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Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.

Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult β-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.

Background Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified. Methods This study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms. Results Immunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs’ mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic. Conclusions Scanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop. Graphical Abstract

Spermatogenesis is a highly specialised process, involving multiple dedicated pathways and regulatory check-points. Defects ultimately lead to male sub-fertility or sterility, and numerous aspects of mammalian sperm formation remain unknown. The predominant expression of the latest globin family member, androglobin (Adgb) in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape and ultrastructural defects in microtubule and mitochondrial organisation. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifide or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates, and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, absence of Adgb leads to mislocalisation of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin (CaM)-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that interdependence between Adgb and Sept10 is required for sperm head shaping via the manchette and proper flagellum formation.

Significance: Red blood cell (RBC)-mediated vasodilation plays an important role in oxygen delivery. This occurs through hemoglobin actions, at least in significant part, to convert heme-bound nitric oxide (NO) (in tense [T]/deoxygenated-state hemoglobin) into vasodilator S-nitrosothiol (SNO) (in relaxed [R]/oxygenated-state hemoglobin), convey SNO through the bloodstream, and release it into tissues to increase blood flow. The coupling of hemoglobin R/T state allostery, both to NO conversion into SNO and to SNO release (along with oxygen), under hypoxia supports the model of a three-gas respiratory cycle (O(2)/NO/CO(2)). Recent Advances: Oxygenation of tissues is dependent on a single, strictly conserved Cys residue in hemoglobin (βCys93). Hemoglobin couples SNO formation/release at βCys93 to O(2) binding/release at hemes (“thermodynamic linkage”). Mice bearing βCys93Ala hemoglobin that is unable to generate SNO-βCys93 establish that SNO-hemoglobin is important for R/T allostery-regulated vasodilation by RBCs that couple blood flow to tissue oxygenation. Critical Issues: The model for RBC-mediated vasodilation originally proposed by Stamler et al. in 1996 has been largely validated: SNO-βCys93 forms in vivo, dilates blood vessels, and is hypoxia-regulated, and RBCs actuate vasodilation proportionate to hypoxia. Numerous compensations in βCys93Ala animals to alleviate tissue hypoxia (discussed herein) are predicted to preserve vasodilatory responses of RBCs but impair linkage to R/T transition in hemoglobin. This is borne out by loss of responsivity of mutant RBCs to oxygen, impaired blood flow responses to hypoxia, and tissue ischemia in βCys93-mutant animals. Future Directions: SNO-hemoglobin mediates hypoxic vasodilation in the respiratory cycle. This fundamental physiology promises new insights in vascular diseases and blood disorders.

β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of −189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding.

Turbot is an important flatfish widely distributed along the European coasts, whose fishery is centered in the North Sea. The commercial value of the species has boosted a successful aquaculture sector in Europe and China. Body growth is the main target of turbot breeding programs and is also a key trait related to local adaptation to temperature and salinity. Differences in growth rate and optimal growth temperature in turbot have been shown to be associated with a hemoglobin polymorphism reported more than 50 years ago. Here, we identified a Gly16Asp amino acid substitution in the β1 globin subunit by searching for genetic variation in the five functional globin genes within the whole annotated turbot genome. We predicted increased stability of the turbot hemoglobin by the replacement of the conserved Gly with the negative charged Asp residue that is consistent with the higher rate of αβ dimer assembly in the human J-Baltimore Gly16β->Asp mutant than in normal HbA. The turbot Hbβ1-Gly16 variant dominated in the northern populations examined, particularly in the Baltic Sea, while the Asp allele showed elevated frequencies in southern populations and was the prevalent variant in the Adriatic Sea. Body weight did not associate with the Hbβ1 genotypes at farming conditions (i.e., high oxygen levels, feeding ad libitum) after analyzing 90 fish with high growth dispersal from nine turbot families. Nevertheless, all data at hand suggest that the turbot hemoglobin polymorphism has an adaptive significance in the variable wild conditions regarding temperature and oxygen availability. Electronic supplementary material The online version of this article (10.1007/s10695-020-00872-y) contains supplementary material, which is available to authorized users.

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.

Significance: Over the past 50 years, the mechanisms for O(2) storage and transport have been determined quantitatively on distance scales from millimeters to tenths of nanometers and timescales from seconds to picoseconds. Recent Advances: In this review, I have described four key conclusions from work done by my group and our close colleagues. (i) O(2) uptake by mammalian red cells is limited by diffusion through unstirred water layers adjacent to the cell surface and across cell-free layers adjacent to vessel walls. (ii) In most vertebrates, hemoglobins (Hbs) and myoglobins (Mbs), the distal histidine at the E7 helical position donates a strong hydrogen bond to bound O(2), which selectively enhances O(2) affinity, prevents carbon monoxide poisoning, and markedly slows autoxidation. (iii) O(2) binding to mammalian Hbs and Mbs occurs by migration of the ligand through a channel created by upward rotation of the His(E7) side chain, capture in the empty space of the distal pocket, and then coordination with the ferroprotoporphyrin IX (heme) iron atom. (iv) The assembly of Mbs and Hbs occurs by formation of molten globule intermediates, in which the N- and C-terminal helices have almost fully formed secondary structures, but the heme pockets are disordered and followed by high-affinity binding of heme. Critical Issues: These conclusions indicate that there are often compromises between O(2) transport function, holoprotein stability, and the efficiency of assembly. Future Directions: However, the biochemical mechanisms underlying these conclusions provide the framework for understanding globin evolution in greater detail and for engineering more efficient and stable globins.

