Search

Cytoglobin (Cygb), neuroglobin (Ngb), and globin X (GbX) belongs to recently discovered members of the vertebrate globin family, they carry a heme prosthetic group that can reversibly bind exogenous ligands such as CO, NO and O2. Although the physiological functions of Cygb, Ngb and GbX are still under debate, several possible physiological functions for these proteins were proposed. Cytoglobin was reported to participate in lipid-based signaling and to stabilize the tumor suppressor p53 upon DNA damage, which imply its anti-cancer role. Neuroglobin was shown to interact with α-subunit of the heterotrimeric G protein as well as cytochrome c which indicates a role in cell apoptosis. Both proteins were also proposed to participate in NO metabolism. Compared to the well-known vertebrate globin, hemoglobin and myoglobin, the new members have several distinct structural characteristics. First, unlike Hb and Mb, the distal histidine coordinates with the heme iron at the sixth axial position in Cygb, Ngb and GbX, forming a hexa-coordinated heme iron and thus regulating kinetics and equilibrium constants for exogenous ligand binding to heme. Second, an oxidation/reduction of an intramolecular disulfide bridge which is found in all three hexa-coordinated globins, also modulates affinity for diatomic ligands such as O2 and CO. Additionally, both Cygb and GbX are found to have extended N- and C- terminals with unclear function, although the N-terminal in GbX proposed to be involved in the protein binding to the membrane. The work presented in this dissertation focuses on investigation of the role of internal ligand (distal histidine) and disulfide bridge on structure-function relationships in GbX, in terms of regulating affinity and kinetics for small diatomic ligands. Indeed, we shown a very weak ligand binding to heme iron in GbX, suggesting its district role among heaxa-coordiante vertebrate globins. In addition, the study of conformation dynamics that affect the heme cavit

Beta-haemoglobinopathies, such as sickle cell disease and beta-thalassaemia are amongst the most common genetic disorders worldwide and are caused by mutations in the adult beta-globin gene. A rare benign condition called the Hereditary Persistence of Foetal Haemoglobin (HPFH), results in reactivation of the developmentally silenced foetal gamma-globin gene, elevating foetal haemoglobin (HbF) levels and alleviating the symptoms of beta-haemoglobinopathies. A cluster of single nucleotide point mutations and a 13 base pair deletion positioned approximately 115 base pairs upstream (5') of the gamma-globin gene transcription start site, cause HPFH and the continued expression of gamma-globin throughout adult life. However despite extensive research, the exact mechanism by which these HPFH mutations elevate HbF has remained elusive. We performed a screen on several key erythroid repressors and identified that the major foetal globin repressor, BCL11A, binds to the -115 site of the gamma-globin proximal promoter and furthermore, that five out of the six reported HPFH mutations located at this site disrupt its binding in vitro. The -113 A>G HPFH mutation failed to disrupt BCL11A binding, however we demonstrated that it operates via a different mechanism, creating a de novo binding site for the transcriptional activator GATA-1. We introduced these naturally occurring HPFH mutations into an erythroid cell line using CRISPR/Cas9 technology and showed that the -114 C>A HPFH mutation disrupts BCL11A binding in vivo to raise gamma-globin expression, while the -113 A>G HPFH mutation creates a de novo GATA-1 binding site, which also elevates gamma-globin expression. These findings demonstrate that BCL11A is a direct repressor of the foetal gamma-globin gene and that the -115 cluster of HPFH mutations either disrupt the binding of a repressor or create a de novo site for a transcriptional activator to elevate HbF.

The globin gene superfamily has been well-characterized in vertebrates, however, there has been limited research in early-diverging lineages, such as phylum Cnidaria. This study aimed to identify globin genes in multiple cnidarian lineages, and use bioinformatic approaches to characterize the evolution, structure, and expression of these genes. Phylogenetic analyses and in silico protein predictions showed that all cnidarians have undergone an expansion of globin genes, which likely have a hexacoordinate protein structure. Our protein modeling has also revealed the possibility of a single pentacoordinate globin lineage in anthozoan species. Some cnidarian globin genes displayed tissue and development specific expression with very few orthologous genes similarly expressed across species. Our phylogenetic analyses also revealed that eumetazoan globin genes form a polyphyletic relationship with vertebrate globin genes. Overall, our analyses suggest that a Ngb-like and GbX-like gene were most likely present in the globin gene repertoire for the last common ancestor of eumetazoans. The identification of a large-scale expansion and subfunctionalization of globin genes in actiniarians provides an excellent starting point to further our understanding of the evolution and function of the globin gene superfamily in early-diverging lineages.

Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

Hemoglobin, one of the most important proteins in the human body, is composed of

Hemoglobin, one of the most important proteins in the human body, is composed of

Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

Hemoglobin, one of the most important proteins in the human body, is composed of

Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

Haemoglobinopathies are autosomal recessive disorders. They are the most common human single-gene disorders and are associated with abnormal globin gene expression, which results in either changes in the quantitative production of the globin chains or their structural aberrations. The majority of amino acid sequence variations are caused by a small number of frequently occurring mutations often with very high regional/ethnic human population dependencies. Pre- and postnatal screening programs are essential for effective diagnosis, counselling, and clinical management of individuals and their families exhibiting haemoglobinopathies. Mass spectrometry is a powerful tool for diagnostic screening programs, but its use for clinical haemoglobinopathy assessment has not been implemented in Australia. This investigation was focused on establishing analytical methods, which allow the unambiguous identification of haemoglobin variants through unique and diagnostically relevant methods for the identification of haemoglobin variant 'signature' peptide(s). In this work, sensitive and rapid analytical methods have been developed for the detection of human haemoglobin disorders using very small volumes (<1 muL) of blood. As an alternative to conventional enzymatic digestion, surfactant-aided enzymatic and microwave-assisted site-specific chemical cleavage methods have been established which allow very rapid proteolysis in less than 10 min. These methods have been applied to fifteen haemoglobin variants with identification of the corresponding haemoglobin variant 'signature' peptides by mass spectrometry. These microwaveassisted and MS-compatible sample preparation methods are expected to significantly contribute to the development of rapid, robust, cost-effective and environmentally benign methods of haemoglobin analysis in population screening studies.

Haemoglobinopathies are autosomal recessive disorders. They are the most common human single-gene disorders and are associated with abnormal globin gene expression, which results in either changes in the quantitative production of the globin chains or their structural aberrations. The majority of amino acid sequence variations are caused by a small number of frequently occurring mutations often with very high regional/ethnic human population dependencies. Pre- and postnatal screening programs are essential for effective diagnosis, counselling, and clinical management of individuals and their families exhibiting haemoglobinopathies. Mass spectrometry is a powerful tool for diagnostic screening programs, but its use for clinical haemoglobinopathy assessment has not been implemented in Australia. This investigation was focused on establishing analytical methods, which allow the unambiguous identification of haemoglobin variants through unique and diagnostically relevant methods for the identification of haemoglobin variant 'signature' peptide(s). In this work, sensitive and rapid analytical methods have been developed for the detection of human haemoglobin disorders using very small volumes (<1 muL) of blood. As an alternative to conventional enzymatic digestion, surfactant-aided enzymatic and microwave-assisted site-specific chemical cleavage methods have been established which allow very rapid proteolysis in less than 10 min. These methods have been applied to fifteen haemoglobin variants with identification of the corresponding haemoglobin variant 'signature' peptides by mass spectrometry. These microwaveassisted and MS-compatible sample preparation methods are expected to significantly contribute to the development of rapid, robust, cost-effective and environmentally benign methods of haemoglobin analysis in population screening studies.

The gene encoding the transcriptional regulator Krüppel-like Factor 3 (Klf3) is expressed highly during erythroid development. To investigate its biological role we have been studying a Klf3 knockout mouse model, where loss of Klf3 leads to numerous erythroid defects and mild anaemia. However, this has been complicated by upregulation of the related transcription factor Klf8 in Klf3 null tissue. These two factors are highly homologous and are able to regulate an overlapping set of target genes, suggesting that Klf8 may functionally compensate for the loss of Klf3. In this study, we have addressed this issue by comparing normal, Klf3, Klf8 and Klf3-Klf8 double mutant mouse lines. This approach has allowed us to determine that Klf3 and Klf8 can both contribute to the repression of embryonic globin gene expression during definitive erythropoiesis. Additionally, we have identified an intriguing new Klf3 target gene, a novel short isoform of the master myeloid regulator Pu.1. This isoform, which we have named Pu.2, is expressed via an internal promoter mapping to a retroviral long terminal repeat present in the mouse Pu.1 locus. We have determined that this and other related promoters are regulated by Klf3. Furthermore, we have found that Pu.2 negatively regulates Pu.1 activity and overexpression of Pu.2 in human myelogenous leukaemia cells induces erythroid differentiation. This study concludes that Klf3 plays a key role in erythroid biology that is achieved in part by regulating expression of factors such as Klf8 and Pu.2.

Neutral evolution is the simplest model of molecular evolution and thus it is most amenable to a comprehensive theoretical investigation. In this paper, we characterize the statistical properties of neutral evolution of proteins under the requirement that the native state remains thermodynamically stable, and compare them to the ones of Kimura's model of neutral evolution. Our study is based on the Structurally Constrained Neutral (SCN) model which we recently proposed. We show that, in the SCN model, the substitution rate decreases as longer time intervals are considered. Fluctuations from one branch of the evolutionary tree to another are strong, leading to a non-Poissonian statistics for the substitution process. Such strong fluctuations are in part due to the fact that neutral substitution rates for individual residues are strongly correlated for most residue pairs. Interestingly, structurally conserved residues, characterized by a much below average substitution rate, are also much less correlated to other residues and evolve in a much more regular way. Our results can improve methods aimed at distinguishing between neutral and adaptive substitutions as well as methods for computing the expected number of substitutions occurred since the divergence of two protein sequences. In particular, we compute the minimal sequence similarity below which no information about the evolutionary divergence of the compared sequences can be obtained.

