Myoglobin Inhibits Breast Cancer Cell Fatty Acid Oxidation and Migration via Heme-dependent Oxidant Production and Not Fatty Acid Binding.

The monomeric heme protein myoglobin (Mb), traditionally thought to be expressed exclusively in cardiac and skeletal muscle, is now known to be expressed in approximately 40% of breast tumors. While Mb expression is associated with better patient prognosis, the molecular mechanisms by which Mb limits cancer progression are unclear. In muscle, Mb's predominant function is oxygen storage and delivery, which is dependent on the protein's heme moiety. However, prior studies demonstrate that the low levels of Mb expressed in cancer cells preclude this function. Recent studies propose a novel fatty acid binding function for Mb via a lysine residue (K46) in the heme pocket. Given that cancer cells can upregulate fatty acid oxidation (FAO) to maintain energy production for cytoskeletal remodeling during cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO to decrease breast cancer cell migration. We demonstrate that the stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb to disrupt Mb fatty acid binding did not reverse Mb-mediated attenuation of FAO or cell migration in these cells. In contrast, cells expressing Apo-Mb, in which heme incorporation was disrupted, showed a reversal of Mb-mediated attenuation of FAO and cell migration, suggesting that Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. To this end, we show that Mb's heme-dependent oxidant generation propagates dysregulated gene expression of migratory genes, and this is reversed by catalase treatment. Collectively, these data demonstrate that Mb decreases breast cancer cell migration, and this effect is due to heme-mediated oxidant production rather than fatty acid binding. The implication of these results will be discussed in the context of therapeutic strategies to modulate oxidant production and Mb in tumors. Highlights: Myoglobin (Mb) expression in MDA-MB-231 breast cancer cells slows migration.Mb expression decreases mitochondrial respiration and fatty acid oxidation.Mb-dependent fatty acid binding does not regulate cell migration or respiration.Mb-dependent oxidant generation decreases mitochondrial metabolism and migration.Mb-derived oxidants dysregulate migratory gene expression.

Associating protein sequence positions with the modulation of quantitative phenotypes.

Although protein sequences encode the information for folding and function, understanding their link is not an easy task. Unluckily, the prediction of how specific amino acids contribute to these features is still considerably impaired. Here, we developed a simple algorithm that finds positions in a protein sequence with potential to modulate the studied quantitative phenotypes. From a few hundred protein sequences, we perform multiple sequence alignments, obtain the per-position pairwise differences for both the sequence and the observed phenotypes, and calculate the correlation between these last two quantities. We tested our methodology with four cases: archaeal Adenylate Kinases and the organisms optimal growth temperatures, microbial rhodopsins and their maximal absorption wavelengths, mammalian myoglobins and their muscular concentration, and inhibition of HIV protease clinical isolates by two different molecules. We found from 3 to 10 positions tightly associated with those phenotypes, depending on the studied case. We showed that these correlations appear using individual positions but an improvement is achieved when the most correlated positions are jointly analyzed. Noteworthy, we performed phenotype predictions using a simple linear model that links per-position divergences and differences in the observed phenotypes. Predictions are comparable to the state-of-art methodologies which, in most of the cases, are far more complex. All of the calculations are obtained at a very low information cost since the only input needed is a multiple sequence alignment of protein sequences with their associated quantitative phenotypes. The diversity of the explored systems makes our work a valuable tool to find sequence determinants of biological activity modulation and to predict various functional features for uncharacterized members of a protein family.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD) associated chronic hemolysis promotes oxidative stress, inflammation and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD related hepatobiliary injury has not been fully elucidated yet. Here, we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane bound CD163. Hemolysis and increase in hepatic heme, hemoglobin and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that HO-1 positively regulates membrane bound CD163 expression independent of NRF2 signaling in SCD liver. We further demonstrate that of the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD associated hepatobiliary injury. Understanding the role of HO-1 in membrane bound CD163 regulation may help identify novel therapeutic targets for hemolysis induced chronic liver injury.

