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Sample records for heme iron polypeptide

  1. Rationale and design of the oral HEMe iron polypeptide Against Treatment with Oral Controlled Release Iron Tablets trial for the correction of anaemia in peritoneal dialysis patients (HEMATOCRIT trial

    Isbel Nicole M

    2009-07-01

    Full Text Available Abstract Background The main hypothesis of this study is that oral heme iron polypeptide (HIP; Proferrin® ES administration will more effectively augment iron stores in erythropoietic stimulatory agent (ESA-treated peritoneal dialysis (PD patients than conventional oral iron supplementation (Ferrogradumet®. Methods Inclusion criteria are peritoneal dialysis patients treated with darbepoietin alpha (DPO; Aranesp®, Amgen for ≥ 1 month. Patients will be randomized 1:1 to receive either slow-release ferrous sulphate (1 tablet twice daily; control or HIP (1 tablet twice daily for a period of 6 months. The study will follow an open-label design but outcome assessors will be blinded to study treatment. During the 6-month study period, haemoglobin levels will be measured monthly and iron studies (including transferring saturation [TSAT] measurements will be performed bi-monthly. The primary outcome measure will be the difference in TSAT levels between the 2 groups at the end of the 6 month study period, adjusted for baseline values using analysis of covariance (ANCOVA. Secondary outcome measures will include serum ferritin concentration, haemoglobin level, DPO dosage, Key's index (DPO dosage divided by haemoglobin concentration, and occurrence of adverse events (especially gastrointestinal adverse events. Discussion This investigator-initiated multicentre study has been designed to provide evidence to help nephrologists and their peritoneal dialysis patients determine whether HIP administration more effectively augments iron stores in ESP-treated PD patients than conventional oral iron supplementation. Trial Registration Australia New Zealand Clinical Trials Registry number ACTRN12609000432213.

  2. Prebiotics increase heme iron bioavailability and do not affect non-heme iron bioavailability in humans.

    Weinborn, Valerie; Valenzuela, Carolina; Olivares, Manuel; Arredondo, Miguel; Weill, Ricardo; Pizarro, Fernando

    2017-05-24

    The aim of this study was to establish the effect of a prebiotic mix on heme and non-heme iron (Fe) bioavailability in humans. To this purpose, twenty-four healthy women were randomized into one of two study groups. One group ate one yogurt per day for 12 days with a prebiotic mix (prebiotic group) and the other group received the same yogurt but without the prebiotic mix (control group). Before and after the intake period, the subjects participated in Fe absorption studies. These studies used 55 Fe and 59 Fe radioactive isotopes as markers of heme Fe and non-heme Fe, respectively, and Fe absorption was measured by the incorporation of radioactive Fe into erythrocytes. The results showed that there were no significant differences in heme and non-heme Fe bioavailability in the control group. Heme Fe bioavailability of the prebiotic group increased significantly by 56% post-prebiotic intake. There were no significant differences in non-heme Fe bioavailability in this group. We concluded that daily consumption of a prebiotic mix increases heme Fe bioavailability and does not affect non-heme iron bioavailability.

  3. Mononuclear non-heme iron(III)

    Home; Journals; Journal of Chemical Sciences; Volume 123; Issue 2. Mononuclear non-heme iron(III) complexes of linear and tripodal tridentate ligands as functional models for catechol dioxygenases: Effect of -alkyl substitution on regioselectivity and reaction rate. Mallayan Palaniandavar Kusalendiran Visvaganesan.

  4. Heme metabolism as an integral part of iron homeostasis

    Paweł Lipiński

    2014-01-01

    Full Text Available Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways – heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S] – is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages.

  5. [Heme metabolism as an integral part of iron homeostasis].

    Lipiński, Paweł; Starzyński, Rafał R; Styś, Agnieszka; Gajowiak, Anna; Staroń, Robert

    2014-01-02

    Heme, a ferrous iron protoporphyrin IX complex, is employed as a prosthetic group in a number of diverse heme proteins that participate in important cellular and systemic physiological processes. Provision of an adequate amount of iron for heme biosynthesis is one of the elemental hallmarks of intracellular iron homeostasis. In the cell the bioavailability of iron for the two main iron biological pathways--heme synthesis and the biogenesis of iron-sulfur clusters ([Fe-S])--is mainly regulated by the IRP/IRE posttranscriptional system. The biogenesis of [Fe-S] centers is crucial for heme synthesis because these co-factors determine the activity of IRP1 and that of ferrochelatase, an enzyme responsible for the insertion of an iron into protoporphyrin IX to produce heme. On the other hand, delivery of iron for heme and hemoglobin synthesis in erythroblasts, precursors of erythrocytes in bone marrow, is an indispensable element of body iron homeostasis. This process relies on the recovery of iron from senescent red blood cells through the enzymatic degradation of heme molecules and recycling of iron to the circulation. Molecular coordination of these processes involves the activity of heme oxygenase 1, IRP1 and IRP2 as well as the functioning of the hepcidin-ferroportin regulatory axis. Recent studies show in mammals the existence of an expanded system of proteins involved in the transport of intact heme molecules at the cellular and systemic levels. The biological role of this system is of particular importance when the concentration of free heme reaches a toxic level in the body (intravascular hemolysis) as well as locally in cells having intensive heme metabolism such as erythroblasts and macrophages.

  6. Heme and non-heme iron transporters in non-polarized and polarized cells

    Yasui Yumiko

    2010-06-01

    Full Text Available Abstract Background Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs, and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs. Results In this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1, and 2 candidate heme transporters--heme carrier protein 1 (HCP1 and heme responsive gene-1 (HRG-1--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase. Conclusions HCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is

  7. Dietary iron controls circadian hepatic glucose metabolism through heme synthesis.

    Simcox, Judith A; Mitchell, Thomas Creighton; Gao, Yan; Just, Steven F; Cooksey, Robert; Cox, James; Ajioka, Richard; Jones, Deborah; Lee, Soh-Hyun; King, Daniel; Huang, Jingyu; McClain, Donald A

    2015-04-01

    The circadian rhythm of the liver maintains glucose homeostasis, and disruption of this rhythm is associated with type 2 diabetes. Feeding is one factor that sets the circadian clock in peripheral tissues, but relatively little is known about the role of specific dietary components in that regard. We assessed the effects of dietary iron on circadian gluconeogenesis. Dietary iron affects circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its cosuppressor nuclear receptor corepressor 1 (NCOR). Loss of regulated heme synthesis was achieved by aminolevulinic acid (ALA) treatment of mice or cultured cells to bypass the rate-limiting enzyme in hepatic heme synthesis, ALA synthase 1 (ALAS1). ALA treatment abolishes differences in hepatic glucose production and in the expression of gluconeogenic enzymes seen with variation of dietary iron. The differences among diets are also lost with inhibition of heme synthesis with isonicotinylhydrazine. Dietary iron modulates levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a transcriptional activator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1α variation observed among the iron diets, suggesting that iron is acting through reactive oxygen species signaling. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  8. Ironing out the Details: Exploring the Role of Iron and Heme in Blood-Sucking Arthropods

    Whiten, Shavonn R.; Eggleston, Heather; Adelman, Zach N.

    2018-01-01

    Heme and iron are essential molecules for many physiological processes and yet have the ability to cause oxidative damage such as lipid peroxidation, protein degradation, and ultimately cell death if not controlled. Blood-sucking arthropods have evolved diverse methods to protect themselves against iron/heme-related damage, as the act of bloodfeeding itself is high risk, high reward process. Protective mechanisms in medically important arthropods include the midgut peritrophic matrix in mosquitoes, heme aggregation into the crystalline structure hemozoin in kissing bugs and hemosomes in ticks. Once heme and iron pass these protective mechanisms they are presumed to enter the midgut epithelial cells via membrane-bound transporters, though relatively few iron or heme transporters have been identified in bloodsucking arthropods. Upon iron entry into midgut epithelial cells, ferritin serves as the universal storage protein and transport for dietary iron in many organisms including arthropods. In addition to its role as a nutrient, heme is also an important signaling molecule in the midgut epithelial cells for many physiological processes including vitellogenesis. This review article will summarize recent advancements in heme/iron uptake, detoxification and exportation in bloodfeeding arthropods. While initial strides have been made at ironing out the role of dietary iron and heme in arthropods, much still remains to be discovered as these molecules may serve as novel targets for the control of many arthropod pests. PMID:29387018

  9. Acquisition of iron from transferrin regulates reticulocyte heme synthesis

    Ponka, P.; Schulman, H.M.

    1985-01-01

    Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59 Fe from [ 59 Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [ 59 Fe]transferrin. Also, Fe-SIH stimulates [2- 14 C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59 Fe incorporation into heme from either [ 59 Fe]transferrin or [ 59 Fe]SIH but does reverse the inhibition of 59 Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes

  10. Heme oxygenase activity correlates with serum indices of iron homeostasis in healthy nonsmokers

    Heme oxygenase (HO) catalyzes the breakdown of heme to carbon monoxide, iron, and biliverdin. While the use of genetically altered animal models in investigation has established distinct associations between HO activity and systemic iron availability, studies have not yet confirm...

  11. Dietary hemoglobin rescues young piglets from severe iron deficiency anemia: Duodenal expression profile of genes involved in heme iron absorption.

    Robert Staroń

    Full Text Available Heme is an efficient source of iron in the diet, and heme preparations are used to prevent and cure iron deficiency anemia in humans and animals. However, the molecular mechanisms responsible for heme absorption remain only partially characterized. Here, we employed young iron-deficient piglets as a convenient animal model to determine the efficacy of oral heme iron supplementation and investigate the pathways of heme iron absorption. The use of bovine hemoglobin as a dietary source of heme iron was found to efficiently counteract the development of iron deficiency anemia in piglets, although it did not fully rebalance their iron status. Our results revealed a concerted increase in the expression of genes responsible for apical and basolateral heme transport in the duodenum of piglets fed a heme-enriched diet. In these animals the catalytic activity of heme oxygenase 1 contributed to the release of elemental iron from the protoporphyrin ring of heme within enterocytes, which may then be transported by the strongly expressed ferroportin across the basolateral membrane to the circulation. We hypothesize that the well-recognized high bioavailability of heme iron may depend on a split pathway mediating the transport of heme-derived elemental iron and intact heme from the interior of duodenal enterocytes to the bloodstream.