Summary Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker of hematopoietic stem cells (HSCs). Although we previously elucidated the functional importance of ESAM in HSCs in stress-induced hematopoiesis in adults, it is unclear how ESAM affects hematopoietic development during fetal life. To address this issue, we analyzed fetuses from conventional or conditional ESAM-knockout mice. Approximately half of ESAM-null fetuses died after mid-gestation due to anemia. RNA sequencing analyses revealed downregulation of adult-type globins and Alas2, a heme biosynthesis enzyme, in ESAM-null fetal livers. These abnormalities were attributed to malfunction of ESAM-null HSCs, which was demonstrated in culture and transplantation experiments. Although crosslinking ESAM directly influenced gene transcription in HSCs, observations in conditional ESAM-knockout fetuses revealed the critical involvement of ESAM expressed in endothelial cells in fetal lethality. Thus, we showed that ESAM had important roles in developing definitive hematopoiesis. Furthermore, we unveiled the importance of endothelial ESAM in this process., Graphical Abstract, Highlights • ESAM deficiency causes life-threatening anemia in fetuses • ESAM deficiency impairs adult-type hemoglobin synthesis • ESAM deficiency downregulates expression of Alas2 and adult-type globins in HSCs • Endothelial ESAM plays roles in the ontogeny of definitive hematopoiesis, Ueda et al. shed light on the contribution of endothelial cell-selective adhesion molecule (ESAM) to the ontogeny of definitive hematopoiesis. ESAM deficiency caused high mortality in fetuses after mid-gestation. Expression of Alas2 and adult-type globin genes decreased in ESAM-null HSCs, which resulted in the impairment of development of adult-type erythropoiesis. The authors also revealed functional involvement of ESAM-expressing endothelial cells.

Globin-X (GbX) is an enigmatic member of the vertebrate globin gene family with a wide phyletic distribution that spans protostomes and deuterostomes. Unlike canonical globins such as hemoglobins and myoglobins, functional data suggest that GbX does not have a primary respiratory function. Instead, evidence suggests that the monomeric, membrane-bound GbX may play a role in cellular signaling or protection against the oxidation of membrane lipids. Recently released genomes from key vertebrates provide an excellent opportunity to address questions about the early stages of the evolution of GbX in vertebrates. We integrate bioinformatics, synteny, and phylogenetic analyses to characterize the diversity of GbX genes in nonteleost ray-finned fishes, resolve relationships between the GbX genes of cartilaginous fish and bony vertebrates, and demonstrate that the GbX genes of cyclostomes and gnathostomes derive from independent duplications. Our study highlights the role that whole-genome duplications (WGDs) have played in expanding the repertoire of genes in vertebrate genomes. Our results indicate that GbX paralogs have a remarkably high rate of retention following WGDs relative to other globin genes and provide an evolutionary framework for interpreting results of experiments that examine functional properties of GbX and patterns of tissue-specific expression. By identifying GbX paralogs that are products of different WGDs, our results can guide the design of experimental work to explore whether gene duplicates that originate via WGDs have evolved novel functional properties or expression profiles relative to singleton or tandemly duplicated copies of GbX.

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1—CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity., Author summary Trematode parasites that invade mammalian tissues have long been believed to produce bioenergy via anaerobic respiration in their definitive hosts. However, recent studies have revealed that these parasites require considerable amounts of oxygen for the generation of hard eggshells during sexual reproduction as well as energy metabolism. Despite these findings, information on the biological mechanisms and relevant molecules responsible for oxygen uptake in the host environment remains largely elusive. Clonorchis sinensis is a carcinogenic trematode parasite that causes clonorchiasis in humans by infecting the bile ducts. Here, we investigated globin genes/proteins in the liver fluke. The genome of C. sinensis encoded at least five globin paralogs (CsMb1, CsMb2, CsMb3, CsNgb, and CsGbX). Temporal expression of these globin genes coincided with the sexual maturation of C. sinensis. Based on the histological localities and induction profiles upon hypoxia, it could be postulated that the oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms by CsMb1 and CsMb3, respectively, for energy metabolism and eggshell formation. Other globin homologs were likely to perform non-respiratory functions. In addition, the oxygenated form of secreted CsMb1 seemed to participate in the scavenging of nitric oxide generated by host immune cells via its nitric oxide dioxygenase activity to increase the survival of the parasite.

The heme propionates in myoglobin (Mb) form a H-bonding network among several residues within its second-sphere coordination, providing a key structural role towards Mb's functional properties. Our work aims to understand the role of the heme propionates on the nitrite reductase (NiR) activity (e.g. reduction of NO2− to NO) of this globin by studying an artificial dimethylester heme-substituted horse heart Mb (DME-Mb). The minor structural change brought about by esterification of the heme propionates causes the NiR rate to increase by more than over two-fold (5.6 ± 0.1 M−1 s−1) relative to wildtype (wt) Mb (2.3 ± 0.1 M−1 s−1). The lower pKa observed in DME-Mb may enhance the tendency of His64 towards protonation, therefore increasing the NiR rate. In addition, the nitrite binding constant (Knitrite) for DME-MbIII is greater than wt MbIII (350 M−1 versus 120 M−1). The disparity in the NiR activity correlates with the differences in electrostatic behavior, which influences the system's reactivity towards the approaching NO2− ion, and thus the formation of the FeII-NO2− intermediate.

