related to myoglobin, and associated with an eye-specific globin from birds. The pattern of globin evolution shows that proteins with clear respiratory roles evolved independently from intracellular globins with uncertain functions. This result suggests either multiple independent functional changes or a yet undefined respiratory role of tissue globins like Ngb and Cygb.

Amino Acid Sequence↗

PubMedDate not supplied

Hypoxia/ischemia and the regulation of neuroglobin and cytoglobin expression.

In analogy to hemoglobin (Hb) and myoglobin (Mb), neuroglobin (Ngb) and cytoglobin (Cygb) are supposed to be involved in oxygen (O2) storage and delivery. The Cygb gene harbours both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation. We examined the relative transcriptional changes of Ngb and Cygb in a situation of chronic hypoxia using real-time quantitative PCR. We could conclude that Cygb is a hypoxia-induced gene, which is transcriptionally upregulated during chronic hypoxia in a hippocampal neuronal cell line and in multiple murine metabolically active tissues. The mechanism of induction of Cygb is HIF-1alpha dependent. HIF-1 is unique among mammalian transcription factors with respect to the specificity and sensitivity of its induction by hypoxia. Ngb expression seems to be regulated using other response elements and is less influenced by hypoxia.

Animals↗

PubMedDate not supplied

Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin.

Neuroglobin (Ngb) and cytoglobin (Cygb) are two recently discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve-globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb is held to facilitate O2 diffusion to the mitochondria and to protect neuronal cells from hypoxic-ischemic insults, may be an oxidative stress-responsive sensor protein for signal transduction, and may carry out enzymatic activities, such as NO/O2 scavenging. Cygb is linked to collagen synthesis, may provide O2 for enzymatic reactions, and may be involved in a ROS(NO)-signaling pathway(s). Ngb and Cgb display the classical three-over-three alpha-helical fold of Hb and Mb, and are endowed with a hexa-coordinate heme-Fe atom, in their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous ligand. Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of Ngb and Cygb. Moreover, Ngb and Cygb display a tunnel/cavity system within the protein matrix held to facilitate ligand channeling to/from the heme, multiple ligand copies storage, multi-ligand reactions, and conformational transitions supporting ligand binding.

Animals↗

PubMed2006

Down-regulation of the cytoglobin gene, located on 17q25, in tylosis with oesophageal cancer (TOC): evidence for trans-allele repression.

Tylosis (focal non-epidermolytic palmoplantar keratoderma) is an autosomal dominant skin disorder that is associated with the early onset of squamous cell oesophageal cancer (SCOC) in three families. Our previous linkage and haplotype analyses have mapped the tylosis with oesophageal cancer (TOC) locus to a 42.5 kb region on chromosome 17q25 that has also been implicated in the aetiology of sporadically occurring SCOC from a number of different geographical populations. Oesophageal cancer is one of the 10 leading causes of cancer mortality worldwide. No inherited disease-causing mutations have been identified in the genes located in the 42.5 kb minimal region. We now show that cytoglobin gene expression in oesophageal biopsies from tylotic patients is dramatically reduced by approximately 70% compared with normal oesophagus. Furthermore, both alleles are equally repressed. Given the autosomal dominant nature of the disease, these results exclude haploinsufficiency as a mechanism of the disease and instead suggest a novel trans-allele interaction. We also show that the promoter is hypermethylated in sporadic oesophageal cancer samples: this may constitute the 'second hit' of a gene previously implicated in this disease by allelic imbalance studies.

Allelic Imbalance↗

PubMed2006

Frequent genetic and epigenetic abnormalities contribute to the deregulation of cytoglobin in non-small cell lung cancer.

Lung cancer demonstrates the highest mortality in the UK. Previous studies have implicated allelic loss at chromosome 17q in the development of non-small cell lung carcinoma (NSCLC), and a number of known and putative tumour-suppressor genes reside within this region. One candidate tumour-suppressor gene is cytoglobin (CYGB), which is contained entirely within the 42.5 kb tylosis with oesophageal cancer (TOC) minimal region. CYGB abnormalities have been demonstrated only in sporadic head and neck cancers. In this study, we investigated the expression, promoter methylation and allelic imbalance status of this gene in 52 paired (normal/tumour) surgically excised lung tissue samples from patients with NSCLC. CYGB expression in tumour tissue was significantly reduced compared with corresponding adjacent normal in 54% of the examined cases (paired t-test, P<0.001). The CYGB promoter was shown by pyrosequencing to be significantly hypermethylated [2-fold increase of methylation index (MtI) in tumours] in 25/52 (48%) tumour samples compared with normal samples. MtI of the CYGB promoter was associated with CYGB mRNA expression (linear regression analysis, P=0.009), suggesting a primary role for the epigenetic events in CYGB silencing. In addition, frequent LOH was detected at the locus 17q25 in 32/48 (67%) tumours examined. It is of note that the loss of expression intensified when both LOH and hypermethylation coincided in samples (Mann-Whitney, P=0.049). These findings provide the first evidence to suggest the implication of CYGB in the pathogenesis of NSCLCs.

Aged↗

PubMed2002

Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues.

Vertebrates possess multiple respiratory globins that differ in terms of structure, function, and tissue distribution. Three types of globins have been described so far: hemoglobin facilitates the transport of oxygen in the blood, myoglobin serves oxygen transport and storage in the muscle, and neuroglobin has a yet unidentified function in nerve cells. Here we report the identification of a fourth and novel type of globin in mouse, man, and zebrafish. It is expressed in apparently all types of human tissue and therefore has been called cytoglobin (CYGB). Mouse and human CYGBs comprise 190 amino acids; the zebrafish CYGB, 174 amino acids. The human CYGB gene is located on chromosome 17q25. The mammalian genes display a unique exon-intron pattern with an additional exon resulting in a C-terminal extension of the protein, which is absent in the fish CYGB. Phylogenetic analyses suggest that the CYGBs had a common ancestor with vertebrate myoglobins. This indicates that the vertebrate myoglobins are in fact a specialized intracellular globin that evolved in adaptation to the special needs of muscle cells.

Amino Acid Sequence↗

PubMed2003

New insight into the haemoglobin superfamily: preliminary crystallographic characterization of human cytoglobin.

Human cytoglobin, present in almost all tissue types, is a newly identified member of the Hb superfamily. A double mutant, having both cysteines replaced by serines, has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant SAD data set has been collected at the haem Fe-atom absorption edge (lambda = 1.720 A) to 2.60 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 46.8, b = 73.1, c = 98.9 A and two molecules per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe-atom sites, thus paving the way for SAD structure determination.

Binding Sites↗

PubMed2006

High-resolution structure of human cytoglobin: identification of extra N- and C-termini and a new dimerization mode.

Cytoglobin (Cgb) is a recently discovered member of the vertebrate haem-containing globin family. The structure of a new crystal form of wild-type human Cgb (space group C2) was determined at a resolution of 1.68 Angstrom. The results show the presence of an additional helix in the N-terminal residues (4-20) prior to the A helix and an ordered loop structure in the C-terminal region (168-188), while these extended peptides were invisible owing to disorder in the previously reported structures using a P3(2)21 crystal at a resolution of 2.4 Angstrom. A detailed comparison of the two crystal structures shows differences in the conformation of the residues (i.e. Arg84) in the haem environment owing to a different dimeric arrangement.

Crystallography, X-Ray↗