Molecular Interactions between Neuroglobin and Cytochrome c: Possible Mechanisms of Antiapoptotic Defense in Neuronal Cells.

Neuroglobin, which is a heme protein from the globin family that is predominantly expressed in nervous tissue, can promote a neuronal survivor. However, the molecular mechanisms underlying the neuroprotective function of Ngb remain poorly understood to this day. The interactions between neuroglobin and mitochondrial cytochrome c may serve as at least one of the mechanisms of neuroglobin-mediated neuroprotection. Interestingly, neuroglobin and cytochrome c possibly can interact with or without electron transfer both in the cytoplasm and within the mitochondria. This review provides a general picture of molecular interactions between neuroglobin and cytochrome c based on the recent experimental and computational work on neuroglobin and cytochrome c interactions.

Nitric Oxide and Globin Glb1 Regulate Fusarium oxysporum Infection of Arabidopsis thaliana.

Plants continuously interact with fungi, some of which, such as Fusarium oxysporum, are lethal, leading to reduced crop yields. Recently, nitric oxide (NO) has been found to play a regulatory role in plant responses to F. oxysporum, although the underlying mechanisms involved are poorly understood. In this study, we show that Arabidopsis mutants with altered levels of phytoglobin 1 (Glb1) have a higher survival rate than wild type (WT) after infection with F. oxysporum, although all the genotypes analyzed exhibited a similar fungal burden. None of the defense responses that were analyzed in Glb1 lines, such as phenols, iron metabolism, peroxidase activity, or reactive oxygen species (ROS) production, appear to explain their higher survival rates. However, the early induction of the PR genes may be one of the reasons for the observed survival rate of Glb1 lines infected with F. oxysporum. Furthermore, while PR1 expression was induced in Glb1 lines very early on the response to F. oxysporum, this induction was not observed in WT plants.

Ligand-Based Regulation of Dynamics and Reactivity of Hemoproteins.

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme-albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins.

GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

Segregation of α- and ß-Globin Gene Cluster in Vertebrate Evolution: Chance or Necessity?

The review is devoted to the patterns of evolution of α- and ß-globin gene domains. A hypothesis is presented according to which segregation of the ancestral cluster of α/ß-globin genes in Amniota occurred due to the performance by α-globins and ß-globins of non-canonical functions not related to oxygen transport.

Low levels of nitric oxide promotes heme maturation into several hemeproteins and is also therapeutic.

Nitric oxide (NO) is a signal molecule and plays a critical role in the regulation of vascular tone, displays anti-platelet and anti-inflammatory properties. While our earlier and current studies found that low NO doses trigger a rapid heme insertion into immature heme-free soluble guanylyl cyclase ß subunit (apo-sGCß), resulting in a mature sGC-αß heterodimer, more recent evidence suggests that low NO doses can also trigger heme-maturation of hemoglobin and myoglobin. This low NO phenomena was not only limited to sGC and the globins, but was also found to occur in all three nitric oxide synthases (iNOS, nNOS and eNOS) and Myeloperoxidase (MPO). Interestingly high NO doses were inhibitory to heme-insertion for these hemeproteins, suggesting that NO has a dose-dependent dual effect as it can act both ways to induce or inhibit heme-maturation of key hemeproteins. While low NO stimulated heme-insertion of globins required the presence of the NO-sGC-cGMP signal pathway, iNOS heme-maturation also required the presence of an active sGC. These effects of low NO were significantly diminished in the tissues of double (n/eNOS-/-) and triple (n/i/eNOS-/-) NOS knock out mice where lung sGC was found be heme-free and the myoglobin or hemoglobin from the heart/lungs were found be low in heme, suggesting that loss of endogenous NO globally impacts the whole animal and that this impact of low NO is both essential and physiologically relevant for hemeprotein maturation. Effects of low NO were also found to be protective against ischemia reperfusion injury on an ex vivo lung perfusion (EVLP) system prior to lung transplant, which further suggests that low NO levels are also therapeutic.

Nitric Oxide Trickle Drives Heme into Hemoglobin and Muscle Myoglobin.