The human ζ-globin gene (HBZ) is transcribed in primitive erythroid cells only during the embryonic stages of development. Reactivation of this embryonic globin synthesis would likely alleviate symptoms both in α-thalassemia and sickle-cell disease. However, the molecular mechanisms controlling ζ-globin expression have remained largely undefined. Moreover, the pharmacologic agent capable of inducing ζ-globin production is currently unavailable. Here, we show that TRIAC, a bioactive thyroid hormone metabolite, significantly induced ζ-globin gene expression during zebrafish embryogenesis. The induction of ζ-globin expression by TRIAC was also observed in human K562 erythroleukemia cell line and primary erythroid cells. Thyroid hormone receptor α (THRA) deficiency abolished the ζ-globin-inducing effect of TRIAC. Furthermore, THRA could directly bind to the distal enhancer regulatory element to regulate ζ-globin expression. Our study provides the first evidence that TRIAC acts as a potent inducer of ζ-globin expression, which might serve as a new potential therapeutic option for patients with severe α-thalassemia or sickle-cell disease. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01108-z.

The present study aimed to elucidate the mechanisms of organismal sensitivity and/or physiological adaptation in the contaminated water environment. Multigenerational cultures (F0, F1, F2) of Daphnia magna in collected stream water (OCSW), contaminated with high fecal coliform, altered the reproductive scenario (changes in first brood size timing, clutch numbers, clutch size etc.), compromised fitness (increase hemoglobin, alteration in behavior), and affected global DNA methylation (hypermethylation) without affecting survival. Using proteomics approach, we found 288 proteins in F0 and 139 proteins in F2 that were significantly differentially upregulated after OCSW exposure. The individual protein expressions, biological processes and molecular functions were mainly related to metabolic processes, development and reproduction, transport (protein/lipid/oxygen), antioxidant activity, increased globin and S-adenosylmethionine synthase protein level etc., which was further found to be connected to phenotype-dependent endpoints. The proteomics pathway analysis evoked proteasome, chaperone family proteins, neuronal disease pathways (such as, Parkinson's disease) and apoptosis signaling pathways in OCSW-F0, which might be the cause of behavioral and developmental alterations in OCSW-F0. Finally, chronic multigenerational exposure to OCSW exhibited slow physiological adaptation in most of the measured effects, including proteomics analysis, from the F0 to F2 generations. The common upregulated proteins in both generations (F0 & F2), such as, globin, vitellinogen, lipid transport proteins etc., were possibly play the pivotal role in the organism's physiological adaptation. Taken together, our results, obtained with a multilevel approach, provide new insight of the molecular mechanism in fecal coliform-induced phenotypic plasticity in Daphnia magna.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller’s sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb–O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb–O2 affinity in (sub)Arctic Steller’s sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Ethylene oxide (EO), a genotoxic industrial chemical and sterilant, forms covalent adducts with DNA and also with nucleophilic amino acids in proteins. The adduct with N-terminal valine in globin [N-(2-hydroxyethyl)valine (HEV)] has been used in biomonitoring of cumulative exposures to EO. Here we studied in rats the fate of EO-adducted N-termini of globin after life termination of the erythrocytes. Rat erythrocytes were incubated with EO to produce the HEV levels in globin at 0.4-13.2 µmol/g as determined after acidic hydrolysis. Alternative hydrolysis of the isolated globin with enzyme pronase afforded N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) and N-(2-hydroxyethyl)-L-valyl-L-histidine (HEVH), the EO-adducted N-terminal dipeptides of rat globin α- and β-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the β-chain. The EO-modified erythrocytes were then given intravenously to the recipient rats. HEVL and HEVH were found to be the ultimate cleavage products excreted in the rat urine. Finally, rats were dosed intraperitoneally with EO, 50 mg/kg. Herein, the initial level of globin-bound HEVL (11.7 ± 1.3 nmol/g) decreased almost linearly over 60 days corresponding to the life span of rat erythrocytes. Daily urinary excretion of HEVL was almost constant for 30-40 days, decreasing faster in the subsequent phase of elimination. Recoveries of the total urinary HEVL from its globin-bound form were 84 ± 6% and 101 ± 17% after administrations of EO and the EO-modified erythrocytes, respectively. In conclusion, urinary HEVL appears to be a promising novel non-invasive biomarker of human exposures to EO.

Here, we report the preparation and photo-physical characterization of hexa-coordinated vertebrate globins, human neuroglobin (hNgb) and cytoglobin (hCygb), with the native iron protoporphyrin IX (FePPIX) cofactor replaced by a fluorescent isostructural analogue, zinc protoporphyrin IX (ZnPPIX). To facilitate insertion of ZnPPIX into hexa-coordinated globins, apoproteins prepared via butanone extraction were unfolded by the addition of GuHCl and subsequently slowly refolded in the presence of ZnPPIX. The absorption/emission spectra of ZnPPIX reconstituted hCygb are similar to those observed for ZnPPIX reconstituted myoglobin whereas the absorption and emission spectra of ZnPPIX reconstituted hNgb are blue shifted by ∼2 nm. Different steady state absorption and emission properties of ZnPPIX incorporated in hCygb and hNgb are consistent with distinct hydrogen bonding interactions between ZnPPIX and the globin matrix. The fluorescence lifetime of ZnPPIX in hexa-coordinated globins is bimodal pointing towards increased heterogeneity of the heme binding cavity in hCygb and hNgb. ZnPPIX reconstituted Ngb binds to cytochrome c with the same affinity as reported for the native protein, suggesting that fluorescent analogues of Cygb and Ngb can be readily employed to monitor interactions between vertebrate hexa-coordinated globins and other proteins.