  12. Studying disorders of vertebrate iron and heme metabolism using zebrafish.

    van der Vorm, Lisa N; Paw, Barry H

    2017-01-01

    Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be - and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Irradiation of bovine meat: effect of heme-iron concentration

    Mistura, Liliana Perazzini Furtado

    2002-01-01

    The irradiation is often used, nowadays, for meat conservation and it is important to know how much this process interferes with the nutritional quality of the meat. In this study round cut meat, ground and steaks (from a local supermarket) was irradiated with doses of O; 1; 2; 3; 4; 5; 7,5 and 10 kGy (JS-7500 Nordium Inc -Canada) and the interference of irradiation and the process of food preparation on heme-iron (H Fe) content was determined. Half of the sample was kept raw and the other half was grilled in a pre-warmed oven at 250 deg C for 9 min and a controlled humidity of 70%. The chemical composition, the total iron (T Fe) (EM) and the heme iron concentration were determined (Hornsey,1956) and the sensorial quality evaluated. The average T Fe concentration of raw and ground , ground and grilled, raw steaks and grilled steak meat, on dry and degreased basis was 113 mug/g, 121 mug/g , 91 mug/g and 77 mug/g; and the H Fe concentration 105 mug/g (93% of T Fe) , 88 mug/g (73% of T Fe), 90 mug/g (99% of T Fe) and 52 mug/g (68% of T Fe) respectively. Data were evaluated by ANOVA with fixed effects and multiple comparisons. The irradiation neither altered the chemical composition nor the proportion of heme iron of meat. The preparation conditions (temperature, cooking time, environment humidity, meat presentation) of the sample interfered more with the heme iron content than the irradiation. With the sensorial analysis we verified that meats irradiated with doses of 3 kGy were better evaluated in softness and succulency attributes than the others. Meat submitted to irradiation doses up to 3 kGy were accepted by the specialists' panel. (author)

  14. The effect of proteins from animal source foods on heme iron bioavailability in humans.

    Pizarro, Fernando; Olivares, Manuel; Valenzuela, Carolina; Brito, Alex; Weinborn, Valerie; Flores, Sebastián; Arredondo, Miguel

    2016-04-01

    Forty-five women (35-45 year) were randomly assigned to three iron (Fe) absorption sub-studies, which measured the effects of dietary animal proteins on the absorption of heme Fe. Study 1 was focused on heme, red blood cell concentrate (RBCC), hemoglobin (Hb), RBCC+beef meat; study 2 on heme, heme+fish, chicken, and beef; and study 3 on heme and heme+purified animal protein (casein, collagen, albumin). Study 1: the bioavailability of heme Fe from Hb was similar to heme only (∼13.0%). RBCC (25.0%) and RBCC+beef (21.3%) were found to be increased 2- and 1.6-fold, respectively, when compared with heme alone (pProteins from animal source foods and their digestion products did not enhance heme Fe absorption. Copyright © 2015. Published by Elsevier Ltd.

  15. Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

    Jiang, Yongying; Trnka, Michael J; Medzihradszky, Katalin F; Ouellet, Hugues; Wang, Yongqiang; Ortiz de Montellano, Paul R

    2009-03-01

    To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.

  16. Wearing red for signaling: the heme-bach axis in heme metabolism, oxidative stress response and iron immunology.

    Igarashi, Kazuhiko; Watanabe-Matsui, Miki

    2014-04-01

    The connection between gene regulation and metabolism is an old issue that warrants revisiting in order to understand both normal as well as pathogenic processes in higher eukaryotes. Metabolites affect the gene expression by either binding to transcription factors or serving as donors for post-translational modification, such as that involving acetylation and methylation. The focus of this review is heme, a prosthetic group of proteins that includes hemoglobin and cytochromes. Heme has been shown to bind to several transcription factors, including Bach1 and Bach2, in higher eukaryotes. Heme inhibits the transcriptional repressor activity of Bach1, resulting in the derepression of its target genes, such as globin in erythroid cells and heme oxygenase-1 in diverse cell types. Since Bach2 is important for class switch recombination and somatic hypermutation of immunoglobulin genes as well as regulatory and effector T cell differentiation and the macrophage function, the heme-Bach2 axis may regulate the immune response as a signaling cascade. We discuss future issues regarding the topic of the iron/heme-gene regulation network based on current understanding of the heme-Bach axis, including the concept of "iron immunology" as the synthesis of the iron metabolism and the immune response.

  17. A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

    Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

    2014-10-01

    Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

  18. TLR Stimulation Dynamically Regulates Heme and Iron Export Gene Expression in Macrophages

    Mary Philip

    2016-01-01

    Full Text Available Pathogenic bacteria have evolved multiple mechanisms to capture iron or iron-containing heme from host tissues or blood. In response, organisms have developed defense mechanisms to keep iron from pathogens. Very little of the body’s iron store is available as free heme; rather nearly all body iron is complexed with heme or other proteins. The feline leukemia virus, subgroup C (FeLV-C receptor, FLVCR, exports heme from cells. It was unknown whether FLVCR regulates heme-iron availability after infection, but given that other heme regulatory proteins are upregulated in macrophages in response to bacterial infection, we hypothesized that macrophages dynamically regulate FLVCR. We stimulated murine primary macrophages or macrophage cell lines with LPS and found that Flvcr is rapidly downregulated in a TLR4/MD2-dependent manner; TLR1/2 and TLR3 stimulation also decreased Flvcr expression. We identified several candidate TLR-activated transcription factors that can bind to the Flvcr promoter. Macrophages must balance the need to sequester iron from systemic circulating or intracellular pathogens with the macrophage requirement for heme and iron to produce reactive oxygen species. Our findings underscore the complexity of this regulation and point to a new role for FLVCR and heme export in macrophages responses to infection and inflammation.

  19. The effect of irradiation and thermal process on beef heme iron concentration and color properties

    Mistura, Liliana Perazzini Furtado; Colli, Celia

    2009-01-01

    The aim of this study was to evaluate the influence of irradiation and thermal process on the heme iron (heme-Fe) concentration and color properties of Brazilian cattle beef. Beef samples (patties and steaks) were irradiated at 0-10 kGy and cooked in a combination oven at 250 deg C for 9 minutes with 70% humidity. Total iron and heme iron (heme-Fe) concentrations were determined. The data were compared by multiple comparisons and fixed- effects ANOVA. Irradiation at doses higher than 5 kGy significantly altered the heme-Fe concentration. However, the sample preparation conditions interfered more in the heme-Fe content than did the irradiation. Depending on the animal species, meat heme iron levels between 35 and 52% of the total iron are used for dietetic calculations. In this study the percentage of heme-iron was, on average, 70% of the total iron showing that humidity is an important factor for its preservation. The samples were analyzed instrumentally for CIE L * , a * , and b * values. (author)

  20. Alteration by irradiation and storage at amount of heme iron in poultry meat

    Souza, A.R.M. de; Arthur, V.; Canniatti-Brazaca, S.G.

    2007-01-01

    Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 °C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author) [pt

  1. Alteration by irradiation and storage at amount of heme iron in poultry meat

    Souza, Adriana Regia Marques de; Arthur, Valter Arthur; Canniatti-Brazaca, Solange Guidolin

    2007-01-01

    Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 deg C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author)

  2. Oxidative stability of a heme iron-fortified bakery product: Effectiveness of ascorbyl palmitate and co-spray-drying of heme iron with calcium caseinate.

    Alemán, Mercedes; Bou, Ricard; Tres, Alba; Polo, Javier; Codony, Rafael; Guardiola, Francesc

    2016-04-01

    Fortification of food products with iron is a common strategy to prevent or overcome iron deficiency. However, any form of iron is a pro-oxidant and its addition will cause off-flavours and reduce a product's shelf life. A highly bioavailable heme iron ingredient was selected to fortify a chocolate cream used to fill sandwich-type cookies. Two different strategies were assessed for avoiding the heme iron catalytic effect on lipid oxidation: ascorbyl palmitate addition and co-spray-drying of heme iron with calcium caseinate. Oxidation development and sensory acceptability were monitored in the cookies over one-year of storage at room temperature in the dark. The addition of ascorbyl palmitate provided protection against oxidation and loss of tocopherols and tocotrienols during the preparation of cookies. In general, ascorbyl palmitate, either alone or in combination with the co-spray-dried heme iron, prevented primary oxidation and hexanal formation during storage. The combination of both strategies resulted in cookies that were acceptable from a sensory point of view after 1year of storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Mononuclear non-heme iron(III) complexes of linear and tripodal ...

    The rate of oxygenation depends on the solvent and the. Lewis acidity of iron(III) ... has been achieved by non-heme iron enzymes and their ..... oxygen atoms of nitrate ion (figure 3). ... enhanced covalency of iron-catecholate interaction and.

  4. Irradiation of bovine meat: effect of heme-iron concentration.; Irradiacao de carne bovina: efeito na concentracao de ferro heme

    Mistura, Liliana Perazzini Furtado

    2002-07-01

    The irradiation is often used, nowadays, for meat conservation and it is important to know how much this process interferes with the nutritional quality of the meat. In this study round cut meat, ground and steaks (from a local supermarket) was irradiated with doses of O; 1; 2; 3; 4; 5; 7,5 and 10 kGy (JS-7500 Nordium Inc -Canada) and the interference of irradiation and the process of food preparation on heme-iron (H Fe) content was determined. Half of the sample was kept raw and the other half was grilled in a pre-warmed oven at 250 deg C for 9 min and a controlled humidity of 70%. The chemical composition, the total iron (T Fe) (EM) and the heme iron concentration were determined (Hornsey,1956) and the sensorial quality evaluated. The average T Fe concentration of raw and ground , ground and grilled, raw steaks and grilled steak meat, on dry and degreased basis was 113 mug/g, 121 mug/g , 91 mug/g and 77 mug/g; and the H Fe concentration 105 mug/g (93% of T Fe) , 88 mug/g (73% of T Fe), 90 mug/g (99% of T Fe) and 52 mug/g (68% of T Fe) respectively. Data were evaluated by ANOVA with fixed effects and multiple comparisons. The irradiation neither altered the chemical composition nor the proportion of heme iron of meat. The preparation conditions (temperature, cooking time, environment humidity, meat presentation) of the sample interfered more with the heme iron content than the irradiation. With the sensorial analysis we verified that meats irradiated with doses of 3 kGy were better evaluated in softness and succulency attributes than the others. Meat submitted to irradiation doses up to 3 kGy were accepted by the specialists' panel. (author)

  5. Factors for the bioavailability of heme iron preparation in female rats

    村上, 亜由美; 岸本, 三香子; 川口, 真規子; 松浦, 寿喜; 市川, 富夫; Ayumi, Murakami; Mikako, Kishimoto; Makiko, Kawaguchi; Toshiki, Matsuura; Tomio, Ichikawa

    1998-01-01

    Factors for iron absorption in small intestine using heme iron preparation (HIP) and ferric citrate (FC) were investigated. We measured the solubility of iron of experimental diets (FC-normal, FC-overload, HIP-normal, HIP-overload) in water (adjusted pH6.8) and the diffusibility of dietary iron after digestion in vitro. The results did not show significantly differences between FC and HIP. Also, we measured microsomal heme oxygenase (HO) activity in intestinal mucosa of female rats fed experi...

  6. Alteration by irradiation and storage at amount of heme iron in poultry meat; Alteracoes provocadas pela irradiacao e armazenamento nos teores de ferro heme em carne de frango

    Souza, Adriana Regia Marques de; Arthur, Valter Arthur [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Lab. de Irradiacao de Alimentos e Radioentomologia; Canniatti-Brazaca, Solange Guidolin [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil). Dept. de Agroindustria, Alimentos e Nutricao]. E-mail: sgcbraza@esalq.usp.br

    2007-04-15

    Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 deg C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author)

  7. PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis.

    Ryu, Moon-Suhn; Zhang, Deliang; Protchenko, Olga; Shakoury-Elizeh, Minoo; Philpott, Caroline C

    2017-05-01

    Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC-binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.

  8. Control of heme synthesis during Friend cell differentiation: role of iron and transferrin

    Laskey, J.D.; Ponka, P.; Schulman, H.M.

