Heme Research Articles

Structural and Functional Insights of an Unprecedented Multiheme Cytochrome from Geobacter Sulfurreducens

Geobacter bacteria have been inspiring the development of many biotechnological applications, which seek improvements in the fields of microbial energy production, bioremediation of contaminated waters and soils, development of sustainable electronic devices and materials, as well as biomedical devices. Geobacter sulfurreducens, the microorganism with the highest current production in microbial fuel cells known to date, possesses over 100 cytochromes that assure an effective electron transfer between the intracellular components and the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA-family, composed of five periplasmic c7 triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome expressed by G. sulfurreducens in Fe(III) reducing conditions, but not expressed at all in cultures grown on fumarate, linking the cytochrome directly with extracellular electron transfer (EET). This cytochrome possesses a low sequence identity with the cytochromes from the PpcA-family and a inevitably different heme-coordination, based on the analysis of its amino acid sequence. This detail, together with the direct linkage with EET pathways, makes GSU0105 an interesting target for functional and structural studies. In this work, several biophysical techniques, including UV-visible, CD, EPR and NMR spectroscopies, were used to characterize the cytochrome. Initial evidences about the secondary structural elements and redox behavior of the cytochrome were probed. NMR and EPR were used to pinpoint the spin-states of the heme groups in different oxidation states. The results obtained, together with BLAST and structural predictions, support the fact that GSU0105 is a member of a new subclass of triheme cytochromes. Acknowledgements: This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through grants SFRH/BD/145039 and PTDC/BIA-BQM/31981/2017 and by UCIBIO, which is financed by national funds from FCT (UIDB/04378/2020).

The Structural Environment Around the Heme-Binding Site in Cyclooxygenase and Its Implication

Cyclooxygenases (COX-1 and COX-2) have a cyclooxygenase activity involved in prostaglandin G2 (PGG2) formation and an associated peroxidase (POX) activity for the reduction of PGG2 to PGH2, which are functionally interdependent. Peroxide-mediated oxidation of the heme group at the POX active site forming an oxyferryl heme radical cation (known as Compund I) initiates the whole catalytic cycle. The heme in COX proteins is ferriprotoporphyrin IX, much of which is lost upon isolation, but can be replaced to convert apo- to holo-enzyme.

Spectroscopic Study of Interaction of Bare, Citrate Capped and BSA Capped Fe3O4 Nanoparticles with Hemoglobin

Background:Fe3O4nanoparticles have great potential in different biomedical applications. The study of the interaction of bare, and capped Fe3O4nanoparticles with common blood proteins is a field of interest for understanding the underlying mechanism and biocompatibility.Objective:This work aims at studying the nature of binding of bare, citrate functionalised and bovine serum albumin coated Fe3O4nanoparticles (Fe3O4NPs, CFe3O4NPs and BFe3O4NPs) with human hemoglobin (Hb), and their instantaneous effect on amino acid group, heme group and secondary structure of Hb.Methods:Nanoparticles were prepared by the chemical route and characterised by TEM, XRD and UV-Visible and FTIR spectroscopy. UV-visible absorbance and fluorescence emission/ excitation spectroscopy and circular dichroism were performed to study the interaction of nanoparticles with Hb.Results:UV-visible absorbance spectroscopy showed no blue or red shift of absorption peaks. Benesi-Hildebrand curves for the amino acid band and soret band of Hb absorbance spectrum were straight lines with positive intercepts; apparent binding constants and Gibbs free energy change were within a moderate level; they were larger for amino acid band in the presence of CFe3O4NPs, and for soret band in the presence of Fe3O4NPs, but noticeably small for both bands in the presence of BFe3O4NPs. Fluorescence emission/excitation spectra showed no noticeable shift of emission/excitation peak position of Hb in the presence of the three nanoparticles. Multiple peak fitting, done for the L-peak of the excitation spectrum of Hb, showed a major increase in the percentage of peak area of Tyr in the presence of CFe3O4NPs. Circular dichroism measurement showed that CFe3O4NPs, Fe3O4NPs and BFe3O4NPs reduced the α-helix content of Hb in decreasing order.Conclusion:Ground state complex formation of human hemoglobin with the studied nanoparticles with 1:1 stoichiometric ratio is suggested. Moreover, it has been observed that CFe3O4NPs may have a stronger interaction with the amino acid group while bare Fe3O4NPs may have a stronger interaction with the heme group of Hb. Hinderance of the energy transfer from tyrosine to tryptophan of Hb in the presence of CFe3O4NPs is also suggested. CFe3O4NPs may also have some effect on the secondary structure of Hb as indicated through reduction of the α-helix content. BFe3O4NPs have shown very weak interaction with Hb in the UV-visible absorbance spectroscopy, fluorescence emission/excitation spectroscopy and circular dichroism experiment.