The multiple effect of pristine graphene (pG) toxicity on cardiovascular developmental defects was assessed using zebrafish as a model. Recently, the nanotoxicity is emerging as a critical issue, and it is more significant in embryogenesis. Especially, graphene and its derivatives have attracted a lot of interest in biomedical applications. However, very little is known about the toxic effects of pG which has been widely used carbon nanomaterial according to concentration and its effects on biological and cardiovascular development. In the present study, we examined the development of zebrafish embryos by exposing to pG (5, 10, 15, 20 and 25 μg/L) under different developmental toxicity end-points such as cardiotoxicity, cardiovascular defect, retardation of cardiac looping, apoptosis and globin expression analysis. For this, the developmental cardiotoxicity of pG at different concentrations and the specific cardiovascular defects thereof were elucidated for the first time. As a result, the exposure to pG was found to be a potential risk factor to cardiovascular system of zebrafish embryos. However, a further study on the variations of physical, molecular properties and mechanisms of nanotoxicity which vary depending on production method and surface functionalization is required. In addition, the potential risks of pG flakes to aquatic organisms and human health should be considered or checked before releasing them to the environment.

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34

The members of the globin superfamily are a classical model system to investigate gene evolution and their fates as well as the diversity of protein function. One of the best-known globins is myoglobin (Mb), which is mainly expressed in heart muscle and transports oxygen from the sarcolemma to the mitochondria. Most vertebrates harbor a single copy of the myoglobin gene, but some fish species have multiple myoglobin genes. Phylogenetic analyses indicate an independent emergence of multiple myoglobin genes, whereby the origin is mostly the last common ancestor of each order. By analyzing different transcriptome data sets, we found at least 15 multiple myoglobin genes in the polypterid gray bichir (Polypterus senegalus) and reedfish (Erpetoichthys calabaricus). In reedfish, the myoglobin genes are expressed in a broad range of tissues but show very different expression values. In contrast, the Mb genes of the gray bichir show a rather scattered expression pattern; only a few Mb genes were found expressed in the analyzed tissues. Both, gray bichir and reedfish possess lungs which enable them to inhabit shallow and swampy waters throughout tropical Africa with frequently fluctuating and low oxygen concentrations. The myoglobin repertoire probably reflects the molecular adaptation to these conditions. The sequence divergence, the substitution rate, and the different expression pattern of multiple myoglobin genes in gray bichir and reedfish imply different functions, probably through sub- and neofunctionalization during evolution.

Cytoglobin (Cygb) is a globin molecule that is ubiquitously expressed in all tissues and has a protective role under oxidative stress. It has also been demonstrated to be effective in the treatment of alcoholic fatty liver disease (AFLD). In order to study the molecular mechanisms underlying its beneficial effects for the treatment of alcoholic liver, two-dimensional electrophoresis and mass spectrometric analysis were performed on serum and liver tissues from an in vivo rat model of AFLD. A total of 26 differentially expressed proteins were identified in the serum and 20 differentially expressed proteins were identified in liver specimens. Using online bioinformatics tools, it was indicated that these differentially expressed proteins were primarily associated with pathways including binding and uptake of ligands by scavenger receptors, response to corticosteroid, plasma lipoprotein remodeling, regulation of complement cascade, hydrogen peroxide catabolic process, as well as response to nutrient and monosaccharide. The present results suggested that recombinant human Cygb exerts its role in the treatment of AFLD primarily through affecting nutrient metabolism, monocarboxylic acid biosynthesis, regulation of glutathione expression, plasma lipoprotein remodeling and removal of metabolic waste from the blood.

Human tauopathies represent a group of neurodegenerative disorders, characterized by abnormal hyperphosphorylation and aggregation of tau protein, which ultimately cause neurodegeneration. The aberrant tau hyperphosphorylation is mostly attributed to the kinases/phosphatases imbalance, which is majorly contributed by the generation of reactive oxygen species (ROS). Globin(s) represent a well-conserved group of proteins which are involved in O2 management, regulation of cellular ROS in different cell types. Similarly, Drosophila globin1 (a homologue of human globin) with its known roles in oxygen management and development of nervous system exhibits striking similarities with the mammalian neuroglobin. Several recent evidences support the hypothesis that neuroglobins are associated with Alzheimer's disease pathogenesis. We herein noted that targeted expression of human-tau induces the cellular level of Glob1 protein in Drosophila tauopathy models. Subsequently, RNAi mediated restored level of Glob1 restricts the pathogenic effect of human-tau by minimizing its hyperphosphorylation via GSK-3β/p-Akt and p-JNK pathways. In addition, it also activates the Nrf2-keap1-ARE cascade to stabilize the tau-mediated increased level of ROS. These two parallel cellular events provide a significant rescue against human tau-mediated neurotoxicity in the fly models. For the first time we report a direct involvement of an oxygen sensing globin gene in tau etiology. In view of the fact that human genome encodes for the multiple Globin proteins including a nervous system specific neuroglobin; and therefore, our findings may pave the way to investigate if the conserved oxygen sensing globin gene(s) can be exploited in devising novel therapeutic strategies against tauopathies.

This work is intended to study the radioprotective effect of palladium α-lipoic acid nano-complex (PLAC) on hemoglobin molecule in vitro. Blood samples were obtained from adult male rats weighing 120–150 g after dissection, using heparinized needles. Each blood sample was divided into four groups; the first group was kept untreated as control, palladium α-lipoic acid (PLAC) was added to the second group at concentration 2% v/v, the third group was exposed to 100 Gy gamma radiation and the forth group was irradiated with the addition of PLAC. Hemoglobin was extracted and prepared for measurement. The effects on the hemoglobin molecule were evaluated by FTIR and UV–visible spectroscopy. The results showed that PLAC increases the optical energy gap of the transition of the amino acid side chains and affects the spatial distribution of the globin part. Gamma radiation affects mainly the globin part, results in unfolding of the protein structure and perturbation in the relative orientation of the transition dipole moments. Addition of PLAC to the blood samples prior to irradiation was shown to provide protective effects which can be attributed to its ability to neutralize the free radicals.

