Mitapivat increases ATP and decreases oxidative stress and erythrocyte mitochondria retention in a SCD mouse model.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.

HIF-1α-Dependent Metabolic Reprogramming, Oxidative Stress, and Bioenergetic Dysfunction in SARS-CoV-2-Infected Hamsters.

The mechanistic interplay between SARS-CoV-2 infection, inflammation, and oxygen homeostasis is not well defined. Here, we show that the hypoxia-inducible factor (HIF-1α) transcriptional pathway is activated, perhaps due to a lack of oxygen or an accumulation of mitochondrial reactive oxygen species (ROS) in the lungs of adult Syrian hamsters infected with SARS-CoV-2. Prominent nuclear localization of HIF-1α and increased expression of HIF-1α target proteins, including glucose transporter 1 (Glut1), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase-1 (PDK1), were observed in areas of lung consolidation filled with infiltrating monocytes/macrophages. Upregulation of these HIF-1α target proteins was accompanied by a rise in glycolysis as measured by extracellular acidification rate (ECAR) in lung homogenates. A concomitant reduction in mitochondrial respiration was also observed as indicated by a partial loss of oxygen consumption rates (OCR) in isolated mitochondrial fractions of SARS-CoV-2-infected hamster lungs. Proteomic analysis further revealed specific deficits in the mitochondrial ATP synthase (Atp5a1) within complex V and in the ATP/ADP translocase (Slc25a4). The activation of HIF-1α in inflammatory macrophages may also drive proinflammatory cytokine production and complement activation and oxidative stress in infected lungs. Together, these findings support a role for HIF-1α as a central mediator of the metabolic reprogramming, inflammation, and bioenergetic dysfunction associated with SARS-CoV-2 infection.

ßCysteine 93 in human hemoglobin: a gateway to oxidative stability in health and disease.

ßcysteine 93 residue plays a key role in oxygen (O2)-linked conformational changes in the hemoglobin (Hb) molecule. This solvent accessible residue is also a target for binding of thiol reagents that can remotely alter O2 affinity, cooperativity, and Hb's sensitivity to changes in pH. In recent years, ßCys93 was assigned a new physiological role in the transport of nitric oxide (NO) through a process of S-nitrosylation as red blood cells (RBCs) travel from lungs to tissues. ßCys93 is readily and irreversibly oxidized in the presence of a mild oxidant to cysteic acid, which causes destabilization of Hb resulting in improper protein folding and the loss of heme. Under these oxidative conditions, ferryl heme (HbFe4+), a higher oxidation state of Hb is formed together with its protein radical (.HbFe4+). This radical migrates to ßCys93 and interacts with other "hotspot" amino acids that are highly susceptible to oxidative modifications. Oxidized ßCys93 may therefore be used as a biomarker of oxidative stress, reflecting the deterioration of Hb within RBCs intended for transfusion or RBCs from patients with hemoglobinopathies. Site specific mutation of a redox active amino acid(s) to reduce the ferryl heme or direct chemical modifications that can shield ßCys93 have been proposed to improve oxidative resistance of Hb and may offer a protective therapeutic strategy.

Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells.

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1ß, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.

Substitutions in the ß subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation.

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, ßE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of "hot-spot" amino acids, including ßCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in ß subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the ß cleft of Hb Providence; and the third had both of these ß substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS ßF41Y and ßF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of ßCys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS ßK82D and ßF41Y/K82D, respectively. Under hypoxia, ßK82D and ßF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS ßF41Y, respectively. Moreover, at 60 °C, rHbS ßK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS ßF41Y/K82D variant. We conclude that the ßK82D and ßF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.

The Providence Mutation (ßK82D) in Human Hemoglobin Substantially Reduces ßCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells.

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (ßK82D) mutation in sickle cell Hb (ßE6V/ßK82D) and in a recombinant crosslinked Hb (rHb0.1/ßK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of ßCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the ßK82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing ßK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying ßK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing ßK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of ßK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that ßK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.

Interactions of an Anti-Sickling Drug with Hemoglobin in Red Blood Cells from a Patient with Sickle Cell Anemia.