    1986-01-01

    In many types of cells the synthesis of σ-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from this laboratory with reticulocytes suggest that the rate of iron uptake from 125 I-transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels or 59 Fe-Tf (20 μM). Furthermore, in induced Friend cells 100 μM Fe-SIH stimulated 2- 14 C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells

  9. X-ray absorption spectroscopic studies of mononuclear non-heme iron enzymes

    Westre, Tami E. [Stanford Univ., CA (United States)

    1996-01-01

    Fe-K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the electronic and geometric structure of the iron active site in non-heme iron enzymes. A new theoretical extended X-ray absorption fine structure (EXAFS) analysis approach, called GNXAS, has been tested on data for iron model complexes to evaluate the utility and reliability of this new technique, especially with respect to the effects of multiple-scattering. In addition, a detailed analysis of the 1s→3d pre-edge feature has been developed as a tool for investigating the oxidation state, spin state, and geometry of iron sites. Edge and EXAFS analyses have then been applied to the study of non-heme iron enzyme active sites.

  10. A central role for heme iron in colon carcinogenesis associated with red meat intake.

    Bastide, Nadia M; Chenni, Fatima; Audebert, Marc; Santarelli, Raphaelle L; Taché, Sylviane; Naud, Nathalie; Baradat, Maryse; Jouanin, Isabelle; Surya, Reggie; Hobbs, Ditte A; Kuhnle, Gunter G; Raymond-Letron, Isabelle; Gueraud, Françoise; Corpet, Denis E; Pierre, Fabrice H F

    2015-03-01

    Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines, and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP + MeIQx, 50 + 25 μg/kg in diet), and NOC (induced by NaNO₂+ NaNO₂; 0.17 + 0.23 g/L of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc-deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions, but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67 ± 39 mm²; 2.5% hemoglobin diet: 114 ± 47 mm², P = 0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation. . ©2015 American Association for Cancer Research.

  11. Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

    Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

    2013-01-01

    Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experi...

  12. Sugars increase non-heme iron bioavailability in human epithelial intestinal and liver cells.

    Tatiana Christides

    Full Text Available Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55 increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

  13. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

  14. Structures of the multicomponent Rieske non-heme iron toluene 2, 3-dioxygenase enzyme system

    Friemann, Rosmarie [Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden); Lee, Kyoung [Department of Microbiology, Changwon National University, Changwon, Kyoungnam 641-773 (Korea, Republic of); Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242 (United States); Brown, Eric N. [Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (United States); Gibson, David T. [Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242 (United States); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden); Ramaswamy, S., E-mail: s-ramaswamy@uiowa.edu [Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (United States); Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden)

    2009-01-01

    The crystal structures of the three-component toluene 2, 3-dioxygenase system provide a model for electron transfer among bacterial Rieske non-heme iron dioxygenases. Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2, 3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe–2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe–2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.

  15. Control of intracellular heme levels: Heme transporters and Heme oxygenases

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology. PMID:21238504

  16. Effects of illumination and packaging on non-heme iron and color attributes of sliced ham.

    Li, H; Li, C B; Xu, X L; Zhou, G H

    2012-08-01

    This study was designed to investigate effects of illumination and packaging on color of cooked cured sliced ham during refrigeration, and the possibility of decomposition of nitrosylheme under light and oxygen exposure. Three illumination levels and three packaging films with different oxygen transmission rates (OTRs) were used in two separate experiments during 35 days storage, and pH value, a* value, nitrosylheme, residual nitrite and non-heme iron were evaluated. Packaging OTRs had significant effects (P0.05) nitrosylheme concentration during storage. For both groups, storage time had a significant effect (P<0.01) on a* value and nitrosylheme. Negative relationships between nitrosylheme and nitrite in the illumination group, and between nitrosylheme and non-heme iron in the packaging group were observed. Therefore, illumination level and packaging OTR had limited effects on overall pigment stability, but more discoloration and loss of redness occurred on the surface of products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Heme iron content in lamb meat is differentially altered upon boiling, grilling, or frying as assessed by four distinct analytical methods.

    Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

    2013-01-01

    Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

  18. Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

    Azin Pourkhalili

    2013-01-01

    Full Text Available Lamb meat is regarded as an important source of highly bioavailable iron (heme iron in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

  19. Tick iron and heme metabolism – New target for an anti-tick intervention

    Hajdušek, Ondřej; Šíma, Radek; Perner, Jan; Loosová, Gabriela; Harcubová, Adéla; Kopáček, Petr

    2016-01-01

    Roč. 7, č. 4 (2016), s. 565-572 ISSN 1877-959X R&D Projects: GA ČR GA13-11043S; GA ČR GP13-27630P; GA ČR GP13-12816P EU Projects: European Commission(XE) 316304 - MODBIOLIN Institutional support: RVO:60077344 Keywords : tick * iron * heme * RNAi * vaccine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.230, year: 2016

  20. Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis.

    Perry, Christopher; de Los Santos, Emmanuel L C; Alkhalaf, Lona M; Challis, Gregory L

    2018-04-13

    Covering: up to the end of 2017The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on experimentally characterised examples. Enzymes belonging to this class are known to catalyse a range of transformations, including oxidative carbocyclisation, N-oxygenation, C-hydroxylation and C-C desaturation. Examples of such enzymes that have yet to be experimentally investigated are also briefly described and their likely functions are discussed.

  1. Silencing of Iron and Heme-Related Genes Revealed a Paramount Role of Iron in the Physiology of the Hematophagous Vector Rhodnius prolixus

    Ana B. Walter-Nuno

    2018-02-01

    Full Text Available Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of Rhodnius prolixus, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP, Heme Oxygenase (HO and the heme exporter Feline Leukemia Virus C Receptor (FLVCR, components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM, where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for R. prolixus physiology and imbalances in the levels of

  2. Heme Iron Concentrate and Iron Sulfate Added to Chocolate Biscuits: Effects on Hematological Indices of Mexican Schoolchildren.

    Quintero-Gutiérrez, Adrián Guillermo; González-Rosendo, Guillermina; Pozo, Javier Polo; Villanueva-Sánchez, Javier

    2016-08-01

    Food fortification is one of the most effective strategies for increasing iron intake in the population. A simple blind trial was conducted to compare the effect of 2 forms of iron fortification and assess the changes in hemoglobin and iron status indices among preschool children from rural communities. Hemoglobin was evaluated in 47 children aged 3-6 years old. For 72 days (10-week period), children ate Nito biscuits. Thirteen pupils with elevated hemoglobin levels were assigned to the biscuit control group, and pupils with hemoglobin equal to 13.5 mg/dL or less were randomly allocated to consume fortified biscuits with a heme iron concentrate (n = 15) or iron sulfate (n = 19). Changes in hemoglobin, plasma ferritin, and other hematological indices were evaluated with analysis of variance (ANOVA) for repeated measurements. Except mean corpuscular hemoglobin concentrations (+1.27 ± 2.25 g/dL), hematological indices increased significantly across the study: Mean corpuscular volume (+2.2 ± 1.0 f/dL), red blood cells (+0.30 ± 0.37 M/μL), mean corpuscular hemoglobin (+1.8 ± 1.74 pg), hemoglobin (+1.68 ± 0.91 g/dL), hematocrit (+3.43% ± 3.03%), and plasma ferritin (+18.38 ± 22.1 μg/L) were all p effect of the iron-fortified chocolate biscuits in the hemoglobin levels was higher than the control group (+1.1 ± 0.2 g/dL) but no difference was found between consumers of fortified biscuits with heme iron concentrate or iron sulfate (+1.9 ± 0.2 g/dL and +2.0 ± 0.2 g/dL, respectively). Heme iron concentrate and iron sulfate were equally effective in increasing Hb levels and hematological indices. Processed foods were shown to be an effective, valuable, and admissible intervention to prevent anemia in preschool children.

  3. Control of intracellular heme levels: Heme transporters and heme oxygenases

    Khan, Anwar A.; Quigley, John G.

    2011-01-01

    Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

  4. Experimental and Computational Evidence for the Mechanism of Intradiol Catechol Dioxygenation by Non-Heme Iron(III) Complexes

    Jastrzebski, Robin; Quesne, Matthew G; Weckhuysen, Bert M; de Visser, Sam P; Bruijnincx, Pieter C A

    2014-01-01

    Catechol intradiol dioxygenation is a unique reaction catalyzed by iron-dependent enzymes and non-heme iron(III) complexes. The mechanism by which these systems activate dioxygen in this important metabolic process remains controversial. Using a combination of kinetic measurements and computational modelling of multiple iron(III) catecholato complexes, we have elucidated the catechol cleavage mechanism and show that oxygen binds the iron center by partial dissociation of the substrate from the iron complex. The iron(III) superoxide complex that is formed subsequently attacks the carbon atom of the substrate by a rate-determining C=O bond formation step. PMID:25322920

  5. Meat, Dietary Heme Iron, and Risk of Type 2 Diabetes Mellitus: The Singapore Chinese Health Study.

    Talaei, Mohammad; Wang, Ye-Li; Yuan, Jian-Min; Pan, An; Koh, Woon-Puay

    2017-10-01

    We evaluated the relationships of red meat, poultry, fish, and shellfish intakes, as well as heme iron intake, with the risk of type 2 diabetes mellitus (T2D).The Singapore Chinese Health Study is a population-based cohort study that recruited 63,257 Chinese adults aged 45-74 years from 1993 to 1998. Usual diet was evaluated using a validated 165-item semiquantitative food frequency questionnaire at recruitment. Physician-diagnosed T2D was self-reported during 2 follow-up interviews in 1999-2004 and 2006-2010. During a mean follow-up of 10.9 years, 5,207 incident cases of T2D were reported. When comparing persons in the highest intake quartiles with those in the lowest, the multivariate-adjusted hazard ratio for T2D was 1.23 (95% confidence interval (CI): 1.14, 1.33) for red meat intake (P for trend meat intake remained significantly associated with T2D risk (multivariate-adjusted hazard ratio = 1.13, 95% CI: 1.01, 1.25; P for trend = 0.02). Heme iron was associated with a higher risk of T2D even after additional adjustment for red meat intake (multivariate-adjusted hazard ratio = 1.14, 95% CI: 1.02, 1.28; P for trend = 0.03). In conclusion, red meat and poultry intakes were associated with a higher risk of T2D. These associations were mediated completely for poultry and partially for red meat by heme iron intake. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Heme degrading protein HemS is involved in oxidative stress response of Bartonella henselae.

    MaFeng Liu

    Full Text Available Bartonellae are hemotropic bacteria, agents of emerging zoonoses. These bacteria are heme auxotroph Alphaproteobacteria which must import heme for supporting their growth, as they cannot synthesize it. Therefore, Bartonella genome encodes for a complete heme uptake system allowing the transportation of this compound across the outer membrane, the periplasm and the inner membranes. Heme has been proposed to be used as an iron source for Bartonella since these bacteria do not synthesize a complete system required for iron Fe³⁺ uptake. Similarly to other bacteria which use heme as an iron source, Bartonellae must transport this compound into the cytoplasm and degrade it to allow the release of iron from the tetrapyrrole ring. For Bartonella, the gene cluster devoted to the synthesis of the complete heme uptake system also contains a gene encoding for a polypeptide that shares homologies with heme trafficking or degrading enzymes. Using complementation of an E. coli mutant strain impaired in heme degradation, we demonstrated that HemS from Bartonella henselae expressed in E. coli allows the release of iron from heme. Purified HemS from B. henselae binds heme and can degrade it in the presence of a suitable electron donor, ascorbate or NADPH-cytochrome P450 reductase. Knocking down the expression of HemS in B. henselae reduces its ability to face H₂O₂ induced oxidative stress.