Carbon Monoxide Poisoning: From Microbes to Therapeutics.

Carbon monoxide (CO) poisoning leads to 50,000-100,000 emergency room visits and 1,500-2,000 deaths each year in the United States alone. Even with treatment, survivors often suffer from long-term cardiac and neurocognitive deficits, highlighting a clear unmet medical need for novel therapeutic strategies that reduce morbidity and mortality associated with CO poisoning. This review examines the prevalence and impact of CO poisoning and pathophysiology in humans and highlights recent advances in therapeutic strategies that accelerate CO clearance and mitigate toxicity. We focus on recent developments of high-affinity molecules that take advantage of the uniquely strong interaction between CO and heme to selectively bind and sequester CO in preclinical models. These scavengers, which employ heme-binding scaffolds ranging from organic small molecules to hemoproteins derived from humans and potentially even microorganisms, show promise as field-deployable antidotes that may rapidly accelerate CO clearance and improve outcomes for survivors of acute CO poisoning.

Cytoglobin regulates NO-dependent cilia motility and organ laterality during development.

Cytoglobin is a heme protein with unresolved physiological function. Genetic deletion of zebrafish cytoglobin (cygb2) causes developmental defects in left-right cardiac determination, which in humans is associated with defects in ciliary function and low airway epithelial nitric oxide production. Here we show that Cygb2 co-localizes with cilia and with the nitric oxide synthase Nos2b in the zebrafish Kupffer's vesicle, and that cilia structure and function are disrupted in cygb2 mutants. Abnormal ciliary function and organ laterality defects are phenocopied by depletion of nos2b and of gucy1a, the soluble guanylate cyclase homolog in fish. The defects are rescued by exposing cygb2 mutant embryos to a nitric oxide donor or a soluble guanylate cyclase stimulator, or with over-expression of nos2b. Cytoglobin knockout mice also show impaired airway epithelial cilia structure and reduced nitric oxide levels. Altogether, our data suggest that cytoglobin is a positive regulator of a signaling axis composed of nitric oxide synthase-soluble guanylate cyclase-cyclic GMP that is necessary for normal cilia motility and left-right patterning.

Engineering neuroglobin nitrite reductase activity based on myoglobin models.

Neuroglobin is a hemoprotein expressed in several nervous system cell lineages with yet unknown physiological functions. Neuroglobin presents a very similar structure to that of the related globins hemoglobin and myoglobin, but shows an hexacoordinate heme as compared to the pentacoordinated heme of myoglobin and hemoglobin. While several reactions of neuroglobin have been characterized in vitro, the relative importance of most of those reactions in vivo is yet undefined. Neuroglobin, like other heme proteins, can reduce nitrite to nitric oxide, providing a possible route to generate nitric oxide in vivo in low oxygen conditions. The reaction kinetics are highly dependent on the nature of the distal residue, and replacement of the distal histidine His64(E7) can increase the reaction rate constants by several orders of magnitude. However, mutation of other distal pocket positions such as Phe28(B10) or Val68(E11) has more limited impact on the rates. Computational analysis using myoglobin as template, guided by the structure of dedicated nitrite reductases like cytochrome cd1 nitrite reductase, has pointed out that combined mutations of the residues B10 and CD1 could increase the nitrite reductase activity of myoglobin, by mimicking the environment of the distal heme pocket in cytochrome cd1 nitrite reductase. As neuroglobin shows high sequence and structural homology with myoglobin, we hypothesized that such mutations (F28H and F42Y in neuroglobin) could also modify the nitrite reductase activity of neuroglobin. Here we study the effect of these mutations. Unfortunately, we do not observe in any case an increase in the nitrite reduction rates. Our results provide some further indications of nitrite reductase regulation in neuroglobin and highlight the minor but critical differences between the structure of penta- and hexacoordinate globins.