Interaction of artemisinin protects the activity of antioxidant enzyme catalase: A biophysical study

The major antioxidant enzyme catalase is downregulated and the enzyme activity is compromised in various disease conditions such as malarial and cancer. Hence, the restoration and protection of catalase is a promising therapeutic strategy in disease management. In the present study, for the first time we have demonstrated the protective role of well-known anti-malarial drug Artemisinin (ART) on the time and temperature-induced degradation of bovine liver catalase (BLC) activity. The findings at different time intervals and at higher temperature showed the protective role of ART on BLC activity. Molecular docking studies suggested specific binding of ART on BLC through heme group interface which was further supported by cyclic voltammetry and dynamic light scattering study. The stabilization of BLC in presence of ART was mediated through forming a BLC-ART complex with reduced and shifted electrochemical peak and increased hydrodynamic diameter. ART substantially prevents the temperature-induced reduction in α-helical content with simultaneous increment in other secondary structures like antiparallel, parallel, β-turn and random coils. Nevertheless, the protective role of ART was accepted from the enhanced thermal stability and increased Tm value of BLC in presence of ART at higher temperatures. Our results uncover the mechanism of interaction between ART with BLC and suggest the protective role of ART towards spatiotemporal alteration of BLC by preventing the structural and molecular change in BLC. Thus, the findings advocate ART as a potential therapeutic drug for diseases associated with reduced catalase activity.

Mechanism of Inhibition of Cytochrome c Oxidase by Triton X-100.

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-β-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2·- by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.

The Destruction of Cytochrome c in a Complex with Cardiolipin during Catalysis of Lipid Peroxidation

The destruction of cytochrome c during its catalysis of the lipoperoxidase reaction, which causes the impairment of mitochondrial membranes in living cells and the release of various pro-apoptotic factors into the cytosol, was studied. Spectrophotometric analysis showed a much more intense destruction of cytochrome c when hydrogen peroxide was added in the presence of tetraoleylcardiolipin at protein : cardiolipin ratios of 1 : 30 and 1 : 60, which were optimal for the formation of a cytochrome c complex with cardiolipin, compared to the sample in which only hydrogen peroxide and cytochrome c were present. In the second case, the destruction of the porphyrite group of heme took the form of a linear function, while in the presence of cardiolipin the dependence was clearly exponential; upon addition of a lipid substrate, phosphatidic acid, the value of the first-order rate constant of the cytochrome c destruction increased. It is proposed that the rapid destruction of cytochrome c during its catalysis of the lipoperoxidase reaction is an evolutionarily developed mechanism for preventing the spontaneous initiation of apoptosis.

Thermodynamics of the P-type Ferryl Form of Bovine Cytochrome c Oxidase.

Several ferryl states of the catalytic heme a3-CuB center of the respiratory cytochrome c oxidases (CcOs) are observed during the reduction of O2 to H2O. One of the P-type ferryl forms, PM, is produced by the reaction of the two-electron reduced CcO with O2. In this state, the heme a3 iron is in the ferryl state and a free radical should be also present at the catalytic center. However, the energetics of the PM formation has not been experimentally established yet. Here, the generation of PM by the reaction of oxidized bovine CcO (O) with one molecule of H2O2 was investigated by the isothermal titration calorimetry and UV-Vis absorption spectroscopy. Two kinetic phases, corresponding to the formation of PM and its endogenous conversion back to O, were resolved by both methods. The ΔH of the entire process (-66 kcal/mol H2O2) was larger than the heat (-50.8 kcal/mol O2) liberated during O2 reduction by ferrocytochrome c (pH 8, 25°C). Interestingly, ΔH of the first phase (-32 kcal/mol ferryl state) far exceeds the enthalpy of the PM production. The data indicate that during the first phase, the radical in PM is quenched and spectrally similar second P-type ferryl form (PR) is produced. Additionally, it was shown that the entropy contribution to the Gibbs energy change (ΔG = -46 kcal/mol O2) during the catalytic reduction of O2 by ferrocytochrome c is negligible (-0.7 cal·mol-1·K-1).