  7. Mono- and binuclear non-heme iron chemistry from a theoretical perspective

    Rokob, T. A.; Chalupský, Jakub; Bím, Daniel; Andrikopoulos, Prokopis C.; Srnec, Martin; Rulíšek, Lubomír

    2016-01-01

    Roč. 21, 5/6 (2016), s. 619-644 ISSN 0949-8257 R&D Projects: GA ČR(CZ) GJ15-10279Y; GA ČR(CZ) GA14-31419S; GA ČR GA15-19143S Grant - others:COST(XE) CM1305 Institutional support: RVO:61388963 ; RVO:61388955 Keywords : non-heme iron * density functional theory * multireference methods * dioxygen activation * reactivity Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.894, year: 2016

  8. Structural and Functional Models of Non-Heme Iron Enzymes : A Study of the 2-His-1-Carboxylate Facial Triad Structural Motif

    Bruijnincx, P.C.A.

    2007-01-01

    The structural and functional modeling of a specific group of non-heme iron enzymes by the synthesis of small synthetic analogues is the topic of this thesis. The group of non-heme iron enzymes with the 2-His-1-carboxylate facial triad has recently been established as a common platform for the

  9. Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

    Park, Kiyoung; Li, Ning; Kwak, Yeonju; Srnec, Martin

    2017-01-01

    Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2 . Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reaction shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.

  10. A functional mimic of natural peroxidases : synthesis and catalytic activity of a non-heme iron peptide hydroperoxide complex

    Choma, CT; Schudde, EP; Kellogg, RM; Robillard, GT; Feringa, BL

    1998-01-01

    Site-selective attachment of unprotected peptides to a non-heme iron complex is achieved by displacing two halides on the catalyst by peptide caesium thiolates, This coupling approach should be compatible with any peptide sequence provided there is only a single reduced cysteine. The oxidation

  11. Practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis

    Donker, A.E.; Raymakers, R.A.P.; Vlasveld, L.T.; Barneveld, T. van; Terink, R.; Dors, N.; Brons, P.P.T.; Knoers, N.V.A.M.; Swinkels, D.W.

    2014-01-01

    During recent years, our understanding of the pathogenesis of inherited microcytic anemias has gained from the identification of several genes and proteins involved in systemic and cellular iron metabolism and heme syntheses. Numerous case reports illustrate that the implementation of these novel

  12. Peroxo-Type Intermediates in Class I Ribonucleotide Reductase and Related Binuclear Non-Heme Iron Enzymes

    Kepp, Kasper Planeta; Bell, Caleb B.; Clay, MIchael D.

    2009-01-01

    We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O str...

  13. Using porphyrin-amino acid pairs to model the electrochemistry of heme proteins: experimental and theoretical investigations.

    Samajdar, Rudra N; Manogaran, Dhivya; Yashonath, S; Bhattacharyya, Aninda J

    2018-04-18

    Quasi reversibility in electrochemical cycling between different oxidation states of iron is an often seen characteristic of iron containing heme proteins that bind dioxygen. Surprisingly, the system becomes fully reversible in the bare iron-porphyrin complex: hemin. This leads to the speculation that the polypeptide bulk (globin) around the iron-porphyrin active site in these heme proteins is probably responsible for the electrochemical quasi reversibility. To understand the effect of such polypeptide bulk on iron-porphyrin, we study the interaction of specific amino acids with the hemin center in solution. We choose three representative amino acids-histidine (a well-known iron coordinator in bio-inorganic systems), tryptophan (a well-known fluoroprobe for proteins), and cysteine (a redox-active organic molecule). The interactions of these amino acids with hemin are studied using electrochemistry, spectroscopy, and density functional theory. The results indicate that among these three, the interaction of histidine with the iron center is strongest. Further, histidine maintains the electrochemical reversibility of iron. On the other hand, tryptophan and cysteine interact weakly with the iron center but disturb the electrochemical reversibility by contributing their own redox active processes to the system. Put together, this study attempts to understand the molecular interactions that can control electrochemical reversibility in heme proteins. The results obtained here from the three representative amino acids can be scaled up to build a heme-amino acid interaction database that may predict the electrochemical properties of any protein with a defined polypeptide sequence.

  14. Dinitrosyl non-heme iron complexes at the gamma radiation treatment of animals

    Aliev, D.I.; Alieva, I.N.; Abilov, Z.G.; Gurbanov, I.S.

    2003-01-01

    Full text: At the present time there are a great number investigations dedicated to revealing of mechanism formation of 2,03 complexes at the some pathologies in an organism. These complexes are represented weakly bounded form of non-heme iron, including into beside iron two nitrogen oxide molecules (NO) and two paired RS- groups of proteins or low-molecular compounds. 2,03 complexes are characterized by an axial symmetrical tensory of the g-factor with g=2,037, g=2,012 and g=2,03. In this study the data testifying 2,03 complexes formation into liver of animal treated by the fatal dose of gamma-radiation are reported. The changing of the ESR signal form was observed. It was shown that the form and intensity of the 2,03 signal in healthy and irradiated animals are differ from each other. The analysis of the 2,03 signal parameters is confirm this fact, too. The conclusion was made that 2,03 complexes ESR signal may be considered as an indicator of integrity of intracellular membranes of the gamma-irradiated animals

  15. Mini Heme-Proteins: Designability of Structure and Diversity of Functions.

    Rai, Jagdish

    2017-08-30

    Natural heme proteins may have heme bound to poly-peptide chain as a cofactor via noncovalent forces or heme as a prosthetic group may be covalently bound to the proteins. Nature has used porphyrins in diverse functions like electron transfer, oxidation, reduction, ligand binding, photosynthesis, signaling, etc. by modulating its properties through diverse protein matrices. Synthetic chemists have tried to utilize these molecules in equally diverse industrial and medical applications due to their versatile electro-chemical and optical properties. The heme iron has catalytic activity which can be modulated and enhanced for specific applications by protein matrix around it. Heme proteins can be designed into novel enzymes for sterio specific catalysis ranging from oxidation to reduction. These designed heme-proteins can have applications in industrial catalysis and biosensing. A peptide folds around heme easily due to hydrophobic effect of the large aromatic ring of heme. The directional property of co-ordinate bonding between peptide and metal ion in heme further specifies the structure. Therefore heme proteins can be easily designed for targeted structure and catalytic activity. The central aromatic chemical entity in heme viz. porphyrin is a very ancient molecule. Its presence in the prebiotic soup and in all forms of life suggests that it has played a vital role in the origin and progressive evolution of living organisms. Porphyrin macrocycles are highly conjugated systems composed of four modified pyrrole subunits interconnected at their α -carbon atoms via methine (=CH-) bridges. Initial minimalist models of hemoproteins focused on effect of heme-ligand co-ordinate bonding on chemical reactivity, spectroscopy, electrochemistry and magnetic properties of heme. The great sensitivity of these spectroscopic features of heme to its surrounding makes them extremely useful in structural elucidation of designed heme-peptide complexes. Therefore heme proteins are

  16. Reversible formation of high-valent-iron-oxo-porphyrin intermediate in heme-based catalysis: revisiting the kinetic model for horseradish peroxidase.

    Haandel, van M.J.H.; Primus, J.L.; Teunis, C.; Boersma, M.G.; Osman, A.M.; Veeger, C.; Rietjens, I.M.C.M.

    1998-01-01

    Many heme-containing biocatalysts exert their catalytic action through the initial formation of so-called high-valent-iron-oxo porphyrin intermediates. For horseradish peroxidase the initial intermediate formed has been identified as a high-valent-iron-oxo porphyrin π-radical cation, called compound

  17. In vivo genotoxic effects of dietary heme iron on rat colon mucosa and ex vivo effects on colon cells monitored by an optimized alkaline comet assay.

    Océane, C Martin

    2015-04-01

    In conclusion, our results offer a suitable protocol to evaluate genotoxicity on in vivo cryopreserved colon mucosa and on in vitro murine colonic cells, with a middle throughput capacity. This protocol confirms the increase of genotoxicity in rat colon mucosa after an heme-iron diet. Moreover, this protocol enables the demonstration that aldehydes from heme-induced lipoperoxidation are responsible for this increase of genotoxicity.

  18. Are meat and heme iron intake associated with pancreatic cancer? Results from the NIH-AARP Diet and Health Cohort

    Taunk, Pulkit; Hecht, Eric; Stolzenberg-Solomon, Rachael

    2015-01-01

    Several studies on pancreatic cancer have reported significant positive associations for intake of red meat but null associations for heme iron. We assessed total, red, white, and processed meat intake, meat cooking methods and doneness, and heme iron and mutagen intake in relation to pancreatic cancer in the NIH-AARP Diet and Health Study cohort. 322,846 participants (187,265 men; 135,581 women) successfully completed and returned the food frequency questionnaire between 1995–1996. After a mean follow-up of 9.2 years (up to 10.17 years), 1,417 individuals (895 men, 522 women) developed exocrine pancreatic cancer. Cox proportional hazard models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI), and trends were calculated using the median value of each quantile. Models incorporated age as the time metric and were adjusted for smoking history, BMI, self-reported diabetes, and energy-adjusted saturated fat. Pancreatic cancer risk significantly increased with intake of total meat (Q5 vs. Q1 HR=1.20, 95% CI 1.02–1.42, p-trend=0.03), red meat (HR=1.22, 95% CI 1.01–1.48, p-trend=0.02), high-temperature cooked meat (HR=1.21, 95% CI 1.00–1.45, p-trend=0.02), grilled/barbequed meat (HR=1.24, 95% CI 1.03–1.50, p-trend=0.007), well/very well done meat (HR=1.32, 95% CI 1.10–1.58, p-trend = 0.005), and heme iron from red meat (Q4 vs. Q1 HR=1.21, 95% CI 1.01–1.45, p-trend=0.04). When stratified by sex, these associations remained significant in men but not women except for white meat intake in women (HR = 1.33, 95% CI 1.02–1.74, p-trend = 0.04). Additional studies should confirm our findings that consuming heme iron from red meat increases pancreatic cancer risk. PMID:26666579

  19. Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

    Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

    2009-01-01

    Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

  20. Convergence of hepcidin deficiency, systemic iron overloading, heme accumulation, and REV-ERBα/β activation in aryl hydrocarbon receptor-elicited hepatotoxicity

    Fader, Kelly A.; Nault, Rance [Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Kirby, Mathew P.; Markous, Gena [Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Matthews, Jason [Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo 0316 (Norway); Zacharewski, Timothy R., E-mail: tzachare@msu.edu [Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States)