The pathophysiology associated with sickle cell disease (SCD) includes hemolytic anemia, vaso-occlusive events, and ultimately end organ damage set off by the polymerization of deoxygenated hemoglobin S (HbS) into long fibers and sickling of red blood cells (RBCs). One approach toward mitigating HbS polymerization is to pharmacologically stabilize the oxygenated (R) conformation of HbS and thereby reduce sickling frequency and SCD pathology. GBT440 is an α-subunit-specific modifying agent that has recently been reported to increase HbS oxygen binding affinity and consequently delay in vitro polymerization. In addition, animal model studies have demonstrated the potential for GBT440 to be a suitable therapeutic for daily oral dosing in humans. Here, we report an optimized method for detecting GBT440 intermediates in human patient hemolysate using a combination of HPLC and mass spectrometry analysis. First, oxygen dissociation curves (ODCs) analyzed from patient blood showed that oxygen affinity increased in a dose dependent manner. Second, HPLC and integrated mass spectrometric analysis collectively confirmed that GBT440 labeling was specific to the α N-terminus thereby ruling out other potential ligand binding sites. Finally, the results from this optimized analytical approach allowed us to detect a stable α-specific GBT440 adduct in the patient's hemolysate in a dose dependent manner. The results and methods presented in this report could therefore potentially help therapeutic monitoring of GBT440 induced oxygen affinity and reveal critical insight into the biophysical properties of GBT440 Hb complexes.

Oxidative pathways in the sickle cell and beyond.

Polymerization of deoxy sickle cell hemoglobin (HbS) is well recognized as the primary event that triggers the classic cycles of sickling/unsickling of patients red blood cells (RBCs). RBCs are also subjected to continuous endogenous and exogenous oxidative onslaughts resulting in hemolytic rate increases which contribute to the evolution of vasculopathies associated with this disease. Compared to steady-state conditions, the occurrences of vaso-occlusive crises increase the levels of both RBC-derived microparticles as well as extracellular Hb in circulation. Common byproduct resulting from free Hb oxidation and from Hb-laden microparticles is heme (now recognized as damage associated molecular pattern (DAMP) molecule) which has been shown to initiate inflammatory responses. This review provides new insights into the interplay between microparticles, free Hb and heme focusing on Hb's pseudoperoxidative activity that drives RBC's cytosolic, membrane changes as well as oxidative toxicity towards the vascular system. Emerging antioxidative strategies that include the use of protein and heme scavengers in controlling Hb oxidative pathways are discussed.

Comprehensive Biochemical and Biophysical Characterization of Hemoglobin-Based Oxygen Carrier Therapeutics: All HBOCs Are Not Created Equally.

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine, and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.

Proceedings of the Food and Drug Administration's public workshop on new red blood cell product regulatory science 2016.

The US Food and Drug Administration (FDA) held a workshop on red blood cell (RBC) product regulatory science on October 6 and 7, 2016, at the Natcher Conference Center on the National Institutes of Health (NIH) Campus in Bethesda, Maryland. The workshop was supported by the National Heart, Lung, and Blood Institute, NIH; the Department of Defense; the Office of the Assistant Secretary for Health, Department of Health and Human Services; and the Center for Biologics Evaluation and Research, FDA. The workshop reviewed the status and scientific basis of the current regulatory framework and the available scientific tools to expand it to evaluate innovative and future RBC transfusion products. A full record of the proceedings is available on the FDA website (http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/ucm507890.htm). The contents of the summary are the authors' opinions and do not represent agency policy.

Engineering oxidative stability in human hemoglobin based on the Hb providence (ßK82D) mutation and genetic cross-linking.

Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; ßK82D), is much more resistant to degradation by H2O2 We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (ßN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrations, all the cross-linked tetramers show smaller rates of autoxidation and hemin loss than HbA, which can dissociate into much less stable dimers and (b) the HbProv ßK82D mutation confers more resistance to degradation by H2O2, by markedly inhibiting oxidation of the ß93 cysteine side chain, particularly in cross-linked tetramers and even in the presence of the destabilizing Hb Presbyterian mutation. These results show that cross-linking and the ßK82D mutation do enhance the resistance of Hb to oxidative degradation, a critical element in the design of a safe and effective oxygen therapeutic.

Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.

BACKGROUND: α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. METHODS: Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. RESULTS: All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. CONCLUSIONS: The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. GENERAL SIGNIFICANCE: The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M.

Determination of extinction coefficients of human hemoglobin in various redox states.

The role of hemoglobin (Hb) redox forms in tissue and organ toxicities remain ambiguous despite the well-documented contribution of Hb redox reactivity to cellular and subcellular oxidative changes. Moreover, several recent studies, in which Hb toxicity were investigated, have shown conflicting outcomes. Uncertainties over the potential role of these species may in part be due to the protein preparation method of choice, the use of published extinction coefficients and the lack of suitable controls for Hb oxidation and heme loss. Highly purified and well characterized redox forms of human Hb were used in this study and the extinction coefficients of each Hb species (ferrous/oxy, ferric/met and ferryl) were determined. A new set of equations were established to improve accuracy in determining the transient ferryl Hb species. Additionally, heme concentrations in solutions and in human plasma were determined using a novel reversed phase HPLC method in conjugation with our photometric measurements. The use of more accurate redox-specific extinction coefficients and method calculations will be an invaluable tool for both in vitro and in vivo experiments aimed at determining the role of Hb-mediated vascular pathology in hemolytic anemias and when Hb is used as oxygen therapeutics.

Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells.

Lung alveoli are lined by alveolar type (AT) 1 cells and cuboidal AT2 cells. The AT1 cells are likely to be exposed to cell-free hemoglobin (Hb) in multiple lung diseases; however, the role of Hb redox (reduction-oxidation) reactions and their precise contributions to AT1 cell injury are not well understood. Using mouse lung epithelial cells (E10) as an AT1 cell model, we demonstrate here that higher Hb oxidation states, ferric Hb (HbFe(3+)) and ferryl Hb (HbFe(4+)) and subsequent heme loss play a central role in the genesis of injury. Exposures to HbFe(2+) and HbFe(3+) for 24 hours induced expression of heme oxygenase (HO)-1 protein in E10 cells and HO-1 translocation in the purified mitochondrial fractions. Both of these effects were intensified with increasing oxidation states of Hb. Next, we examined the effects of Hb oxidation and free heme on mitochondrial bioenergetic function by measuring changes in the mitochondrial transmembrane potential and oxygen consumption rate. In contrast to HbFe(2+), HbFe(3+) reduced basal oxygen consumption rate, indicating compromised mitochondrial activity. However, HbFe(4+) exposure not only induced early expression of HO-1 but also caused mitochondrial dysfunction within 12 hours when compared with HbFe(2+) and HbFe(3+). Exposure to HbFe(4+) for 24 hours also caused mitochondrial depolarization in E10 cells. The deleterious effects of HbFe(3+) and HbFe(4+) were reversed by the addition of scavenger proteins, haptoglobin and hemopexin. Collectively, these data establish, for the first time, a central role for cell-free Hb in lung epithelial injury, and that these effects are mediated through the redox transition of Hb to higher oxidation states.

Sickle Cell Hemoglobin in the Ferryl State Promotes ßCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10).

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized ßCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.

Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function.

There is growing evidence that extracellular haemoglobin and haem mediate inflammatory and oxidative damage in sickle cell disease. Haptoglobin (Hp), the scavenger for free haemoglobin, is depleted in most patients with sickle cell disease due to chronic haemolysis. Although single infusions of Hp can ameliorate vaso-occlusion in mouse models of sickle cell disease, prior studies have not examined the therapeutic benefits of more chronic Hp dosing on sickle cell disease manifestations. In the present study, we explored the effect of Hp treatment over a 3-month period in sickle mice at two dosing regimens: the first at a moderate dose of 200 mg/kg thrice weekly and the second at a higher dose of 400 mg/kg thrice weekly. We found that only the higher dosing regimen resulted in increased haem-oxygenase-1 and heavy chain ferritin (H-ferritin) expression and decreased iron deposition in the kidney. Despite the decreased kidney iron deposition following Hp treatment, there was no significant improvement in kidney function. However, there was a nearly significant trend towards decreased liver infarction.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4ß1, or αVß3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and ß subunits of adult Hb (HbA) Bristol-Alesha (ß67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in ß subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, ß, and γ subunits with respect to redox reactivity and may have direct implications to α/ß hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.

Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease.

Sickle cell disease (SCD), a hereditary hemolytic disorder is characterized by chronic hemolysis, oxidative stress, vaso-occlusion and end-organ damage. Hemolysis releases toxic cell-free hemoglobin (Hb) into circulation. Under physiologic conditions, plasma Hb binds to haptoglobin (Hp) and forms Hb-Hp dimers. The dimers bind to CD163 receptors on macrophages for further internalization and degradation. However, in SCD patients plasma Hp is depleted and free Hb is cleared primarily by proximal tubules of kidneys. Excess free Hb in plasma predisposes patients to renal damage. We hypothesized that administration of exogenous Hp reduces Hb-mediated renal damage. To test this hypothesis, human renal proximal tubular cells (HK-2) were exposed to HbA (50µM heme) for 24h. HbA increased the expression of heme oxygenase-1 (HO-1), an enzyme which degrades heme, reduces heme-mediated oxidative toxicity, and confers cytoprotection. Similarly, infusion of HbA (32µM heme/kg) induced HO-1 expression in kidneys of SCD mice. Immunohistochemistry confirmed the increased HO-1 expression in the proximal tubules of the kidney. Exogenous Hp attenuated the HbA-induced HO-1 expression in vitro and in SCD mice. Our results suggest that Hb-mediated oxidative toxicity may contribute to renal damage in SCD and that Hp treatment reduces heme/iron toxicity in the kidneys following hemolysis.