β-Oxochlorin cobalt(II) complexes catalyze the electrochemical reduction of CO2.

Inspired by the architecture of the macrocycle of heme d1, a series of synthetic mono-, di- and tri-β-oxo-substituted porphyrinoid cobalt(ii) complexes were evaluated as electrocatalytic CO2 reducers, identifying complexes of unusually high efficiencies in generating multi-electron reduction products, including CH4.

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

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

Reverse Phase-high-performance Liquid Chromatography (RP-HPLC) Analysis of Globin Chains from Human Erythroid Cells

β-hemoglobinopathies are severe genetic disorders characterized either by the abnormal synthesis of the adult β-globin chains of the hemoglobin (Hb) tetramer (βS-globin chains) in sickle cell disease (SCD) or by the reduced β-globin production in β-thalassemia. The identification and quantification of globin chains are crucial for the diagnosis of these diseases and for testing new therapeutic approaches aimed at correcting the β-hemoglobinopathy phenotype. Conventional techniques to detect the different Hb molecules include cellulose-acetate electrophoresis (CEA), capillary electrophoresis (CE), isoelectric focusing (IEF), and cation-exchange-HPLC (CE-HPLC). However, these methods cannot distinguish the different globin chains and precisely determine their relative expression. We have set up a high-resolution and reproducible reverse phase-HPLC (RP-HPLC) to detect and identify the globin chains composing the hemoglobin tetramers based on their different hydrophobic properties. RP-HPLC mobile phases are composed of acetonitrile (ACN) that creates a hydrophobic environment and trifluoroacetic acid (TFA), which breaks the heme group within the Hb tetramers releasing individual globin chains. Hb-containing lysates are loaded onto the AerisTM 3.6-µm WIDEPORE C4 200 Å LC Column and a gradient of increasing hydrophobicity of the mobile phase over time allows globin chain separation. The relative amount of globin chains is measured at a wavelength (λ) of 220 nm. This protocol is designed for evaluating globin chains in (i) red blood cells (RBCs) obtained from human peripheral blood, (ii) RBCs in vitro differentiated from hematopoietic stem/progenitor cells (HSPCs), and (iii) burst-forming unit-erythroid (BFU-E), i.e., erythroid progenitors obtained in vitro from human peripheral blood or in vitro cultured HSPCs. This technique allows to precisely identify the different globin chains and obtain a relative quantification. RP-HPLC can be used to confirm the diagnosis of β-hemoglobinopathies, to evaluate the disease severity and validate novel approaches for the treatment of these diseases.

Protoporfiria eritropoética: apresentação clínica em criança com diagnóstico prévio de lúpus eritematoso cutâneo

Erythropoietic protoporphyria (EPP) is a metabolic error in the biosynthesis of the heme group, the enzyme ferrochelatase (FEHC) deficiency culminates in the excessive accumulation of free protoporphyrin in erythroid cells, plasma, skin, and liver. The excess of this metabolite generates symptoms like skin pain, burning, pruritus during the sun exposure, that arise from this the childhood. This article reports the case of a 4-year and 6-month patient who, after prolonged sun exposure, opened a typical photosensitivity picture of erythropoietic protoporphyria. Erythema malar led to a misdiagnosis of cutaneous erythematosus lupus. EPP diagnosis was confirmed by the measurement of serum protoporphyria and corroborated by the genetic test that shows defect in the gene that transcribes the FEHC enzyme. The treatment is based on measures of photoprotection, recently medications such as afamelanotide and hematin have been shown to be effective for the control of the disease. In order to elucidate the difference between the diagnosis of cutaneous lupus and PPE, this article brings the clinical difference between these pathologies, in addition to the EPP laboratory diagnosis.