Thiol-catalyzed formation of NO-ferroheme regulates intravascular NO signaling.

Nitric oxide (NO) is an endogenously produced signaling molecule that regulates blood flow and platelet activation. However, intracellular and intravascular diffusion of NO are limited by scavenging reactions with several hemoproteins, raising questions as to how free NO can signal in hemoprotein-rich environments. We explore the hypothesis that NO can be stabilized as a labile ferrous heme-nitrosyl complex (Fe2+-NO, NO-ferroheme). We observe a reaction between NO, labile ferric heme (Fe3+) and reduced thiols to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation occurs when heme is solubilized in lipophilic environments such as red blood cell membranes or bound to serum albumin. The resulting NO-ferroheme resists oxidative inactivation, is soluble in cell membranes and is transported intravascularly by albumin to promote potent vasodilation. We therefore provide an alternative route for NO delivery from erythrocytes and blood via transfer of NO-ferroheme and activation of apo-soluble guanylyl cyclase.

Thiol catalyzed formation of NO-ferroheme regulates canonical intravascular NO signaling.

Nitric oxide (NO) is an endogenously produced physiological signaling molecule that regulates blood flow and platelet activation. However, both the intracellular and intravascular diffusion of NO is severely limited by scavenging reactions with hemoglobin, myoglobin, and other hemoproteins, raising unanswered questions as to how free NO can signal in hemoprotein-rich environments, like blood and cardiomyocytes. We explored the hypothesis that NO could be stabilized as a ferrous heme-nitrosyl complex (Fe 2+ -NO, NO-ferroheme) either in solution within membranes or bound to albumin. Unexpectedly, we observed a rapid reaction of NO with free ferric heme (Fe 3+ ) and a reduced thiol under physiological conditions to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation reaction occurs readily when the hemin is solubilized in lipophilic environments, such as red blood cell membranes, or bound to serum albumin. NO-ferroheme albumin is stable, even in the presence of excess oxyhemoglobin, and potently inhibits platelet activation. NO-ferroheme-albumin administered intravenously to mice dose-dependently vasodilates at low- to mid-nanomolar concentrations. In conclusion, we report the fastest rate of reductive nitrosylation observed to date to generate a NO-ferroheme molecule that resists oxidative inactivation, is soluble in cell membranes, and is transported intravascularly by albumin to promote potent vasodilation.

Cell-free and alkylated hemoproteins improve survival in mouse models of carbon monoxide poisoning.

I.v. administration of a high-affinity carbon monoxide-binding (CO-binding) molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to discover how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating 3 readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb); N-ethylmaleimide modified hemoglobin (NEMHb); and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values, and they scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.

Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations.

Cytoglobin is a hemoprotein widely expressed in fibroblasts and related cell lineages with yet undefined physiological function. Cytoglobin, as other heme proteins, can reduce nitrite to nitric oxide (NO) providing a route to generate NO in vivo in low oxygen conditions. In addition, cytoglobin can also bind lipids such as oleic acid and cardiolipin with high affinity. These two processes are potentially relevant to cytoglobin function. Little is known about how specific amino acids contribute to nitrite reduction and lipid binding. Here we investigate the role of the distal histidine His81 (E7) and several surface residues on the regulation of nitrite reduction and lipid binding. We observe that the replacement of His81 (E7) greatly increases heme reactivity towards nitrite, with nitrite reduction rate constants of up to 1100 M-1s-1 for the His81Ala mutant. His81 (E7) mutation causes a small decrease in lipid binding affinity, however experiments on the presence of imidazole indicate that His81 (E7) does not compete with the lipid for the binding site. Mutations of the surface residues Arg84 and Lys116 largely impair lipid binding. Our results suggest that dissociation of His81 (E7) from the heme mediates the formation of a hydrophobic cavity in the proximal heme side that can accommodate the lipid, with important contributions of the hydrophobic patch around residues Thr91, Val105, and Leu108, whereas the positive charges from Arg84 and Lys116 stabilize the carboxyl group of the fatty acid. Gain and loss-of-function mutations described here can serve as tools to study in vivo the physiological role of these putative cytoglobin functions.