    2017-04-15

    Persistent aryl hydrocarbon receptor (AhR) agonists elicit dose-dependent hepatic lipid accumulation, oxidative stress, inflammation, and fibrosis in mice. Iron (Fe) promotes AhR-mediated oxidative stress by catalyzing reactive oxygen species (ROS) production. To further characterize the role of Fe in AhR-mediated hepatotoxicity, male C57BL/6 mice were orally gavaged with sesame oil vehicle or 0.01–30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days. Duodenal epithelial and hepatic RNA-Seq data were integrated with hepatic AhR ChIP-Seq, capillary electrophoresis protein measurements, and clinical chemistry analyses. TCDD dose-dependently repressed hepatic expression of hepcidin (Hamp and Hamp2), the master regulator of systemic Fe homeostasis, resulting in a 2.6-fold increase in serum Fe with accumulating Fe spilling into urine. Total hepatic Fe levels were negligibly increased while transferrin saturation remained unchanged. Furthermore, TCDD elicited dose-dependent gene expression changes in heme biosynthesis including the induction of aminolevulinic acid synthase 1 (Alas1) and repression of uroporphyrinogen decarboxylase (Urod), leading to a 50% increase in hepatic hemin and a 13.2-fold increase in total urinary porphyrins. Consistent with this heme accumulation, differential gene expression suggests that heme activated BACH1 and REV-ERBα/β, causing induction of heme oxygenase 1 (Hmox1) and repression of fatty acid biosynthesis, respectively. Collectively, these results suggest that Hamp repression, Fe accumulation, and increased heme levels converge to promote oxidative stress and the progression of TCDD-elicited hepatotoxicity. - Highlights: • TCDD represses hepatic hepcidin expression, leading to systemic iron overloading. • Dysregulation of heme biosynthesis is consistent with heme and porphyrin accumulation. • Heme-activated REV-ERBα/β repress circadian-regulated hepatic lipid metabolism. • Disruption of iron

  1. A non-heme iron-mediated chemical demethylation in DNA and RNA.

    Yi, Chengqi; Yang, Cai-Guang; He, Chuan

    2009-04-21

    DNA methylation is arguably one of the most important chemical signals in biology. However, aberrant DNA methylation can lead to cytotoxic or mutagenic consequences. A DNA repair protein in Escherichia coli, AlkB, corrects some of the unwanted methylations of DNA bases by a unique oxidative demethylation in which the methyl carbon is liberated as formaldehyde. The enzyme also repairs exocyclic DNA lesions--that is, derivatives in which the base is augmented with an additional heterocyclic subunit--by a similar mechanism. Two proteins in humans that are homologous to AlkB, ABH2 and ABH3, repair the same spectrum of lesions; another human homologue of AlkB, FTO, is linked to obesity. In this Account, we describe our studies of AlkB, ABH2, and ABH3, including our development of a general strategy to trap homogeneous protein-DNA complexes through active-site disulfide cross-linking. AlkB uses a non-heme mononuclear iron(II) and the cofactors 2-ketoglutarate (2KG) and dioxygen to effect oxidative demethylation of the DNA base lesions 1-methyladenine (1-meA), 3-methylcytosine (3-meC), 1-methylguanine (1-meG), and 3-methylthymine (3-meT). ABH3, like AlkB, works better on single-stranded DNA (ssDNA) and is capable of repairing damaged bases in RNA. Conversely, ABH2 primarily repairs lesions in double-stranded DNA (dsDNA); it is the main housekeeping enzyme that protects the mammalian genome from 1-meA base damage. The AlkB-family proteins have moderate affinities for their substrates and bind DNA in a non-sequence-specific manner. Knowing that these proteins flip the damaged base out from the duplex DNA and insert it into the active site for further processing, we first engineered a disulfide cross-link in the active site to stabilize the Michaelis complex. Based on the detailed structural information afforded by the active-site cross-linked structures, we can readily install a cross-link away from the active site to obtain the native-like structures of these complexes

  2. Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent

    Christian A. Schaer

    2013-01-01

    Full Text Available The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb, which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM. We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1 response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

  3. Practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis.

    Donker, Albertine E; Raymakers, Reinier A P; Vlasveld, L Thom; van Barneveld, Teus; Terink, Rieneke; Dors, Natasja; Brons, Paul P T; Knoers, Nine V A M; Swinkels, Dorine W

    2014-06-19

    During recent years, our understanding of the pathogenesis of inherited microcytic anemias has gained from the identification of several genes and proteins involved in systemic and cellular iron metabolism and heme syntheses. Numerous case reports illustrate that the implementation of these novel molecular discoveries in clinical practice has increased our understanding of the presentation, diagnosis, and management of these diseases. Integration of these insights into daily clinical practice will reduce delays in establishing a proper diagnosis, invasive and/or costly diagnostic tests, and unnecessary or even detrimental treatments. To assist the clinician, we developed evidence-based multidisciplinary guidelines on the management of rare microcytic anemias due to genetic disorders of iron metabolism and heme synthesis. These genetic disorders may present at all ages, and therefore these guidelines are relevant for pediatricians as well as clinicians who treat adults. This article summarizes these clinical practice guidelines and includes background on pathogenesis, conclusions, and recommendations and a diagnostic flowchart to facilitate using these guidelines in the clinical setting. © 2014 by The American Society of Hematology.

  4. Heme Synthesis and Acquisition in Bacterial Pathogens

    Choby, Jacob E.; Skaar, Eric P.

    2016-01-01

    Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host source...

  5. A Non-Heme Iron Photocatalyst for Light-Driven Aerobic Oxidation of Methanol

    Chen, Juan; Stepanovic, Stepan; Draksharapu, Apparao; Gruden, Maja; Browne, Wesley R

    2018-01-01

    Non-heme (L)FeIIIand (L)FeIII-O-FeIII(L) complexes (L=1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)ethan-1-amine) underwent reduction under irradiation to the FeIIstate with concomitant oxidation of methanol to methanal, without the need for a secondary photosensitizer. Spectroscopic and DFT

  6. Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1

    Bolloskis, Michael P.; Carvalho, Fabiana P.; Loo, George

    2016-01-01

    Some of the health-promoting properties of cruciferous vegetables are thought to be partly attributed to isothiocyanates. These phytochemicals can upregulate the expression of certain cytoprotective stress genes, but it is unknown if a particular nutrient is involved. Herein, the objective was to ascertain if adequate iron is needed for enabling HCT116 cells to optimally express heme oxygenase-1 (HO-1) when induced by phenethyl isothiocyanate (PEITC). PEITC increased HO-1 expression and also nuclear translocation of Nrf2, which is a transcription factor known to activate the HO-1 gene. However, in HCT116 cells that were made iron-deficient by depleting intracellular iron with deferoxamine (DFO), PEITC was less able to increase HO-1 expression and nuclear translocation of Nrf2. These suppressive effects of DFO were overcome by replenishing the iron-deficient cells with the missing iron. To elucidate these findings, it was found that PEITC-induced HO-1 upregulation can be inhibited with thiol antioxidants (glutathione and N-acetylcysteine). Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin) and a superoxide scavenger (Tiron) each inhibited PEITC-induced HO-1 upregulation. In doing so, diphenyleneiodonium was the most potent and also inhibited nuclear translocation of redox-sensitive Nrf2. Collectively, the results imply that the HO-1 upregulation by PEITC involves an iron-dependent, oxidant signaling pathway. Therefore, it is concluded that ample iron is required to enable PEITC to fully upregulate HO-1 expression in HCT116 cells. As such, it is conceivable that iron-deficient individuals may not reap the full health benefits of eating PEITC-containing cruciferous vegetables that via HO-1 may help protect against multiple chronic diseases. - Highlights: • PEITC increased HO-1 expression in HCT116 cells. • PEITC-induced HO-1 upregulation was impaired in iron-depleted HCT116 cells. • Impairment of PEITC-induced HO-1 upregulation was

  7. X-ray absorption spectroscopy of soybean lipoxygenase-1 : Influence of lipid hydroperoxide activation and lyophilization on the structure of the non-heme iron active site

    Vliegenthart, J.F.G.; Heijdt, L.M. van der; Feiters, M.C.; Navaratnam, S.; Nolting, H.-F.; Hermes, C.; Veldink, G.A.

    1992-01-01

    X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 23 eV

  8. Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors.

    Hanna, David A; Martinez-Guzman, Osiris; Reddi, Amit R

    2017-04-04

    Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.

  9. 21 CFR 862.1410 - Iron (non-heme) test system.

    2010-04-01

    ... characterized by pigmentation of the skin), and chronic renal disease. (b) Classification. Class I. ... diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated...

  10. Mononuclear non-heme iron enzymes with the 2-His-1-carboxylate facial triad: recent developments in enzymology and modeling studies.

    Bruijnincx, Pieter C A; van Koten, Gerard; Klein Gebbink, Robertus J M

    2008-12-01

    Iron-containing enzymes are one of Nature's main means of effecting key biological transformations. The mononuclear non-heme iron oxygenases and oxidases have received the most attention recently, primarily because of the recent availability of crystal structures of many different enzymes and the stunningly diverse oxidative transformations that these enzymes catalyze. The wealth of available structural data has furthermore established the so-called 2-His-1-carboxylate facial triad as a new common structural motif for the activation of dioxygen. This superfamily of mononuclear iron(ii) enzymes catalyzes a wide range of oxidative transformations, ranging from the cis-dihydroxylation of arenes to the biosynthesis of antibiotics such as isopenicillin and fosfomycin. The remarkable scope of oxidative transformations seems to be even broader than that associated with oxidative heme enzymes. Not only are many of these oxidative transformations of key biological importance, many of these selective oxidations are also unprecedented in synthetic organic chemistry. In this critical review, we wish to provide a concise background on the chemistry of the mononuclear non-heme iron enzymes characterized by the 2-His-1-carboxylate facial triad and to discuss the many recent developments in the field. New examples of enzymes with unique reactivities belonging to the superfamily have been reported. Furthermore, key insights into the intricate mechanistic details and reactive intermediates have been obtained from both enzyme and modeling studies. Sections of this review are devoted to each of these subjects, i.e. the enzymes, biomimetic models, and reactive intermediates (225 references).

  11. A dual component heme biosensor that integrates heme transport and synthesis in bacteria.

    Nobles, Christopher L; Clark, Justin R; Green, Sabrina I; Maresso, Anthony W

    2015-11-01

    Bacterial pathogens acquire host iron to power cellular processes and replication. Heme, an iron-containing cofactor bound to hemoglobin, is scavenged by bacterial proteins to attain iron. Methods to measure intracellular heme are laborious, involve complex chemistry, or require radioactivity. Such drawbacks limit the study of the mechanistic steps of heme transport and breakdown. Hypothesizing heme homeostasis could be measured with fluorescent methods, we coupled the conversion of heme to biliverdin IXα (a product of heme catabolism) by heme oxygenase 1 (HO1) with the production of near-infrared light upon binding this verdin by infrared fluorescent protein (IFP1.4). The resultant heme sensor, IFP-HO1, was fluorescent in pathogenic E. coli exposed to heme but not in the absence of the heme transporter ChuA and membrane coupling protein TonB, thereby validating their long-standing proposed role in heme uptake. Fluorescence was abolished in a strain lacking hemE, the central gene in the heme biosynthetic pathway, but stimulated by iron, signifying the sensor reports on intracellular heme production. Finally, an invasive strain of E. coli harboring the sensor was fluorescent during an active infection. This work will allow researchers to expand the molecular toolbox used to study heme and iron acquisition in culture and during infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Photoenhanced Oxidative DNA Cleavage with Non-Heme Iron(II) Complexes

    Li, Qian; Browne, Wesley R.; Roelfes, Gerard

    2010-01-01

    The DNA cleavage activity of iron(II) complexes of a series of monotopic pentadentate N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py)-derived ligands (1-5) was investigated under laser irradiation at 473, 400.8, and 355 nm in the absence of a reducing agent and compared to that under

