Distinct proteomes and allergen profiles appear across the life-cycle stages of Alternaria alternata.

BACKGROUND: Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus. OBJECTIVE: We sought to determine differences between spore and hyphae proteomes and how allergens are distributed in Aalternata. METHODS: Data-independent acquisition mass spectrometry was used to quantitatively compare the proteomes of asexual spores (nongerminating and germinating) with vegetative hyphae. RESULTS: We identified 4515 proteins in nongerminating spores, germinating spores, and hyphae; most known allergens are more abundant in nongerminating spores. On comparing significant protein fold-change differences between nongerminating spores and hyphae, we found that 174 proteins were upregulated in nongerminating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. On comparing nongerminating and germinating spores, 25 proteins were found to be upregulated in nongerminating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media. CONCLUSIONS: Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in nongerminating spores.

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.

Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease.

Intracellular oxidative stress and oxidative modification of sickle hemoglobin (HbS) play a role in sickle cell disease (SCD) pathogenesis. Recently, we reported that Hb-dependent oxidative stress induced post-translational modifications (PTMs) of Hb and red blood cell (RBC) membrane proteins of transgenic SCD mice. To identify the mechanistic basis of these protein modifications, we followed in vitro oxidative changes occurring in intracellular Hb obtained from RBCs and RBC-derived microparticles (MPs) from the blood of 23 SCD patients (HbSS) of which 11 were on, and 12, off hydroxyurea (HU) treatment, and 5 ethnic matched controls. We used mass spectrometry-based proteomics to characterize these oxidative PTMs on a cross-sectional group of these patients (n = 4) and a separate subgroup of patients (n = 2) studied prior to initiation and during HU treatment. Collectively, these data indicated that band-3 and its interaction network involved in MPs formation exhibited more protein phosphorylation and ubiquitination in SCD patients than in controls. HU treatment reversed these oxidative PTMs back to level observed in controls. These PTMs were also confirmed using orthogonal immunoprecipitation experiments. Moreover, we observed specific markers reflective of oxidative stress, including irreversible oxidation of ßCys93 and ubiquitination of Hb ßLys145 (and ßLys96). Overall, these studies strongly suggest that extensive erythrocyte membrane protein phosphorylation and ubiquitination are involved in SCD pathogenesis and provide further insight into the multifaceted effects of HU treatment.

Antisickling Drugs Targeting ßCys93 Reduce Iron Oxidation and Oxidative Changes in Sickle Cell Hemoglobin.

Sickle cell disease is a genetic blood disorder caused by a single point mutation in the ß globin gene where glutamic acid is replaced by valine at the sixth position of the ß chain of hemoglobin (Hb). At low oxygen tension, the polymerization of deoxyHbS into fibers occurs in red blood cells (RBCs) leading to an impaired blood vessel transit. Sickle cell hemoglobin (HbS), when oxidized with hydrogen peroxide (H2O2), stays longer in a highly oxidizing ferryl (Fe4+) form causing irreversible oxidation of ßCys93 to a destabilizing cysteic acid. We have previously reported that an antisickling drug can be designed to bind specifically to ßCys93 and effectively protect against its irreversible oxidation by H2O2. Here, we report oxygen dissociation, oxidation, and polymerization kinetic reactions for four antisickling drugs (under different preclinical/clinical developmental stages) that either site-specifically target ßCys93 or other sites on the HbS molecule. Molecules that specifically bind to or modify ßCys93, such as 4,4'-di(1,2,3-triazolyl) disulfide (TD-3) and hydroxyurea (HU) were contrasted with molecules that target other sites on Hb including 5-hydroxymethyl-2-furfural (5-HMF) and L-glutamine. All reagents induced a left shift in the oxygen dissociation curve (ODC) except L-glutamine. In the presence of H2O2 (2.5:1, H2O2:heme), both TD-3 and HU reduced the ferryl heme by 22 and 37%, respectively, which corresponded to a 3- to 2-fold reduction in the levels of ßCys93 oxidation as verified by mass spectrometry. Increases in the delay times prior to polymerization of HbS under hypoxia were in the following order: TD-3 > HU > 5-HMF = L-glutamine. Designing antisickling agents that can specifically target ßCys93 may provide a dual antioxidant and antisickling therapeutic benefits in treating this disease.