This study evaluated the effects of globin and spray-dried porcine plasma (SDPP) on growth performance, digestibility, nitrogen retention, energy retention efficiency (ERE) and intestinal morphology of broiler chickens. A total of 336-day-old male broiler chickens were reared from 1 to 40 days of age and fed 3 diets (8 replicates/diet, 14 birds/replicate) during 3 feeding phases: starter (1-12 days), grower (12-25 days) and finisher (25-40 days). Isonitrogenous diets were formulated by replacing gluten protein isolate contained in the control diet (C diet) with 2% (starter) or 1% (grower and finisher) spray-dried porcine plasma in the plasma diet (SDPP diet). The globin diet (G diet) was obtained by adding globin on the top of C diet at a dose of 0.08% for the whole rearing period. Total tract apparent digestibility (aD), nitrogen retention and ERE were assessed during the three growing phases. At 12 and 40 days of age, one bird per pen was slaughtered to sample gut, liver, spleen and bursa of Fabricius for histomorphological investigations. The SDPP diet increased body weights of chickens at 12 (+60 g; p

Neuroglobin (Ngb), the third member of the globin family, was discovered in human and murine brains in 2000. This monomeric globin is structurally similar to myoglobin (Mb) and hemoglobin (Hb) α and β subunits, but it hosts a bis-histidyl six-coordinated heme-Fe atom. Therefore, the heme-based reactivity of Ngb is modulated by the dissociation of the distal HisE7-heme-Fe bond, which reflects in turn the redox state of the cell. The high Ngb levels (~100–200 μM) present in the retinal ganglion cell layer and in the optic nerve facilitate the O2 buffer and delivery. In contrast, the very low levels of Ngb (~1 μM) in most tissues and organs support (pseudo-)enzymatic properties including NO/O2 metabolism, peroxynitrite and free radical scavenging, nitrite, hydroxylamine, hydrogen sulfide reduction, and the nitration of aromatic compounds. Here, structural and (pseudo-)enzymatic properties of Ngb, which are at the root of tissue and organ protection, are reviewed, envisaging a possible role in the protection from neuronal degeneration of the retina and the optic nerve.

Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of β-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human β-globin gene (β-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in β-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of β-hemoglobinopathies.

Background How vascular systems and their respiratory pigments evolved is still debated. While many animals present a vascular system, hemoglobin exists as a blood pigment only in a few groups (vertebrates, annelids, a few arthropod and mollusk species). Hemoglobins are formed of globin sub-units, belonging to multigene families, in various multimeric assemblages. It was so far unclear whether hemoglobin families from different bilaterian groups had a common origin. Results To unravel globin evolution in bilaterians, we studied the marine annelid Platynereis dumerilii, a species with a slow evolving genome. Platynereis exhibits a closed vascular system filled with extracellular hemoglobin. Platynereis genome and transcriptomes reveal a family of 19 globins, nine of which are predicted to be extracellular. Extracellular globins are produced by specialized cells lining the vessels of the segmental appendages of the worm, serving as gills, and thus likely participate in the assembly of a previously characterized annelid-specific giant hemoglobin. Extracellular globin mRNAs are absent in smaller juveniles, accumulate considerably in growing and more active worms and peak in swarming adults, as the need for O2 culminates. Next, we conducted a metazoan-wide phylogenetic analysis of globins using data from complete genomes. We establish that five globin genes (stem globins) were present in the last common ancestor of bilaterians. Based on these results, we propose a new nomenclature of globins, with five clades. All five ancestral stem-globin clades are retained in some spiralians, while some clades disappeared early in deuterostome and ecdysozoan evolution. All known bilaterian blood globin families are grouped in a single clade (clade I) together with intracellular globins of bilaterians devoid of red blood. Conclusions We uncover a complex “pre-blood” evolution of globins, with an early gene radiation in ancestral bilaterians. Circulating hemoglobins in various bilaterian groups evolved convergently, presumably in correlation with animal size and activity. However, all hemoglobins derive from a clade I globin, or cytoglobin, probably involved in intracellular O2 transit and regulation. The annelid Platynereis is remarkable in having a large family of extracellular blood globins, while retaining all clades of ancestral bilaterian globins.

Antonini and Brunori’s 1971 book “Hemoglobin and Myoglobin in Their Reactions with Ligands” was a truly remarkable publication that summarized almost 100 years of research on O(2) binding to these globins. Over the ensuing 50 years, ultra-fast laser photolysis techniques, high-resolution and time resolved X-ray crystallography, molecular dynamics simulations, and libraries of recombinant myoglobin (Mb) and hemoglobin (Hb) variants have provided structural interpretations of O(2) binding to these proteins. The resultant mechanisms provide quantitative descriptions of the stereochemical factors that govern overall affinity, including proximal and distal steric restrictions that affect iron reactivity and favorable positive electrostatic interactions that preferentially stabilize bound O(2). The pathway for O(2) uptake and release by Mb and subunits of Hb has been mapped by screening libraries of site-directed mutants in laser photolysis experiments. O(2) enters mammalian Mb and the α and β subunits of human HbA through a channel created by upward and outward rotation of the distal His at the E7 helical position, is non-covalently captured in the interior of the distal cavity, and then internally forms a bond with the heme Fe(II) atom. O(2) dissociation is governed by disruption of hydrogen bonding interactions with His(E7), breakage of the Fe(II)-O(2) bond, and then competition between rebinding and escape through the E7-gate. The structural features that govern the rates of both the individual steps and overall reactions have been determined and provide the framework for: (1) defining the physiological functions of specific globins and their evolution; (2) understanding the clinical features of hemoglobinopathies; and (3) designing safer and more efficient acellular hemoglobin-based oxygen carriers (HBOCs) for transfusion therapy, organ preservation, and other commercially relevant O(2) transport and storage processes.