  13. The detection of iron protoporphyrin (heme b) in phytoplankton and marine particulate material by electrospray ionisation mass spectrometry – comparison with diode array detection

    Gledhill, Martha, E-mail: m.gledhill@geomar.de

    2014-09-02

    Highlights: • Mass spectrometry was applied to the analysis of heme b in biological material. • Optimal conditions involved selective reactant monitoring of the heme b product ion. • The isotopic signature for this iron tetrapyrrole further improved selectivity. • Mass spectrometry and spectrophotometry were compared for heme b analysis. • Combining techniques made a powerful tool for analysis of heme in marine microbes. - Abstract: A mass spectrometric (MS) method for the identification of iron protoporphyrin (IX) (FePTP, heme b) in marine particulate material and phytoplankton is described. Electrospray ionisation of FePTP produced the molecular Fe(III)PTP{sup +} ion (m/z = 616) or the pseudomolecular [Fe(II)PTP + H]{sup +} ion (m/z = 617), depending on the oxidation state of the central iron ion. Collision induced dissociation (CID) in the ion trap mass spectrometer resulted in a single detected product ion (m/z = 557) indicative of loss of ethanoic acid from a carboxylic acid side chain. Widening the isolation width to 616 ± 3 resulted in production of a mass spectrum demonstrating the distinctive isotopic ratio of the iron containing fragment, further increasing the specificity of the analysis. Selective reactant monitoring (SRM) of the fragment ion (m/z = 557) was applied to the detection of FePTP after chromatography of ammoniacal OGP extracts of marine samples. The detection limit for FePTP analysed by SRM after chromatography was 1.2 ± 0.5 fmol. For phytoplankton samples, reasonably good agreement was achieved between results obtained with SRM and those obtained by monitoring absorbance at λ = 400 nm using a diode array detector (DAD). Use of SRM for analysis of particulate material obtained from the high latitude North Atlantic allowed for the analysis of FePTP in the presence of a co-eluting compound that interfered with detection by DAD. Simultaneous collection of mass spectra from m/z = 300 to 1500 resulted in identification of the

  14. Design and synthesis of a tetradentate '3-amine-1-carboxylate' ligand to mimic the metal binding environment at the non-heme iron(II) oxidase active site.

    Dungan, Victoria J; Ortin, Yannick; Mueller-Bunz, Helge; Rutledge, Peter J

    2010-04-07

    Non-heme iron(II) oxidases (NHIOs) catalyse a diverse array of oxidative chemistry in Nature. As part of ongoing efforts to realize biomimetic, iron-mediated C-H activation, we report the synthesis of a new 'three-amine-one-carboxylate' ligand designed to complex with iron(II) and mimic the NHIO active site. The tetradentate ligand has been prepared as a single enantiomer in nine synthetic steps from N-Cbz-L-alanine, pyridine-2,6-dimethanol and diphenylamine, using Seebach oxazolidinone chemistry to control the stereochemistry. X-Ray crystal structures are reported for two important intermediates, along with variable temperature NMR experiments to probe the hindered interconversion of conformational isomers of several key intermediates, 2,6-disubstituted pyridine derivatives. The target ligand and an N-Cbz-protected precursor were each then complexed with iron(II) and tested for their ability to promote alkene dihydroxylation, using hydrogen peroxide as the oxidant.

  15. Heme Synthesis and Acquisition in Bacterial Pathogens.

    Choby, Jacob E; Skaar, Eric P

    2016-08-28

    Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host sources, particularly hemoglobin, and both heme acquisition and synthesis are important for pathogenesis. Paradoxically, excess heme is toxic to bacteria and pathogens must rely on heme detoxification strategies. Heme is a key nutrient in the struggle for survival between host and pathogen, and its study has offered significant insight into the molecular mechanisms of bacterial pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Red and processed meat, nitrite, and heme iron intakes and postmenopausal breast cancer risk in the NIH-AARP Diet and Health Study

    Inoue-Choi, Maki; Sinha, Rashmi; Gierach, Gretchen L.; Ward, Mary H.

    2015-01-01

    Previous studies have shown inconsistent associations between red and processed meat intake and breast cancer risk. N-nitroso compounds and heme iron have been hypothesized as contributing factors. We followed 193,742 postmenopausal women in the NIH-AARP Diet and Health Study and identified 9,305 incident breast cancers (1995–2006). Dietary intake was assessed using a food frequency questionnaire at baseline. We adjusted daily intakes of meat, nitrite, and heme iron for energy intake using the nutrient density method. We estimated multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) by quintiles of dietary exposures for all breast cancer, by stage (in-situ, localized, regional/distant), and by estrogen/progesterone receptor (ER/PR) status using Cox proportional hazards regression. Total red meat intake was positively associated with risk of regional/distant cancer (p-trend=0.02). The risk was 25% higher in the highest vs. lowest intake quintile (95%CI=1.03–1.52). Higher processed red meat intake (Q5 vs. Q1) was associated with 27% higher risk of localized breast cancer (95%CI=1.01–1.27, p-trend=0.03) and a 19% higher risk of regional/distant cancer (95%CI=0.98–1.44, p-trend=0.10). In addition, higher nitrite intake from processed red meat was positively associated with localized cancer (HR for Q5 vs. Q1=1.23, 95%CI=1.09–1.39, p-trendmeat and processed meat may increase risk of postmenopausal breast cancer. Added nitrite and heme iron may partly contribute to these observed associations. PMID:26505173

  17. The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS.

    Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina; Hunter, Gregory A; Gakh, Oleksandr; Isaya, Grazia; Ferreira, Gloria C; Al-Karadaghi, Salam

    2016-05-27

    Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Natural selection on HFE in Asian populations contributes to enhanced non-heme iron absorption.

    Ye, Kaixiong; Cao, Chang; Lin, Xu; O'Brien, Kimberly O; Gu, Zhenglong

    2015-06-10

    HFE, a major regulator of iron (Fe) homeostasis, has been suggested to be under positive selection in both European and Asian populations. While the genetic variant under selection in Europeans (a non-synonymous mutation, C282Y) has been relatively well-studied, the adaptive variant in Asians and its functional consequences are still unknown. Identifying the adaptive HFE variants in Asians will not only elucidate the evolutionary history and the genetic basis of population difference in Fe status, but also assist the future practice of genome-informed dietary recommendation. Using data from the International HapMap Project, we confirmed the signatures of positive selection on HFE in Asian populations and identified a candidate adaptive haplotype that is common in Asians (52.35-54.71%) but rare in Europeans (5.98%) and Africans (4.35%). The T allele at tag SNP rs9366637 (C/T) captured 95.8% of this Asian-common haplotype. A significantly reduced HFE expression was observed in individuals carrying T/T at rs9366637 compared to C/C and C/T, indicating a possible role of gene regulation in adaptation. We recruited 57 women of Asian descent and measured Fe absorption using stable isotopes in those homozygous at rs9366637. We observed a 22% higher absorption in women homozygous for the Asian-common haplotype (T/T) compared to the control genotype (C/C). Additionally, compared with a group of age-matched Caucasian women, Asian women exhibited significantly elevated Fe absorption. Our results indicate parallel adaptation of HFE gene in Europeans and Asians with different genetic variants. Moreover, natural selection on HFE may have contributed to elevated Fe absorption in Asians. This study regarding population differences in Fe homeostasis has significant medical impact as high Fe level has been linked to an increased disease risk of metabolic syndromes.

  19. Mortality from different causes associated with meat, heme iron, nitrates, and nitrites in the NIH-AARP Diet and Health Study: population based cohort study

    Sinha, Rashmi; Ward, Mary H; Graubard, Barry I; Inoue-Choi, Maki; Dawsey, Sanford M; Abnet, Christian C

    2017-01-01

    Objective To determine the association of different types of meat intake and meat associated compounds with overall and cause specific mortality. Design Population based cohort study. Setting Baseline dietary data of the NIH-AARP Diet and Health Study (prospective cohort of the general population from six states and two metropolitan areas in the US) and 16 year follow-up data until 31 December 2011. Participants 536 969 AARP members aged 50-71 at baseline. Exposures Intake of total meat, processed and unprocessed red meat (beef, lamb, and pork) and white meat (poultry and fish), heme iron, and nitrate/nitrite from processed meat based on dietary questionnaire. Adjusted Cox proportional hazards regression models were used with the lowest fifth of calorie adjusted intakes as reference categories. Main outcome measure Mortality from any cause during follow-up. Results An increased risk of all cause mortality (hazard ratio for highest versus lowest fifth 1.26, 95% confidence interval 1.23 to 1.29) and death due to nine different causes associated with red meat intake was observed. Both processed and unprocessed red meat intakes were associated with all cause and cause specific mortality. Heme iron and processed meat nitrate/nitrite were independently associated with increased risk of all cause and cause specific mortality. Mediation models estimated that the increased mortality associated with processed red meat was influenced by nitrate intake (37.0-72.0%) and to a lesser degree by heme iron (20.9-24.1%). When the total meat intake was constant, the highest fifth of white meat intake was associated with a 25% reduction in risk of all cause mortality compared with the lowest intake level. Almost all causes of death showed an inverse association with white meat intake. Conclusions The results show increased risks of all cause mortality and death due to nine different causes associated with both processed and unprocessed red meat, accounted for, in part, by

  20. Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons.

    Singh, Neha; Bhatla, Satish C

    2016-02-29

    Nitric oxide is a significant component of iron signaling in plants. Heme is one of the iron sensors in plants. Free heme is highly toxic and can cause cell damage as it catalyzes the formation of reactive oxygen species (ROS). Its catabolism is carried out by heme oxygenase (HOs; EC 1.14.99.3) which uses heme both as a prosthetic group and as a substrate. Two significant events, which accompany adaptation to salt stress in sunflower seedlings, are accumulation of ROS and enhanced production of nitric oxide (NO) in roots and cotyledons. Present investigations on the immunolocalization of heme oxygenase distribution in sunflower seedling cotyledons by confocal laser scanning microscopic (CLSM) imaging provide new information on the differential spatial distribution of the inducible form of HO (HO-1) as a long distance in response to NaCl stress. The enzyme is abundantly distributed in the specialized cells around the secretory canals (SCs) in seedling cotyledons. Abundance of tyrosine nitrated proteins has also been observed in the specialized cells around the secretory canals in cotyledons derived from salt stressed seedlings. The spatial distribution of tyrosine nitrated proteins and HO-1 expression further correlates with the abundance of mitochondria in these cells. Present findings, thus, highlight a link among distribution of HO-1 expression, abundance of tyrosine nitrated proteins and mitochondria in specialized cells around the secretory canal as a long distance mechanism of salt stress tolerance in sunflower seedlings. Enhanced spatial distribution of HO-1 in response to NaCl stress in seedling cotyledons is in congruence with the observed increase in specific activity of HO-1 in NaCl stressed conditions. The enzyme activity is further enhanced by hemin (HO-1 inducer) both in the absence or presence of NaCl stress and inhibited by zinc protoporphyrin. Western blot analysis of cotyledon homogenates using anti-HO-1 polyclonal antibody shows one major band (29

  1. Heme Mobilization in Animals: A Metallolipid's Journey.

    Reddi, Amit R; Hamza, Iqbal

    2016-06-21

    Heme is universally recognized as an essential and ubiquitous prosthetic group that enables proteins to carry out a diverse array of functions. All heme-dependent processes, from protein hemylation to heme signaling, require the dynamic and rapid mobilization of heme to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitates that heme mobilization is carefully controlled at the cellular and systemic level. However, the molecules and mechanisms that mediate heme homeostasis are poorly understood. In this Account, we provide a heuristic paradigm with which to conceptualize heme trafficking and highlight the most recent developments in the mechanisms underlying heme trafficking. As an iron-containing tetrapyrrole, heme exhibits properties of both transition metals and lipids. Accordingly, we propose its transport and trafficking will reflect principles gleaned from the trafficking of both metals and lipids. Using this conceptual framework, we follow the flow of heme from the final step of heme synthesis in the mitochondria to hemoproteins present in various subcellular organelles. Further, given that many cells and animals that cannot make heme can assimilate it intact from nutritional sources, we propose that intercellular heme trafficking pathways must exist. This necessitates that heme be able to be imported and exported from cells, escorted between cells and organs, and regulated at the organismal level via a coordinated systemic process. In this Account, we highlight recently discovered heme transport and trafficking factors and provide the biochemical foundation for the cell and systems biology of heme. Altogether, we seek to reconceptualize heme from an exchange inert cofactor buried in hemoprotein active sites to an exchange labile and mobile metallonutrient.