Liver-to-lung microembolic NETs promote gasdermin D-dependent inflammatory lung injury in sickle cell disease.

Acute lung injury, referred to as the acute chest syndrome, is a major cause of morbidity and mortality in patients with sickle cell disease (SCD), which often occurs in the setting of a vaso-occlusive painful crisis. P-selectin antibody therapy reduces hospitalization of patients with SCD by ∼50%, suggesting that an unknown P-selectin-independent mechanism promotes remaining vaso-occlusive events. In patients with SCD, intraerythrocytic polymerization of mutant hemoglobin promotes ischemia-reperfusion injury and hemolysis, which leads to the development of sterile inflammation. Using intravital microscopy in transgenic, humanized mice with SCD and in vitro studies with blood from patients with SCD, we reveal for the first time that the sterile inflammatory milieu in SCD promotes caspase-4/11-dependent activation of neutrophil-gasdermin D (GSDMD), which triggers P-selectin-independent shedding of neutrophil extracellular traps (NETs) in the liver. Remarkably, these NETs travel intravascularly from liver to lung, where they promote neutrophil-platelet aggregation and the development of acute lung injury. This study introduces a novel paradigm that liver-to-lung embolic translocation of NETs promotes pulmonary vascular vaso-occlusion and identifies a new GSDMD-mediated, P-selectin-independent mechanism of lung injury in SCD.

Endogenous Hemoprotein-Dependent Signaling Pathways of Nitric Oxide and Nitrite.

Interdisciplinary research at the interface of chemistry, physiology, and biomedicine have uncovered pivotal roles of nitric oxide (NO) as a signaling molecule that regulates vascular tone, platelet aggregation, and other pathways relevant to human health and disease. Heme is central to physiological NO signaling, serving as the active site for canonical NO biosynthesis in nitric oxide synthase (NOS) enzymes and as the highly selective NO binding site in the soluble guanylyl cyclase receptor. Outside of the primary NOS-dependent biosynthetic pathway, other hemoproteins, including hemoglobin and myoglobin, generate NO via the reduction of nitrite. This auxiliary hemoprotein reaction unlocks a "second axis" of NO signaling in which nitrite serves as a stable NO reservoir. In this Forum Article, we highlight these NO-dependent physiological pathways and examine complex chemical and biochemical reactions that govern NO and nitrite signaling in vivo. We focus on hemoprotein-dependent reaction pathways that generate and consume NO in the presence of nitrite and consider intermediate nitrogen oxides, including NO2, N2O3, and S-nitrosothiols, that may facilitate nitrite-based signaling in blood vessels and tissues. We also discuss emergent therapeutic strategies that leverage our understanding of these key reaction pathways to target NO signaling and treat a wide range of diseases.

Mechanistic insights into cell-free hemoglobin-induced injury during septic shock.