Vertebrate hemoglobin (Hb) and myoglobin (Mb) were among the first proteins whose structures and sequences were determined over 50 years ago. In the subsequent pregenomic period, numerous related proteins came to light in plants, invertebrates and bacteria, that shared the myoglobin fold, a signature sequence motif characteristic of a 3-on-3 α-helical sandwich. Concomitantly, eukaryote and bacterial globins with a truncated 2-on-2 α-helical fold were discovered. Genomic information over the last 20 years has dramatically expanded the list of known globins, demonstrating their existence in a limited number of archaeal genomes, a majority of bacterial genomes and an overwhelming majority of eukaryote genomes. In vertebrates, 6 additional globin types were identified, namely neuroglobin (Ngb), cytoglobin (Cygb), globin E (GbE), globin X (GbX), globin Y (GbY) and androglobin (Adgb). Furthermore, functions beyond the familiar oxygen transport and storage have been discovered within the vertebrate globin family, including NO metabolism, peroxidase activity, scavenging of free radicals, and signaling functions. The extension of the knowledge on globin functions suggests that the original roles of bacterial globins must have been enzymatic, involved in defense against NO toxicity, and perhaps also as sensors of O2, regulating taxis away or towards high O2 concentrations. In this review, we aimed to discuss the evolution and remarkable functional diversity of vertebrate globins with particular focus on the variety of non-canonical expression sites of mammalian globins and their according impressive variability of atypical functions.

Animals depend on the sequential oxidation of organic molecules to survive; thus, oxygen-carrying/transporting proteins play a fundamental role in aerobic metabolism. Globins are the most common and widespread group of respiratory proteins. They can be divided into three types: circulating intracellular, noncirculating intracellular, and extracellular, all of which have been reported in annelids. The diversity of oxygen transport proteins has been underestimated across metazoans. We probed 250 annelid transcriptomes in search of globin diversity in order to elucidate the evolutionary history of this gene family within this phylum. We report two new globin types in annelids, namely androglobins and cytoglobins. Although cytoglobins and myoglobins from vertebrates and from invertebrates are referred to by the same name, our data show they are not genuine orthologs. Our phylogenetic analyses show that extracellular globins from annelids are more closely related to extracellular globins from other metazoans than to the intracellular globins of annelids. Broadly, our findings indicate that multiple gene duplication and neo-functionalization events shaped the evolutionary history of the globin family.

β-thalassemia is a lethal inherited disease resulting from β-globin gene mutations. Severe β-thalassemia requires regular blood transfusions. Other active interventions, including iron chelating, stem cell transplantation and gene therapy, have remarkably improved the quality of life and prolonged the survival of patients with transfusion-dependent β-thalassemia, but all with significant limitations and complications. MicroRNAs (miRNAs), encoded by a class of endogenous genes, are found to play important roles in regulating globin expression. Among the miRNAs of particular interest related to β-thalassemia, miR-15a/16-1, miR-486-3p, miR-26b, miR-199b-5p, miR-210, miR-34a, miR-138, miR-326, let-7, and miR-17/92 cluster elevate γ-globin expression, while miR-96, miR-146a, miR-223-3p, and miR-144 inhibit γ-globin expression. A couple of miRNAs, miR-144 and miR-150, repress α-globin expression, whereas miR-451 induces α-, β- and γ-globin expression. Single nucleotide polymorphism in miRNA genes or their targeted genes might also contribute to the abnormal expression of hemoglobin. Moreover, changes in the expression of miR-125b, miR-210, miR-451, and miR-609 reflect the severity of anemia and hemolysis in β-thalassemia patients. These results suggest that miRNAs are potential biomarkers for the diagnosis and prognosis of β-thalassemia, and miRNA-based therapeutic strategy might be used as a coordinated approach for effectively treating β-thalassemia.

Neuroglobin is the third member of the globin family to be identified in 2000 in neurons of both human and mouse nervous systems. Neuroglobin is an oxygen-binding globin found in neurons within the central nervous system as well as in peripheral neurons, that produces a protective effect against hypoxic/ischemic damage induced by promoting oxygen availability within the mitochondria. Numerous investigations have demonstrated that impaired neuroglobin functioning is implicated in the pathogenesis of multiple neurodegenerative disorders. Several in vitro and animal studies have reported the potential of neuroglobin upregulation in improving the neuroprotection through modulation of mitochondrial functions, such as ATP production, clearing reactive oxygen species (ROS), promoting the dynamics of mitochondria, and controlling apoptosis. Neuroglobin acts as a stress-inducible globin, which has been associated hypoxic/ischemic insults where it acts to protect the heart and brain, providing a wide range of applicability in the treatment of human disorders. This review article discusses normal physiological functions of neuroglobin in mitochondria-associated pathways, as well as outlining how dysregulation of neuroglobin is associated with the pathogenesis of neurodegenerative disorders.