Rutin present in Alibertia edulis extract acts on human platelet aggregation through inhibition of cyclooxygenase/thromboxane.

Alibertia edulis leaf extract is commonly used in folk medicine, with rutin caffeic and vanillic acids being its major compounds. The Alibertia edulis leaf extract was investigated for its pharmacological effects via platelet aggregation, calcium mobilization, cyclic nucleotides levels, vasodilator-stimulated phosphoprotein Ser157 and Ser239 and protein kinase Cβ2 phosphorylation, thromboxane B2, cyclooxygenases 1 and 2, docking and molecular dynamics. Alibertia edulis leaf extract significantly inhibited (100-1000 μg mL-1) platelet aggregation induced by different agonists. Arachidonic acid increased levels of calcium and thromboxane B2, phosphorylation of vasodilator-stimulated phosphoprotein Ser157 and Ser239, and protein kinase Cβ, which were significantly reduced by Alibertia edulis leaf extract, rutin, and caffeic acid as well mixtures of rutin/caffeic acid. Cyclooxygenase 1 activity was inhibited for Alibertia edulis leaf extract, rutin and caffeic acid. These inhibitions were firsrtly explored by specific stabilization of rutin and caffeic acid compared to diclofenac at the catalytic site from docking score and free-energy dissociation profiles. Then, simulations detailed the rutin interactions close to the heme group and Tyr385, responsible for catalyzing the conversion of arachidonic acid to its products. Our results reveal the antiplatelet aggregation properties of Alibertia edulis leaf extract, rutin and caffeic acid providing pharmacological information about its origin from cyclooxygenase 1 inhibition and its downstream pathway.

Immune Modulation by Inhibitors of the HO System.

The heme oxygenase (HO) system involves three isoforms of this enzyme, HO-1, HO-2, and HO-3. The three of them display the same catalytic activity, oxidating the heme group to produce biliverdin, ferrous iron, and carbon monoxide (CO). HO-1 is the isoform most widely studied in proinflammatory diseases because treatments that overexpress this enzyme promote the generation of anti-inflammatory products. However, neonatal jaundice (hyperbilirubinemia) derived from HO overexpression led to the development of inhibitors, such as those based on metaloproto- and meso-porphyrins inhibitors with competitive activity. Further, non-competitive inhibitors have also been identified, such as synthetic and natural imidazole-dioxolane-based, small synthetic molecules, inhibitors of the enzyme regulation pathway, and genetic engineering using iRNA or CRISPR cas9. Despite most of the applications of the HO inhibitors being related to metabolic diseases, the beneficial effects of these molecules in immune-mediated diseases have also emerged. Different medical implications, including cancer, Alzheimer´s disease, and infections, are discussed in this article and as to how the selective inhibition of HO isoforms may contribute to the treatment of these ailments.

Integration of an Inhibitor-like Rule and Structure-based Virtual Screening for the Discovery of Novel Myeloperoxidase Inhibitors.

Myeloperoxidase (MPO) is an attractive therapeutic target against inflammation. Herein, we developed an inhibitor-like rule, based on known MPO inhibitors, and generated a target database containing 6546 molecules with privileged inhibitory properties. Using a structure-based approach validated by decoys, robust statistical metrics, redocking, and cross-docking, we selected 10 putative MPO inhibitors with high chemical diversity. At 20 μM, six of these 10 compounds (i.e., 60% success rate) inhibited more than 20% of the chlorinating activity of the enzyme. Additionally, we found that compound ZINC9089086 forms hydrogen bonds with Arg233 and with the hemic carboxylate. It makes a π-stacking interaction with the heme group and displays a high affinity for the enzyme active site. When incubated with purified MPO, ZINC9089086 inhibited the chlorinating activity of the enzyme with an IC50 of 2.2 ± 0.1 μM in a reversible manner. Subsequent experiments revealed that ZINC9089086 inhibited hypochlorous acid production in dHL-60 cells and human neutrophils. Furthermore, the theoretical ADME/Tox profile indicated that this compound exhibits low toxicity risks and adequate pharmacokinetic parameters, thus making ZINC9089086 a very promising candidate for preclinical anti-inflammatory studies. Overall, our study shows that integrating an inhibitor-like rule with a validated structure-based methodology is an excellent approach for improving the success rate and molecular diversity of novel MPO inhibitors with good pharmacokinetics and toxicological profiles. By combining these tools, it was possible to increase the assurance rate, which ultimately diminishes the costs and time needed for the acquisition, synthesis, and evaluation of new compounds.