Cell-free hemoglobin (CFH) levels are elevated in septic shock and are higher in nonsurvivors. Whether CFH is only a marker of sepsis severity or is involved in pathogenesis is unknown. This study aimed to investigate whether CFH worsens sepsis-associated injuries and to determine potential mechanisms of harm. Fifty-one, 10-12 kg purpose-bred beagles were randomized to receive Staphylococcus aureus intrapulmonary challenges or saline followed by CFH infusions (oxyhemoglobin >80%) or placebo. Animals received antibiotics and intensive care support for 96 h. CFH significantly increased mean pulmonary arterial pressures and right ventricular afterload in both septic and nonseptic animals, effects that were significantly greater in nonsurvivors. These findings are consistent with CFH-associated nitric oxide (NO) scavenging and were associated with significantly depressed cardiac function, and worsened shock, lactate levels, metabolic acidosis, and multiorgan failure. In septic animals only, CFH administration significantly increased mean alveolar-arterial oxygenation gradients, also to a significantly greater degree in nonsurvivors. CFH-associated iron levels were significantly suppressed in infected animals, suggesting that bacterial iron uptake worsened pneumonia. Notably, cytokine levels were similar in survivors and nonsurvivors and were not predictive of outcome. In the absence and presence of infection, CFH infusions resulted in pulmonary hypertension, cardiogenic shock, and multiorgan failure, likely through NO scavenging. In the presence of infection alone, CFH infusions worsened oxygen exchange and lung injury, presumably by supplying iron that promoted bacterial growth. CFH elevation, a known consequence of clinical septic shock, adversely impacts sepsis outcomes through more than one mechanism, and is a biologically plausible, nonantibiotic, noncytokine target for therapeutic intervention.NEW & NOTEWORTHY Cell-free hemoglobin (CFH) elevations are a known consequence of clinical sepsis. Using a two-by-two factorial design and extensive physiological and biochemical evidence, we found a direct mechanism of injury related to nitric oxide scavenging leading to pulmonary hypertension increasing right heart afterload, depressed cardiac function, worsening circulatory failure, and death, as well as an indirect mechanism related to iron toxicity. These discoveries alter conventional thinking about septic shock pathogenesis and provide novel therapeutic approaches.

P-selectin deficiency promotes liver senescence in sickle cell disease mice.

Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.

Stressed erythrophagocytosis induces immunosuppression during sepsis through heme-mediated STAT1 dysregulation.

Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent RBCs (sRBCs). Macrophages are also essential in defense against microbial threats, but pathological states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype after intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, in that K. pneumoniae mutants lacking siderophore function recapitulated the findings observed with the WT strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and IFN-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulated the STAT1 pathway, with implications in severe infection.

Redox sensor properties of human cytoglobin allosterically regulate heme pocket reactivity.

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.

No evidence of hemoglobin damage by SARS-CoV-2 infection.

SARS-CoV-2 disease (COVID-19) has affected over 22 million patients worldwide as of August 2020. As the medical community seeks better understanding of the underlying pathophysiology of COVID-19, several theories have been proposed. One widely shared theory suggests that SARS-CoV-2 proteins directly interact with human hemoglobin (Hb) and facilitate removal of iron from the heme prosthetic group, leading to the loss of functional hemoglobin and accumulation of iron. Herein, we refute this theory. We compared clinical data from 21 critically ill COVID-19 patients to 21 non-COVID-19 ARDS patient controls, generating hemoglobin-oxygen dissociation curves from venous blood gases. This curve generated from the COVID-19 cohort matched the idealized oxygen-hemoglobin dissociation curve well (Pearson correlation, R2 = 0.97, P.

Tandem P-selectin glycoprotein ligand immunoglobulin prevents lung vaso-occlusion in sickle cell disease mice.

Sickle cell disease (SCD) is a monogenic disorder estimated to affect more than three million people worldwide. Acute systemic painful vaso-occlusive episode (VOE) is the primary reason for emergency medical care among SCD patients. VOE may also progress to acute chest syndrome (ACS), a type of acute lung injury and one of the primary reasons for mortality among SCD patients. Recently, P-selectin monoclonal antibodies were found to attenuate VOE in SCD patients and lung vaso-occlusion in transgenic humanized SCD mice, highlighting the therapeutic benefit of P-selectin inhibition in SCD. Here, we use quantitative fluorescence intravital lung microscopy (qFILM) to illustrate that tandem P-selectin-glycoprotein ligand-immunoglobulin (TSGL-Ig) fusion molecule containing four P-selectin binding sites, significantly attenuated intravenous (IV) oxyhemoglobin triggered lung vaso-occlusion in SCD mice. These findings highlight the therapeutic potential of TSGL-Ig in preventing VOE and ACS in SCD.