Ever since the days of NO being proclaimed as the "molecule of the year", the molecular effects of this miracle gas on the globins have remained elusive. While its vasodilatory role in the cardiopulmonary system and the vasculature is well recognized, the molecular underpinnings of the NO-globin axis are incompletely understood. We show, by transwell co-culture of nitric oxide (NO) generating, HEK eNOS/nNOS cells, and K562 erythroid or C2C12 muscle myoblasts, that low doses of NO can effectively insert heme into hemoglobin (Hb) and myoglobin (Mb), making NO not only a vasodilator, but also a globin heme trigger. We found this process to be dependent on the NO flux, occurring at low NO doses and fading at higher doses. This NO-triggered heme insertion occurred into Hb in just 30 min in K562 cells and into muscle Mb in C2C12 myoblasts between 30 min and 1 h, suggesting that the classical effect of NO on upregulation of globin (Hb or Mb) is just not transcriptional, but may involve sufficient translational events where NO can cause heme-downloading into the apo-globins (Hb/Mb). This effect of NO is unexpected and highlights its significance in maintaining globins in its heme-containing holo-form, where such heme insertions might be required in the circulating blood or in the muscle cells to perform spontaneous functions.

Role of Nuclear Receptors in Controlling Erythropoiesis.

Nuclear receptors (NRs), are a wide family of ligand-regulated transcription factors sharing a common modular structure composed by an N-terminal domain and a ligand-binding domain connected by a short hinge linker to a DNA-binding domain. NRs are involved in many physiological processes, including metabolism, reproduction and development. Most of them respond to small lipophilic ligands, such as steroids, retinoids, and phospholipids, which act as conformational switches. Some NRs are still "orphan" and the search for their ligands is still ongoing. Upon DNA binding, NRs can act both as transcriptional activators or repressors of their target genes. Theoretically, the possibility to modulate NRs activity with small molecules makes them ideal therapeutic targets, although the complexity of their signaling makes drug design challenging. In this review, we discuss the role of NRs in erythropoiesis, in both homeostatic and stress conditions. This knowledge is important in view of modulating red blood cells production in disease conditions, such as anemias, and for the expansion of erythroid cells in culture for research purposes and for reaching the long-term goal of cultured blood for transfusion.

Challenges in the Diagnosis of Beta-thalassemia Syndrome: The Importance of Molecular Diagnosis.

Patients with heterozygous ß-thalassemia are generally asymptomatic. However, the intermediate phenotype is uncommon, and patients require further investigation to confirm the diagnosis. We describe a 32-year-old woman (gravida 3, para 2) with heterozygous ß-thalassemia who presented with symptomatic anemia and had a history of frequent blood transfusion in each pregnancy. Physical examination was unremarkable. Laboratory results at presentation showed hypochromic microcytic anemia with reticulocytosis. Molecular study revealed intermedia phenotypes resulting from coinheritance of heterozygous ß-globin chain mutation (IVS1-5) and a rare heterozygous α-globin triplication (αααanti-3.7). In this case report, we discuss the laboratory diagnostic approaches and the challenges faced in investigating this case.

Improving the cell-membrane-penetrating activity of globins by introducing positive charges on protein surface: A case study of sperm whale myoglobin.

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.

The X-ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand-binding sites.

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells.

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.

Kinetic mechanisms for O2 binding to myoglobins and hemoglobins.

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 O2 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 O2 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 O2. The pathway for O2 uptake and release by Mb and subunits of Hb has been mapped by screening libraries of site-directed mutants in laser photolysis experiments. O2 enters mammalian Mb and the α and ß 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. O2 dissociation is governed by disruption of hydrogen bonding interactions with His (E7), breakage of the Fe(II)-O2 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 O2 transport and storage processes.

CRISPR-Cas9 to induce fetal hemoglobin for the treatment of sickle cell disease.

Genome editing is potentially a curative technique available to all individuals with ß-hemoglobinopathies, including sickle cell disease (SCD). Fetal hemoglobin (HbF) inhibits sickle hemoglobin (HbS) polymerization, and it is well described that naturally occurring hereditary persistence of HbF (HPFH) alleviates disease symptoms; therefore, reawakening of developmentally silenced HbF in adult red blood cells (RBCs) has long been of interest as a therapeutic strategy. Recent advances in genome editing platforms, particularly with the use of CRISPR-Cas9, have paved the way for efficient HbF induction through the creation of artificial HPFH mutations, editing of transcriptional HbF silencers, and modulating epigenetic intermediates that govern HbF expression. Clinical trials investigating BCL11A enhancer editing in patients with ß-hemoglobinopathies have demonstrated promising results, although follow-up is short and the number of patients treated to date is low. While practical, economic, and clinical challenges of genome editing are well recognized by the scientific community, potential solutions to overcome these hurdles are in development. Here, we review the recent progress and obstacles yet to be overcome for the most effective and feasible HbF reactivation practice using CRISPR-Cas9 genome editing as a curative strategy for patients with SCD.