The experimental work presented in this thesis pertains to the regulation of gene expression and focuses on the regulation of the B globin genes. The expression of the globin genes is restricted to the erythropoietic tissue and is regulated during development. Therefore the study of globin gene expression can yield valuable insights in how organisms regulate their genes in a coordinated tissue and developmental manner. Two main issues will be addressed in the chapters of this thesis. The first concerns the mechanism by which a transcriptionally competent domain is formed in the chromosomal environment. This topic is the subject of Chapter 2. Once transcriptionally competent domains are formed, gene transcription can take place and it is orchestrated by factors that interact specifically with the regulatory elements of the gene itself. This interaction determines tissue specificity and developmental regulation of a gene. The second issue addressed concerns the transcriptional regulation of the ~ globin gene via their CCAAT boxes studied through NFE6, a factor that binds to these elements (Chapters 3 and 4). NF-E6 interaction with its target sequence participates in the change in globin gene expression concomitant with the progressive changes that occur during the development of an embryo to a fetus.

Three test systems-heme synthesis by rat liver homogenates and rabbit reticulocyte lystaes, globin synthesis by a cell-free rabbit reticulocyte lysate system and erythroid cell proliferation in a plasma clot culture system have been established and standardized. For the enzyme systems, studies have been done to show the stability, of the enzymes at -70 C. Vehicles used for delivery of the benzene and toluene derivatives were examined for their effects on the globin translation and erythroid culture systems. The effects of benzene, the dinitrobenzenes, the dinitrotoluenes, and the trisubstituted toluenes on heme synthesis and globin synthesis were studied. Benzene was shown to have little deleterious effect on either the enzymes of heme synthsis or on globin translation. Ortho- and para-dinitrobenzene were shown to affect heme and globin synthesis more than meta-dinitrobenzene. Dose-toxicity studies of the effect of 23 benzene and toluene derivatives on ALA synthetase and heme synthetase were examined. The most pronounced effects were seen with the dinitrobenzenes, the dinitrotoluenes, the trinitrotoluenes, the amino-dinitrotoluenes, and hexachlorobenzene., Prepared in cooperation with George Washington Univ., Washington, DC. School of Medicine. Contract DAMD17-77-C-7045. Sponsored in part by IA0-7705.

Murine erythroleukemia cells (MELC) are transformed erythroid cell precursors that may be induced, by a variety of agents, including DMSO, to initiate the program of terminal cell differentiation and cell division characteristic of normal ertyropoiesis. We can provide increasing insight into the changes in MELC that accompany globin gene expression induced by polar chemicals, such as DMSO, and other agents. These transformed, CFUe-like erythroid precursor cells exhibit in their uninduced state, a DNA methylation pattern and globin gene chromatin configuration (DNase I sensitivity) that is compatible with actual or potential gene transcription. Such features may reflect alterations in chromatin configuration that have occurred at a stage prior to leukemic transformation, during the differentiation of earlier erythroid precursor cells and associated with the restriction in developmental potential characteristic of progression to the CFUe (or MELC) stage of erythropoiesis. Uninduced MELC display a low level of globin gene transcription, producing globin mRNA or mRNA precursors whose processing or stabilization is the target of action of hemin., This article is from 'Conference on Biological Actions and Medical Applications of Dimethyl Sulfoxide (DMSO), 15-17 September 1982,' AD-A133 175. p141-149.

Basic ferritin (liver-type) was measured in erythrocytes of subjects with alpha- and beta-thalassemia trait, thalassemia intermedia and Cooley's disease, and compared with normals and patients with abnormal iron metabolism without erythrocyte metabolic defect (iron deficiency anemia and idiopathic hemochromatosis). In all the thalassemic syndromes considered, erythrocyte ferritin was significantly higher than in normals (p less than 0.001) and increased progressively with the increasing 'severity' of the thalassemic disorder. In both thalassemic and non-thalassemic subjects, erythrocyte ferritin levels were related to body iron status, but in the thalassemic group, the increased erythrocyte ferritin values seemed also to be closely related to the intracellular metabolic abnormality. The severity of the defect in globin chain synthesis seemed to play an important role in determining ferritin accumulation in red cells of thalassemic subjects.

Liu, Y., Croft, K. D., Hodgson, J. M., Mori, T., & Ward, N. C. (2020). Mechanisms of the protective effects of nitrate and nitrite in cardiovascular and metabolic diseases. Nitric Oxide, 96, 35-43. https://doi.org/10.1016/j.niox.2020.01.006