Redox states of hemoglobin determine left ventricle pressure recovery and activity of mitochondrial complex IV in hypoxic rat hearts.

Cardiovascular effects were reported to occur in humans and in animal models during transfusion with hemoglobin (Hb)-based oxygen therapeutics. The effects of Hb's iron redox states on cardiac parameters during hypoxia/reoxygenation are however poorly defined. We hypothesize that acute exposures to ferric Hb during hypoxia leads to cardiomyocyte injury and an impaired left ventricular response accompanied by cardiac mitochondrial bioenergetic dysfunction. Recovery of left ventricular functions in an isolated rat heart Langendorff perfusion system was observed following perfusion with ferrous but not with ferric Hb. Ferric Hb induced the development of heart lesions, and impairment of the respiratory chain complex activity. Under normoxia, a sharp decline in cardiac parameters was observed following co-perfusion of low (20 µM) and high (100 µM) ascorbic acid (Asc) with ferrous Hb. This trend continued with ferric Hb co-perfusion, but only at the higher concentration of Asc. These observations suggest that perfusion of the hypoxic heart with ferric Hb increases oxidative stress thereby resulting in cardiac dysfunction. Intervention with Asc to reduce ferric Hb may offer a strategy to control Hb toxicity; however, timing of administration, and dosage of Asc may require individual optimization to target specific redox forms of Hb.

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.

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.

Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent ß-globin posttranslational modifications, including the irreversible oxidation of ßCys93 and the ubiquitination of ßLys96 and ßLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, ßCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin.

Redox active cysteine residues including ßCys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of ßCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.

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.

Heterogeneity in non-epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B Neisseria meningitidis.

PorB is a well-characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface-exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non-epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non-adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane-associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB-containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion.

Exploring Oxidative Reactions in Hemoglobin Variants Using Mass Spectrometry: Lessons for Engineering Oxidatively Stable Oxygen Therapeutics.

SIGNIFICANCE: Worldwide demand has driven the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) as potential acellular oxygen therapeutics. HBOCs have the potential to provide an oxygen bridge to patients and minimize current problems associated with supply and storage of donated blood. However, to date, safety and efficacy issues have hampered the approval of viable HBOCs in the United States. These previous efforts have underscored the need for a better molecular understanding of toxicity to design safe and oxidatively stable HBOCs. Recent Advances: High-resolution accurate mass (HRAM) mass spectrometry (MS) has recently become a versatile tool in characterizing oxidative post-translational modifications that occur in Hb. When integrated with other analytical techniques, HRAM data have been invaluable in providing mechanistic insight into the extent of oxidative modification by quantifying oxidation in amino acids near the reactive heme or at specific "oxidative hotspots." CRITICAL ISSUES: In addition to providing a deeper understanding of Hb oxidative toxicity, HRAM MS studies are currently being used toward developing suitable HBOCs using a "two-prong" strategy that involves (i) understanding the mechanism of Hb toxicity by evaluating mutant Hbs identified in patients with hemoglobinopathies and (ii) utilizing this information toward designing against (or for) these reactions in acellular oxygen therapeutics that will result in oxidatively stable protein. FUTURE DIRECTIONS: Future HRAM studies are aimed at fully characterizing engineered candidate HBOCs to determine the most oxidatively stable protein while retaining oxygen carrying function in vivo. Antioxid. Redox Signal. 26, 777-793.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Oxidative instability of hemoglobin E (ß26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/ß-thalassemia.