Conservative and Atypical Ferritins of Sponges.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

Structural modeling of a novel membrane-bound globin-coupled sensor in Geobacter sulfurreducens.

Globin-coupled sensors (GCS) usually consist of three domains: a sensor/globin, a linker, and a transmitter domain. The globin domain (GD), activated by ligand binding and/or redox change, induces an intramolecular signal transduction resulting in a response of the transmitter domain. Depending on the nature of the transmitter domain, GCSs can have different activities and functions, including adenylate and di-guanylate cyclase, histidine kinase activity, aerotaxis and/or oxygen sensing function. The gram-negative delta-proteobacterium Geobacter sulfurreducens expresses a protein with a GD covalently linked to a four transmembrane domain, classified, by sequence similarity, as GCS (GsGCS). While its GD is fully characterized, not so its transmembrane domain, which is rarely found in the globin superfamily. In the present work, GsGCS was characterized spectroscopically and by native ion mobility-mass spectrometry in combination with cryo-electron microscopy. Although lacking high resolution, the oligomeric state and the electron density map were valuable for further rational modeling of the full-length GsGCS structure. This model demonstrates that GsGCS forms a transmembrane domain-driven tetramer with minimal contact between the GDs and with the heme groups oriented outward. This organization makes an intramolecular signal transduction less likely. Our results, including the auto-oxidation rate and redox potential, suggest a potential role for GsGCS as redox sensor or in a membrane-bound e-/H+ transfer. As such, GsGCS might act as a player in connecting energy production to the oxidation of organic compounds and metal reduction. Database searches indicate that GDs linked to a four or seven helices transmembrane domain occur more frequently than expected.

[Sickle cell disease]. / Sichelzellkrankheit.

The term "sickle cell disease" covers a group of genetic blood disorders caused by sickle hemoglobin (HbS) alone or in combination with other variants of the ß­globin locus. Sickle cell disease occurs frequently in sub-Saharan Africa, but is also common in Turkey, Greece, Sicily, the Middle East, India, and the Americas. Polymerization of deoxygenated sickle hemoglobin leads to decreased deformability of red blood cells. These altered erythrocytes can obstruct small blood vessels and cause acute episodes of pain, hemolytic anemia, and organ damage. Complications can vary between the different genotypes and it is important to be aware of the special features of the disease. Hydroxycarbamide has been shown to reduce the morbidity and mortality of patients with sickle cell disease. New drugs and novel treatment approaches such as gene therapy are currently being tested.

A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments.

BACKGROUND: Despite the ease of conventional splicing by overlap-extension (SOEing) PCR technique in theory, when splicing more than two fragments, and especially if one of the complementary sequences is A-T rich, the attachment of the fragments would be challenging. A new rapid and highly efficient SOEing PCR assay was developed for simultaneous splicing of multiple DNA fragments and induction of site-directed mutagenesis in a single tube. METHODS: The method was adapted for splicing human beta-globin UTRs to OCT4, SOX2, KLF4, C-MYC, LIN28A, and destabilized GFP for the construction of chimeric DNA fragments for in vitro transcription. In addition, the native Kozak sequence of beta-globin (K1) was replaced by the strongest Kozak sequence (K2) using site-directed mutagenesis to enhance the expression of target genes. RESULTS: ChimericGFPd2/K1, GFPd2/K2, OCT4, and KLF4 were created by the optimized conventional SOEing PCR. The single tube method was able to create the chimeric SOX2, C-MYC, and LIN28A in high quality and quantity in comparison with the conventional SOEing PCR. Moreover, using single tube SOEing PCR, the reaction time and materials that are required in the conventional SOEing PCR were significantly reduced. Fluorescent microscopy and flow cytometry examinations indicated highly efficient translation of K2 sequence in comparison with the K1sequence. CONCLUSION: Single tube SOEing PCR is a valuable method to construct more multiple fragments with high yield. The method can successfully be applied for construction of various kinds of complex chimeric genes.

Regulation of Nitric Oxide Metabolism and Vascular Tone by Cytoglobin.

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.

Heterocellular Contact Can Dictate Arterial Function.

RATIONALE: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. OBJECTIVE: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. METHODS AND RESULTS: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. CONCLUSIONS: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.