  2. Synthesis of a Non-Heme Template for Attaching Four Peptides : An Approach to Artificial Iron(II)-Containing Peroxidases

    Heuvel, Marco van den; Berg, Tieme A. van den; Kellogg, Richard M.; Choma, Christin T.; Feringa, Bernard

    2004-01-01

    We are developing all-synthetic model cofactor-protein complexes in order to define the parameters controlling non-natural cofactor activity. The long-term objective is to establish the theoretical and practical basis for designing novel enzymes. A non-heme pentadentate ligand (N4Py) is being

  3. Mechanisms of heme utilization by Francisella tularensis.

    Helena Lindgren

    Full Text Available Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.

  4. Determination of lanthanum and rare earth elements in bovine whole blood reference material by ICP-MS after coprecipitation preconcentration with heme-iron as coprecipitant

    Fujimori, Eiji; Hayashi, Tatsuya; Inagaki, Kazumi; Haraguchi, H.

    1999-01-01

    An analytical method for the determination of lanthanide elements in the bovine whole blood reference material (IAEA A-13) has been investigated by inductively coupled plasma mass spectrometry (ICP-MS). The bovine whole blood reference material was digested with HNO 3 and HClO 4 , and then the pH of the digested solution was adjusted to 12 with 3 M sodium hydroxide aqueous solution. In this experimental procedure, lanthanide elements in the blood sample were coprecipitated with iron mainly derived from heme-iron in blood itself. In order to minimize matrix effects due to iron, excess iron in the analysis solution was removed by solvent extraction using methyl isobutyl ketone (MIBK) prior to the determination of lanthanide elements by ICP-MS. The recoveries of all lanthanide elements were almost quantitative in the recovery test. In consequence, it has been found that all lanthanide elements in bovine whole blood reference material are at the wide concentration range of 0.90 pg/g for Tm ∝1880 pg/g for Ce. (orig.)

  5. The DFT-DVM theoretical study of the differences of quadrupole splitting and the iron electronic structure for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin

    Yuryeva, E. I. [Institute of Solid State Chemistry of the Ural Branch of the Russian Academy of Sciences (Russian Federation); Oshtrakh, M. I., E-mail: oshtrakh@mail.utnet.ru [Ural State Technical University-UPI, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation)

    2008-01-15

    Quantum chemical calculations of the iron electron structure and {sup 57}Fe quadrupole splitting were made by density functional theory and X{alpha} discrete variation method for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin accounting stereochemical differences of the active sites in native proteins. The calculations revealed differences of quadrupole splitting temperature dependences for three models indicating sensitivity of quadrupole splitting and Fe(II) electronic structure to small variations of iron stereochemistry.

  6. Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

    Joubert, Laetitia; Dagieu, Jean-Baptiste; Fernandez, Annabelle; Derré-Bobillot, Aurélie; Borezée-Durant, Elise; Fleurot, Isabelle; Gruss, Alexandra; Lechardeur, Delphine

    2017-01-16

    Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

  7. Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron(II and 2-oxoglutarate-dependent dioxygenase EctD.

    Klaus Reuter

    2010-05-01

    Full Text Available As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD is a member of the non-heme iron(II-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11. These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe(3+ at a resolution of 1.85 A. Like other non-heme iron(II and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family.

  8. Influence of food tannins on certain aspects of iron metabolism : Part 3 -- Heme synthesis and haematopoiesis in normal and anemic rats

    Roy, S N [Albert Einstein Coll. of Medicine, Bronx, NY (USA); Mukherjee, S [Calcutta Univ. (India). Dept. of Applied Chemistry

    1979-06-01

    Tannin from various fruits and vegetables at a dose level of 0.5 mg/kg wt/day helps approximately 65% recovery of the blood hemoglobin concentration in hemolytic anemic rats within 7 days resulting in normal levels of haematological parameters. While in vitro tannin at low doses (5-10 ..mu..g/mg protein) stimulates iron incorporation into protoporphyrin IX by rat liver subcellular fractions, at higher doses (15-40 ..mu..g/mg protein) it inhibits the heme synthesis in liver, the inhibition being complete at 40 ..mu..g/mg protein. In vivo studies indicate that the administration of tannin (0.5 mg/kg) exhibits significant increase in incorporation of label into hemin of anemic rats compared to that of anemic control and tannin-fed normal groups. In rats receiving supplements of tannin (0.5 mg/k.o.), incorporation of the label into hemin of anemic ones is comparatively greater when /sup 59/Fe is given by intravenous route instead of oral administration of radio-iron. The total labelling of /sup 59/Fe in red blood cells is significantly greater in tannin-fed anemic rats than anemic control. These results suggest that tannin (0.5 mg/kg) from fruits and vegetables may help iron utilization more effectively for greater haematopoiesis in hemolytic anemia.

  9. Influence of food tannins on certain aspects of iron metabolism : Part 3 -- Heme synthesis and haematopoiesis in normal and anemic rats

    Roy, S.N.; Mukherjee, S.

    1979-01-01

    Tannin from various fruits and vegetables at a dose level of 0.5 mg/kg wt/day helps approximately 65% recovery of the blood hemoglobin concentration in hemolytic anemic rats within 7 days resulting in normal levels of haematological parameters. While in vitro tannin at low doses (5-10 μg/mg protein) stimulates iron incorporation into protoporphyrin IX by rat liver subcellular fractions, at higher doses (15-40 μg/mg protein) it inhibits the heme synthesis in liver, the inhibition being complete at 40 μg/mg protein. In vivo studies indicate that the administration of tannin (0.5 mg/kg) exhibits significant increase in incorporation of label into hemin of anemic rats compared to that of anemic control and tannin-fed normal groups. In rats receiving supplements of tannin (0.5 mg/k.o.), incorporation of the label into hemin of anemic ones is comparatively greater when 59 Fe is given by intravenous route instead of oral administration of radio-iron. The total labelling of 59 Fe in red blood cells is significantly greater in tannin-fed anemic rats than anemic control. These results suggest that tannin (0.5 mg/kg) from fruits and vegetables may help iron utilization more effectively for greater haematopoiesis in hemolytic anemia. (auth.)

  10. A role for heme in Alzheimer's disease: Heme binds amyloid β and has altered metabolism

    Atamna, Hani; Frey, William H.

    2004-01-01

    Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-b (P < 0.01) and 26% less heme-a (P = 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in he...

  11. Experimental and Computational Evidence for the Mechanism of Intradiol Catechol Dioxygenation by Non- Heme Iron(III) Complexes

    Jastrzebski, Robin; Quesne, Matthew G.; Weckhuysen, Bert M.; de Visser, Sam P.; Bruijnincx, Pieter C. A.

    2014-01-01

    Catechol intradiol dioxygenation is a unique reaction catalyzed by iron-dependent enzymes and nonheme iron(III) complexes. The mechanism by which these systems activate dioxygen in this important metabolic process remains controversial. Using a combination of kinetic measurements and computational

  12. Estimation of perimortal percent carboxy-heme in nonstandard postmortem specimens using analysis of carbon monoxide by GC/MS and iron by flame atomic absorption spectrophotometry.

    Middleberg, R A; Easterling, D E; Zelonis, S F; Rieders, F; Rieders, M F

    1993-01-01

    In decomposed, formalin-fixed, embalmed, exhumed, and some fire-dried cases in which normal blood is unavailable, the usual methods for determination of carboxyhemoglobin saturation frequently fail. To address these specimens, a method utilizing both gas chromatography/mass spectrometric (GC/MS) determination of carbon monoxide (CO) and flame atomic absorption spectrophotometry (FAAS) determination of iron (Fe), in the same specimen, was developed. The method is reported here, along with its application to seven pertinent forsensic death investigations. The CO analytical methodology involves acid liberation of the gas from the specimen aliquot in a headspace vial. After heating and equilibrating, a sample of the headspace vapor is injected into the GC/MS system with a gastight syringe. Quantitation is achieved by standard addition comparison utilizing the ideal gas law equation. Iron is quantified by FAAS analysis of the same aliquot used for the CO determination, following nitric acid digestion. The concentration is determined by comparison to a standard curve. A formula for determining the minimum percent carboxy-heme saturation was derived by using the ratio of the amount of CO to the amount of Fe in the aliquot analyzed. Tissue types analyzed include spleen, liver, muscle, dried blood, and unspecified decomposed tissue.

  13. Effects of dietary heme iron and exercise training on abdominal fat accumulation and lipid metabolism in high-fat diet-fed mice.

    Katsumura, Masanori; Takagi, Shoko; Oya, Hana; Tamura, Shohei; Saneyasu, Takaoki; Honda, Kazuhisa; Kamisoyama, Hiroshi

    2017-08-01

    Animal by-products can be recycled and used as sources of essential nutrients. Water-soluble heme iron (WSHI), a functional food additive for supplementing iron, is produced by processing animal blood. In this study, we investigated the effects of dietary supplementation of 3% WSHI and exercise training for 4 weeks on the accumulation of abdominal fat and lipid metabolism in mice fed high-fat diet. Exercise-trained mice had significantly less perirenal adipose tissue, whereas WSHI-fed mice tended to have less epididymal adipose tissue. In addition, total weight of abdominal adipose tissues was significantly decreased in the Exercise + WSHI group. Dietary WSHI significantly increased the messenger RNA (mRNA) levels of lipoprotein lipase and hormone-sensitive lipase. WSHI-fed mice also tended to show increased mRNA levels of adipose triglyceride lipase in their epididymal adipose tissue. Dietary WSHI also significantly decreased the mRNA levels of fatty acid oxidation-related enzymes in the liver, but did not influence levels in the Gastrocnemius muscle. Exercise training did not influence the mRNA levels of lipid metabolism-related enzymes in the epididymal adipose tissue, liver or the Gastrocnemius muscle. These findings suggest that the accumulation of abdominal fat can be efficiently decreased by the combination of dietary WSHI and exercise training in mice fed high-fat diet. © 2016 Japanese Society of Animal Science.