We have recently reported that, after in vivo hematopoietic stem cell/progenitor (HSPC) transduction with HDAd5/35++ vectors, SB100x transposase-mediated γ-globin gene addition achieved 10%–15% γ-globin of adult mouse globin, resulting in significant but incomplete phenotypic correction in a thalassemia intermedia mouse model. Furthermore, genome editing of a γ-globin repressor binding site within the γ-globin promoter by CRISPR-Cas9 results in efficient reactivation of endogenous γ-globin. Here, we aimed to combine these two mechanisms to obtain curative levels of γ-globin after in vivo HSPC transduction. We generated a HDAd5/35++ adenovirus vector (HDAd-combo) containing both modules and tested it in vitro and after in vivo HSPC transduction in healthy CD46/β-YAC mice and in a sickle cell disease mouse model (CD46/Townes). Compared to HDAd vectors containing either the γ-globin addition or the CRISPR-Cas9 reactivation units alone, in vivo HSC transduction of CD46/Townes mice with the HDAd-combo resulted in significantly higher γ-globin in red blood cells, reaching 30% of that of adult human α and β(S) chains and a complete phenotypic correction of sickle cell disease.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues., Globin loci harbor genes that are expressed embryonically and silenced postnatally. Here the authors show that zeta-globin silencing depends upon selective hypoacetylation of its TAD subdomain, which blocks its interaction with the alpha-globin super-enhancer, and zeta-globin can be reactivated by acetylation.

The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.

Novel aminonaphthylcysteine (ANC) adducts, formed via naphthylnitrenium ions and/or their metabolic precursors in the biotransformation of naphthylamines (NA) and nitronaphthalenes (NN), were identified and quantified in globin of rats dosed intraperitoneally with 0.16 mmol/kg b.w. of 1-NA, 1-NN, 2-NA and 2-NN. Using HPLC-ESI-MS2 analysis of the globin hydrolysates, S-(1-amino-2-naphthyl)cysteine (1A2NC) together with S-(4-amino-1-naphthyl)cysteine (4A1NC) were found in rats given 1-NA or 1-NN, and S-(2-amino-1-naphthyl)cysteine (2A1NC) in those given 2-NA or 2-NN. The highest level of ANC was produced by the most mutagenic and carcinogenic isomer 2-NA (35.8 ± 5.4 nmol/g globin). The ratio of ANC adduct levels for 1-NA, 1-NN, 2-NA and 2-NN was 1:2:100:3, respectively. Notably, the ratio of 1A2NC:4A1NC in globin of rats dosed with 1-NA and 1-NN differed significantly (2:98 versus 16:84 respectively), indicating differences in mechanism of the adduct formation. Moreover, aminonaphthylmercapturic acids, formed via conjugation of naphthylnitrenium ions and/or their metabolic precursors with glutathione, were identified in the rat urine. Their amounts excreted after dosing rats with 1-NA, 1-NN, 2-NA and 2-NN were in the ratio 1:100:40:2, respectively. For all four compounds tested, haemoglobin binding index for ANC was several-fold higher than that for the sulphinamide adducts, generated via nitrosoarene metabolites. Due to involvement of electrophilic intermediates in their formation, ANC adducts in globin may become toxicologically more relevant biomarkers of cumulative exposure to carcinogenic or non-carcinogenic arylamines and nitroarenes than the currently used sulphinamide adducts.

Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (HbF) is a promising approach for the management of β-thalassemia and sickle cell disease (SCD). We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules that could induce HbF, which resulted in identification of the hit compound 1. Exploration of structure–activity relationships and optimization of ADME properties led to 2-azaspiro[3.3]heptane derivative 18, which is more rigid and has a unique structure. In vivo using cynomolgus monkeys, compound 18 induced a significant dose-dependent increase in globin switching, with developable properties. Moreover, compound 18 showed no genotoxic effects and was much safer than hydroxyurea. These findings could facilitate the development of effective new therapies for the treatment of β-hemoglobinopathies, including SCD.

Background The tissue regeneration process requires high oxygen and energy levels. Cytoglobin (Cygb) is a member of the globin family, which has the ability to bind oxygen, plays a role in dealing with oxidative stress, and carries oxygen into the mitochondria. Energy production for tissue regeneration is associated with mitochondria—especially mitochondrial biogenesis. The peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha protein helps to regulate mitochondrial biogenesis. House geckos (Hemidactylus platyurus) are reptiles that have the ability to regenerate the tissue in their tails. House geckos were selected as the animal models for this study in order to analyze the association of Cygb with oxygen supply and the association of PGC-1α with energy production for tissue regeneration. Results The growth of house gecko tails showed a slow growth at the wound healing phase, then followed by a fast growth after wound healing phase of the regeneration process. While Cygb mRNA expression reached its peak at the wound healing phase and slowly decreased until the end of the observation. PGC-1α mRNA was expressed and reached its peak earlier than Cygb. Conclusions The expressions of both the Cygb and PGC-1α genes were relatively high compared to the control group. We therefore suggest that Cygb and PGC-1α play an important role during the tissue regeneration process.

Significance: Cytoglobin (Cygb) was discovered as a new addition to the globin superfamily and subsequently identified to have potent nitric oxide (NO) dioxygenase function. Cygb plays a critical role in the oxygen-dependent regulation of NO levels and vascular tone. Recent Advances: In recent years, the mechanism of the Cygb-mediated NO dioxygenation has been studied in isolated protein, smooth muscle cell, isolated blood vessel, and in vivo animal model systems. Studies in Cygb(−/−) mice have demonstrated that Cygb plays a critical role in regulating blood pressure and vascular tone. This review summarizes advances in the knowledge of NO dioxygenation/metabolism regulated by Cygb. Advances in measurement of NO diffusion dynamics across blood vessels and kinetic modeling of Cygb-mediated NO dioxygenation are summarized. The oxygen-dependent regulation of NO degradation by Cygb is also reviewed along with how Cygb paradoxically generates NO from nitrite under anaerobic conditions. The important role of Cygb in the regulation of vascular function and disease is reviewed. Critical Issues: Cygb is a more potent NO dioxygenase (NOD) than previously known globins with structural differences in heme coordination and environment, conferring it with a higher rate of reduction and more rapid process of NO dioxygenation with unique oxygen dependence. Various cellular reducing systems regenerate the catalytic oxyferrous Cygb species, supporting a high rate of NO dioxygenation. Future Directions: There remains a critical need to further characterize the factors and processes that modulate Cygb-mediated NOD function, and to develop pharmacological or other approaches to modulate Cygb function and expression.