When adding peroxide (H2O2), ß subunits of hemoglobin (Hb) bear the burden of oxidative changes due in part to the direct oxidation of its Cys93. The presence of unpaired α subunits within red cells and/or co-inheritance of another ß subunit mutant, HbE (ß26 Glu→Lys) have been implicated in the pathogenesis and severity of ß thalassemia. We have found that although both HbA and HbE autoxidize at initially comparable rates, HbE loses heme at a rate almost 2 fold higher than HbA due to unfolding of the protein. Using mass spectrometry and the spin trap, DMPO, we were able to quantify irreversible oxidization of ßCys93 to reflect oxidative instability of ß subunits. In the presence of free α subunits and H2O2, both HbA and HbE showed ßCys93 oxidation which increased with higher H2O2 concentrations. In the presence of Alpha-hemoglobin stabilizing protein (AHSP), which stabilizes the α-subunit in a redox inactive hexacoordinate conformation (thus unable to undergo the redox ferric/ferryl transition), Cys93 oxidation was substantially reduced in both proteins. These experiments establish two important features that may have relevance to the mechanistic understanding of these two inherited hemoglobinopathies, i.e. HbE/ß thalassemia: First, a persistent ferryl/ferryl radical in HbE is more damaging to its own ß subunit (i.e., ßCys93) than HbA. Secondly, in the presence of excess free α-subunit and under the same oxidative conditions, these events are substantially increased for HbE compared to HbA, and may therefore create an oxidative milieu affecting the already unstable HbE.

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on k(cat) were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K(i) for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.

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.

Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.

In the presence of excess hydrogen peroxide (H2O2), ferrous (Fe(+2)) human hemoglobin (Hb) (α2ß2) undergoes a rapid conversion to a higher oxidation ferryl state (Fe(+4)) which rapidly autoreduces back to the ferric form (Fe(+3)) as H2O2 is consumed in the reaction. In the presence of additional H2O2 the ferric state can form both ferryl Hb and an associated protein radical in a pseudoperoxidative cycle that results in the loss of radicals and heme degradation. We examined whether adult HbA (ß2α2) exhibits a different pseudoenzymatic activity than fetal Hb (γ2α2) due to the switch of γ to ß subunits. Rapid mixing of the ferric forms of both proteins with excess H2O2 resulted in biphasic kinetic time courses that can be assigned to γ/ß and α, respectively. Although there was a 1.5 fold increase in the fast reacting γ /ß subunits the slower reacting phases (attributed to α subunits of both proteins) were essentially the same. However, the rate constant for the auto-reduction of ferryl back to ferric for both proteins was found to be 76% higher for HbF than HbA and in the presence of the mild reducing agent, ascorbate there was a 3-fold higher reduction rate in ferryl HbF as opposed to ferryl HbA. Using quantitative mass spectrometry in the presence of H2O2 we found oxidized γ/ß Cys93, to be more abundantly present in HbA than HbF, whereas higher levels of nitrated ß Tyr35 containing peptides were found in HbA samples treated with nitrite. The extraordinary stability of HbF reported here may explain the evolutionary advantage this protein may confer onto co-inherited hemoglobinopathies and can also be utilized in the engineering of oxidatively stable Hb-based oxygen carriers.

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.

Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis.

Posttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular-the methylation of guanosine at position 518 (G518) of the 16S rRNA in Mycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidB mutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidB mutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility of M. tuberculosis to streptomycin (SM). Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidB mutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidB mutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed in M. tuberculosis strains that contain a gidB mutation.

A coordinated proteomic approach for identifying proteins that interact with the E. coli ribosomal protein S12.

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to ß-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader , M. B. ; Costantino , N. ; Elkins , C. A. ; Chen , C. Y. ; Patel , I. ; Makusky , A. J. ; Choy , J. S. ; Court , D. L. ; Markey , S. P. ; Kowalak , J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011 , 10 , M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography-tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.