β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies.

Hemoglobin is a tetrameric protein composed of two α and two β chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and β-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two β subunits (α2β2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in β-globin production cause two of the most common monogenic-inherited disorders: β-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of β-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.

Effect of Membrane Environment on the Ligand-Binding Properties of the Terminal Oxidase Cytochrome bd-I from Escherichia coli.

Cytochrome bd-I is a terminal oxidase of the Escherichia coli respiratory chain. This integral membrane protein contains three redox-active prosthetic groups (hemes b558, b595, and d) and couples the electron transfer from quinol to molecular oxygen to the generation of proton motive force, as one of its important physiological functions. The study was aimed at examining the effect of the membrane environment on the ligand-binding properties of cytochrome bd-I by absorption spectroscopy. The membrane environment was found to modulate the ligand-binding characteristics of the hemoprotein in both oxidized and reduced states. Absorption changes upon the addition of exogenous ligands, such as cyanide or carbon monoxide (CO), to the detergent-solubilized enzyme were much more significant and heterogeneous than those observed with the membrane-bound enzyme. In the native membranes, both cyanide and CO interacted mainly with heme d. An additional ligand-binding site (heme b558) appeared in the isolated enzyme, as was evidenced by more pronounced changes in the absorption in the Soret band. This additional reactivity could also be detected after treatment of E. coli membranes with a detergent. The observed effect did not result from the enzyme denaturation, since reconstitution of the isolated enzyme into azolectin liposomes restored the ligand-binding pattern close to that observed for the intact membranes.

Effect of ascorbic acid and hemin on experimental colon carcinogenesis in mice

Colon cancer is known as the third most widespread cancer in the world. The interaction of heme-iron and ascorbic acid (AA) in colon carcinogenesis is not evident. Hemin (ferric chloride heme) is an iron-containing porphyrin with chlorine that can be formed from a heme group. The purpose of this study was to investigate the protective effect of AA on the formation of pre-neoplastic lesions induced by azoxymethane (AOM)/dextran sodium sulfate (DSS) plus hemin in mice. Forty-five ICR male mice were divided into three experimental groups; AOM/ DSS treatment (control group), hemin (2 g hemin/kg of b.w.), hemin + AA (1.0% in drinking water). The mice had three s.c. injections (0–2nd weeks of the experiment) of AOM (10 mg/kg b.w.) weekly and 2% DSS as drinking water for the next one week and the animals fed on AIN-76A purified rodent diet for 6 weeks. The numbers of aberrant crypt foci (ACF) and aberrant crypts (ACs) in colonic mucosa were counted after methylene blue staining. Lipid peroxidation in feces was measured by the thiobarbituric acid-reactive substances (TBARS) assay. The numbers of ACF and ACs per colon significantly increased in Hemin group compared to the control group. However, the numbers of ACF and ACs per colon notably decreased in hemin + AA group compared to the control group or hemin group (p<0.05). In feces, the TBARS value of hemin group was higher than the control group (p<0.01). The TBARS value of hemin + AA group was slightly decreased compared to Hemin group. These results indicate that hemin can promote the experimental colon carcinogenesis in ICR mice. On the other hand, additional supplement of AA via drinking water has a protective effect against the colon carcinogenesis. The related mechanisms need to be illustrated by further studies in future.

Natural Antioxidant and Anti-Inflammatory Compounds in Foodstuff or Medicinal Herbs Inducing Heme Oxygenase-1 Expression.

Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme that catalyzes heme group degradation. Decreased level of HO-1 is correlated with disease progression, and HO-1 induction suppresses development of metabolic and neurological disorders. Natural compounds with antioxidant activities have emerged as a rich source of HO-1 inducers with marginal toxicity. Here we discuss the therapeutic role of HO-1 in obesity, hypertension, atherosclerosis, Parkinson’s disease and hepatic fibrosis, and present important signaling pathway components that lead to HO-1 expression. We provide an updated, comprehensive list of natural HO-1 inducers in foodstuff and medicinal herbs categorized by their chemical structures. Based on the continued research in HO-1 signaling pathways and rapid development of their natural inducers, HO-1 may serve as a preventive and therapeutic target for metabolic and neurological disorders.

Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4+ T cells

BackgroundNeutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown.MethodsTo assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot.ResultsFifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4+ from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs.ConclusionThe ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.

RNA Seq Profiles and Bioinformatics Validation in a Large Sample of Sickle Cell Disease Patients

Background Sickle Cell Disease (SCD) is a genetic disorder caused by a single amino acid substitution in the ß-hemoglobin chain. Clinical manifestations of SCD are multisystemic and heterogeneous, with a wide range of organ damage between patients despite identical genetic mutations. The sub-phenotypes of SCD reflect damage to different organs arising from the pathophysiology of the disease. Biomarkers, such as N-terminal-pro-Brain natriuretic peptide (NT-proBNP), tricuspid regurgitant velocity (TRV), and hemolytic parameters provide readouts of the degree of different organ damage and are known risk markers. Dissecting its pathophysiology along known biological pathways has led to development of therapeutic targets, but we still do not fully understand the pathogenesis for much of the organ damage. Given the complexity of the pathophysiology of SCD that is likely to involve multiple overlapping and interacting biological pathways, an agnostic, network-based approach using gene expression data with annotated risk markers provides an attractive alternative. We assessed the plausibility of using such an approach with bilirubin levels as a phenotype. Bilirubin is a breakdown product of red blood cells (RBCs) that is measured in routine labs and a validated hemolytic and disease severity marker of SCD. Methods Using RNA-Seq data from 224 patients with SCD (80% HbSS and HbSBeta0 thalassemia combined, 15% HbSC, 5% HbSBeta+ thalassemia, and 0.4% HbSD), we performed a differential gene expression (DGE) analysis and weighted gene co-expression network analysis (WGCNA) of 12,450 genes. For the DGE analysis we used total bilirubin levels as a continuous variable for the main phenotype of interest, adjusting for age, sex, and hemoglobin genotype. We then conducted WGCNA to identify modules of genes with similar coexpression patterns, which we functionally annotated (via the ClusterProfiler R package) for interesting biological themes. Results Of the 12,450 genes, 10,604 were protein coding, and 1,560 were lncRNAs, and 286 were other non-coding transcripts. 3,509 (2,975 coding, 447 lncRNA, and 87 other non-coding) were considered differentially expressed (unadjusted p-value &amp;lt; 0.05) according to bilirubin level. Through the WCGNA, the genes clustered into 11 co-expression modules, representing a range of biological pathways. Four of these modules had representative eigengenes that were significantly correlated with bilirubin (p &amp;lt; 0.05), and we focused on 2 of these that had biologically interesting functions. The first module of interest (turquoise) consisted of 3,636 genes (3,371 protein-coding and 210 lncRNA) (25% differentially expressed, and 62% upregulated). Genes in this module have been associated with cellular functioning of younger RBCs, validating the presence of the significant Gene Ontology (GO) biological pathways listed in the bar graph (mitochondria gene expression, ribonucleotide processes, etc.), and aligning with expected processes as the young RBCs replace the degraded RBCs that had caused the high bilirubin. Notably, this module contained the biliverdin reductase A (BLVRA) gene (differentially expressed at p &amp;lt; 0.008), which reduces biliverdin to bilirubin. The other highlighted module (red) consisted of 914 genes (846 protein-coding and 57 lncRNA) (42% differentially expressed, and 97% upregulated). Multiple pathways in the red module were related to catabolic processes. As previously stated, bilirubin is the byproduct of hemoprotein catabolism, suggesting an interesting correlation. Additionally, this module contained 2 other pathways associated with porphyrin, which is a component of RBCs that contains the heme group and is involved in heme biosynthesis. Conclusion Taken together, these results provide interesting insights into the biological pathways driving one facet of sickle cell disease's many clinical manifestations. Figure 1 Disclosures No relevant conflicts of interest to declare.