In the freezing waters of the Southern Ocean, Antarctic teleost fish, the Notothenioidei, have developed unique adaptations to cope with cold, including, at the extreme, the loss of hemoglobin in icefish. As a consequence, icefish are thought to be the most vulnerable of the Antarctic fish species to ongoing ocean warming. Some icefish also fail to express myoglobin but all appear to retain neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X. Despite the lack of the inducible heat shock response, Antarctic notothenioid fish are endowed with physiological plasticity to partially compensate for environmental changes, as shown by numerous physiological and genomic/transcriptomic studies over the last decade. However, the regulatory mechanisms that determine temperature/oxygen-induced changes in gene expression remain largely unexplored in these species. Proteins such as globins are susceptible to environmental changes in oxygen levels and temperature, thus playing important roles in mediating Antarctic fish adaptations. In this study, we sequenced the full-length transcripts of myoglobin, neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X from the Antarctic red-blooded notothenioid Trematomus bernacchii and the white-blooded icefish Chionodraco hamatus and evaluated transcripts levels after exposure to high temperature and low oxygen levels. Basal levels of globins are similar in the two species and both stressors affect the expression of Antarctic fish globins in brain, retina and gills. Temperature up-regulates globin expression more effectively in white-blooded than in red-blooded fish while hypoxia strongly up-regulates globins in red-blooded fish, particularly in the gills. These results suggest globins function as regulators of temperature and hypoxia tolerance. This study provides the first insights into globin transcriptional changes in Antarctic fish.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

Background Hemoglobin (Hb) subunits are composed of the specific functional prosthetic group "heme'' and a protein moiety "globin". Bird Hbs are functionally similar to mammalian Hbs but they are structurally dissimilar with mammalian. The insufficient structural studies on avian Hbs limit us to understand their degree of adaptation to such critical environments. The Great Cormorant (GCT) can fly and swim, the dual characteristic of GCT leads to study the sturcture of hemoglobin. Objective To determine the crystal structure of Great Cormorant Hemoglobin and to compare its three dimensional structure with other high and low oxygen affinity hemoglobin species to understand its characteristic features of high oxygen affinity. Method The GCT hemoglobin has been purified, crystallized and data sets were processed using iMosflm. The integrated data has been solved using Molecular replacement method using Graylag hemoglobin (1FAW) as the template. The structure has been deposited in Protein Data Bank with PDB code: 3WR1. Results In order to characterize the tertiary and quaternary structural differences, the structure of cormorant hemoglobin is compared with GLG, BHG and human Hb. The larger variation observed between GCT and human Hb indicates that GCT Hb differs remarkably from human. The α1β1 interface of Great cormorant Hb is similar to bar-headed goose Hb with few amino acid substitutions. It has been found that the interaction which is common among avian hemoglobins (α119 Pro- β55Leu) is altered by Ala 119 in GCT. This intra-dimer contact (α119 Pro - β 55 Leu) disruption leads to high oxygen affinity in BGH Hb. In cormorant, GLG and human the proline is unchanged but interestingly, in cormorant Hb, the β55 position was found to be Thr instead of Leu. Similar kind of substitutions (β 55 Leu - Ser) observed in Andean goose Hb structure leads to elevated oxygen affinity between Hb-O2. To our surprise, such type of substitution at β 55 (Thr) in cormorant Hb confirms that it is comparable with Andean goose Hb structure. Thus the sequence, structural differences at alpha, beta heme pocket and interface contacts confirms that GCT adopts high oxygen affinity conformation. Conclusion The three dimensional structure of Great cormorant hemoglobin has been investigated to understand its unique structural features to adopt during hypoxia condition. By comparing the sequence and overall structural similarities with high and low oxygen affinity species, it appears that GCT has more possibilities to subsist with low oxygen demand.

The intermolecular interaction of flufenamic acid (Hfluf) with two model proteins i.e., hemoglobin and lysozyme was explored using fluorescence, UV-vis, circular dichroism, DLS, and molecular docking techniques. The corroborative spectroscopic techniques suggested efficient binding of Hfluf to both the proteins. The S-V plot in Hb-Hfluf system showed positive deviation highlighting the presence of both static and dynamic quenching. Hence, ground state complex model and sphere of action quenching model were used for the study. In Lyz-Hfluf system, a linear S-V plot was obtained indicating the presence of a single quenching mechanism. FRET study suggested a high probability of energy transfer from Hb/Lyz to Hfluf. Our thermodynamic results revealed that binding reaction in both the systems was exothermic and spontaneous. The UV-vis spectroscopy demonstrated that the binding of Hfluf affected the globin, Soret and oxy-bands of Hb along with globin band and polypeptide backbone of Lyz. CD spectra revealed the enhancement of ɑ-helicity in Lyz and decrease in case of Hb whereas the Rh values of proteins from DLS experiment corroborated the CD findings. 3-D fluorescence spectra highlighted the conformational changes upon binding whereas docking studies predicted the active binding site of both the proteins as the binding site of Hfluf.