  14. Heme oxygenase activity increases after exercise in healthy volunteers

    AbstractHeme oxygenase (HO) is an essential, rate-limiting protein which participates in the catabolism of heme to iron, carbon monoxide (CO), and biliverdin. The alpha methene bridge carbon of the heme is eliminated as CO which can be measured as blood carboxyhemoglobin (COHb)....

  15. 4-Hydroxy-2(E)-nonenal metabolism differs in Apc(+/+) cells and in Apc(Min/+) cells: it may explain colon cancer promotion by heme iron.

    Baradat, Maryse; Jouanin, Isabelle; Dalleau, Sabine; Taché, Sylviane; Gieules, Mathilde; Debrauwer, Laurent; Canlet, Cécile; Huc, Laurence; Dupuy, Jacques; Pierre, Fabrice H F; Guéraud, Françoise

    2011-11-21

    Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.

  16. Models for cytochrome P450 prosthetic heme alkylation. Reaction of diazoacetophenone with (tetraphenylporphyrinato)iron(II) chloride

    Komives, E.A.; Tew, D.; Olmstead, M.M.; Ortiz de Montellano, P.R.

    1988-01-01

    The reaction of diazoacetophenone with (tetraphenylporphyrinato)iron(II) yields [N-(2-phenyl-2-oxoethyl)tetraphenylporphyrinato]iron(II) chloride. The structure of this product has been established by spectroscopic methods and by x-ray crystallography. The crystal structure shows that the first carbon of the N-alkyl group is 2.94 angstrom from the iron atom and that the oxygen of the N-alkyl group points away from the iron. No evidence is seen for the Fe-C-N product expected from insertion of the diazo carbon into the metalloporphyrin iron-nitrogen bond or for intermediates in which the oxygen of the N-(2-phenyl-2-oxoethyl) group is coordinated to the iron. These results suggest it is unlikely that carbene-insertion or oxygen-coordinated intermediates will be detected during the N-alkylation of cytochrome P450 by diazo ketones. The results also rationalize the failure to detect iron-chelated enol species during N-alkylation of the prosthetic group of cytochrome P450 by catalytically activated phenylacetylene. 43 references, 5 figures, 4 tables

  17. Synthesis and characterization of an iron complex bearing a cyclic tetra-N-heterocyclic carbene ligand: An artifical heme analogue?

    Anneser, Markus R.

    2015-04-20

    An iron(II) complex with a cyclic tetradentate ligand containing four N-heterocyclic carbenes was synthesized and characterized by means of NMR and IR spectroscopies, as well as by single-crystal X-ray structure analysis. The iron center exhibits an octahedral coordination geometry with two acetonitrile ligands in axial positions, showing structural analogies with porphyrine-ligated iron complexes. The acetonitrile ligands can readily be substituted by other ligands, for instance, dimethyl sulfoxide, carbon monoxide, and nitric oxide. Cyclic voltammetry was used to examine the electronic properties of the synthesized compounds. © 2015 American Chemical Society.

  18. The mechanism of stereospecific C-H oxidation by Fe(Pytacn) complexes: bioinspired non-heme iron catalysts containing cis-labile exchangeable sites.

    Prat, Irene; Company, Anna; Postils, Verònica; Ribas, Xavi; Que, Lawrence; Luis, Josep M; Costas, Miquel

    2013-05-17

    A detailed mechanistic study of the hydroxylation of alkane C-H bonds using H2O2 by a family of mononuclear non heme iron catalysts with the formula [Fe(II)(CF3SO3)2(L)] is described, in which L is a tetradentate ligand containing a triazacyclononane tripod and a pyridine ring bearing different substituents at the α and γ positions, which tune the electronic or steric properties of the corresponding iron complexes. Two inequivalent cis-labile exchangeable sites, occupied by triflate ions, complete the octahedral iron coordination sphere. The C-H hydroxylation mediated by this family of complexes takes place with retention of configuration. Oxygen atoms from water are incorporated into hydroxylated products and the extent of this incorporation depends in a systematic manner on the nature of the catalyst, and the substrate. Mechanistic probes and isotopic analyses, in combination with detailed density functional theory (DFT) calculations, provide strong evidence that C-H hydroxylation is performed by highly electrophilic [Fe(V)(O)(OH)L] species through a concerted asynchronous mechanism, involving homolytic breakage of the C-H bond, followed by rebound of the hydroxyl ligand. The [Fe(V)(O)(OH)L] species can exist in two tautomeric forms, differing in the position of oxo and hydroxide ligands. Isotopic-labeling analysis shows that the relative reactivities of the two tautomeric forms are sensitively affected by the α substituent of the pyridine, and this reactivity behavior is rationalized by computational methods. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Heme and erythropoieis: more than a structural role

    Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own ...

  20. Influence of Ligand Architecture in Tuning Reaction Bifurcation Pathways for Chlorite Oxidation by Non-Heme Iron Complexes

    Barman, Prasenjit; Faponle, Abayomi S; Vardhaman, Anil Kumar; Angelone, Davide; Löhr, Anna-Maria; Browne, Wesley R; Comba, Peter; Sastri, Chivukula V; de Visser, Sam P

    2016-01-01

    Reaction bifurcation processes are often encountered in the oxidation of substrates by enzymes and generally lead to a mixture of products. One particular bifurcation process that is common in biology relates to electron transfer versus oxygen atom transfer by high-valent iron(IV)-oxo complexes,

  1. Heme transport and erythropoiesis

    Yuan, Xiaojing; Fleming, Mark D.; Hamza, Iqbal

    2013-01-01

    In humans, systemic heme homeostasis is achieved via coordinated regulation of heme synthesis, transport and degradation. Although the heme biosynthesis and degradation pathways have been well characterized, the pathways for heme trafficking and incorporation into hemoproteins remains poorly understood. In the past few years, researchers have exploited genetic, cellular and biochemical tools, to identify heme transporters and, in the process, reveal unexpected functions for this elusive group of proteins. However, given the complexity of heme trafficking pathways, current knowledge of heme transporters is fragmented and sometimes contradictory. This review seeks to focus on recent studies on heme transporters with specific emphasis on their functions during erythropoiesis. PMID:23415705

  2. Single or functionalized fullerenes interacting with heme group

    Costa, Wallison Chaves; Diniz, Eduardo Moraes, E-mail: eduardo.diniz@ufma.br [Departamento de Física, Universidade Federal do Maranhão, Avenida dos Portugueses, 1966, CEP 65080-805, São Luís - MA (Brazil)

    2014-09-15

    The heme group is responsible for iron transportation through the bloodstream, where iron participates in redox reactions, electron transfer, gases detection etc. The efficiency of such processes can be reduced if the whole heme molecule or even the iron is somehow altered from its original oxidation state, which can be caused by interactions with nanoparticles as fullerenes. To verify how such particles alter the geometry and electronic structure of heme molecule, here we report first principles calculations based on density functional theory of heme group interacting with single C{sub 60} fullerene or with C{sub 60} functionalized with small functional groups (−CH{sub 3}, −COOH, −NH{sub 2}, −OH). The calculations shown that the system heme + nanoparticle has a different spin state in comparison with heme group if the fullerene is functionalized. Also a functional group can provide a stronger binding between nanoparticle and heme molecule or inhibit the chemical bonding in comparison with single fullerene results. In addition heme molecule loses electrons to the nanoparticles and some systems exhibited a geometry distortion in heme group, depending on the binding energy. Furthermore, one find that such nanoparticles induce a formation of spin up states in heme group. Moreover, there exist modifications in density of states near the Fermi energy. Although of such changes in heme electronic structure and geometry, the iron atom remains in the heme group with the same oxidation state, so that processes that involve the iron might not be affected, only those that depend on the whole heme molecule.

  3. Heme Recognition By a Staphylococcus Aureus IsdE

    Grigg, J.C.; Vermeiren, C.L.; Heinrichs, D.E.; Murphy, M.E.P.

    2009-06-03

    Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single {alpha}-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met{sup 78} and His{sup 229}. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His{sup 299} is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

  4. The effect of soy products in the diet on retention of non-heme iron from radiolabeled test meals fed to marginally iron-deficient young rats

    Thompson, D.B.

    1984-01-01

    Diets based either on casein or soy products and containing about 25 ppm iron were fed to weanling rats for 13 days. Rats were fasted overnight and fed a 59 Fe-radiolabeled casein test meal the morning of day 14. On day 21 less 59 Fe was retained by rats fed various diets based on selected soy products than by rats fed the casein-based diet. A similar adverse effect of diet components on 59 Fe retention from a casein test meal was observed for lactalbumin and for psyllium husk. No adverse effect of diet on 59 Fe retention was observed for the fiber of soy cotyledons or for rapeseed protein concentrate. For a commercial soy protein isolated (SPI) fed throughout the 21-day experiment, the adverse effect of diet on 59 Fe retention was observed to the sum of the effect of dietary SPI previous to the 59 Fe-radiolabeled casein test meal fed on day 14 and the effect of dietary SPI subsequent to the casein test meal. An effect of dietary soy products on 59 Fe retention from a casein test meal was not observed with diets containing higher iron levels (83 ppm) or when diets were fed for a longer period prior to the test meal (56 days). The present work shows that in some circumstances the concept of iron bioavailability must be expanded to include not only the influence of meal composition, but also the influence of diet previous to and subsequent to a meal

  5. Heme synthesis in normal mouse liver and mouse liver tumors

    Stout, D.L.; Becker, F.F.

    1990-01-01

    Hepatic cancers from mice and rats demonstrate decreased levels of delta-aminolevulinic acid synthase, the rate-limiting enzyme in the heme synthetic pathway, and increased heme oxygenase, the heme-catabolizing enzyme. These findings suggest that diminution of P-450, b5, and catalase in these lesions may result from a heme supply that is limited by decreased heme synthesis and increased heme catabolism. Heme synthesis was measured in mouse liver tumors (MLT) and adjacent tumor-free lobes (BKG) by administering the radiolabeled heme precursors 55 FeCl3 and [2- 14 C]glycine and subsequently extracting the heme for determination of specific activity. Despite reduced delta-aminolevulinic acid synthase activity in MLT, both tissues incorporated [2-14C]glycine into heme at similar rates. At early time points, heme extracted from MLT contained less 55Fe than that from BKG. This was attributed to the findings that MLT took up 55Fe at a slower rate than BKG and had larger iron stores than BKG. The amount of heme per milligram of protein was also similar in both tissues. These findings militate against the hypothesis that diminished hemoprotein levels in MLT result from limited availability of heme. It is probable, therefore, that decreased hemoprotein levels in hepatic tumors are linked to a general program of dedifferentiation associated with the cancer phenotype. Diminution of hemoprotein in MLT may result in a relatively increased intracellular heme pool. delta-Aminolevulinic acid synthase and heme oxygenase are, respectively, negatively and positively regulated by heme. Thus, their alteration in MLT may be due to the regulatory influences of the heme pool

  6. Heme and erythropoieis: more than a structural role.

    Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

    2014-06-01

    Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own synthesis and regulates the expression of several erythroid-specific genes. Heme is synthesized in developing erythroid progenitors by the stage of proerythroblast, through a series of eight enzymatic reactions divided between mitochondria and cytosol. Defects of heme synthesis in the erythroid lineage result in sideroblastic anemias, characterized by microcytic anemia associated to mitochondrial iron overload, or in erythropoietic porphyrias, characterized by porphyrin deposition in erythroid cells. Here, we focus on the heme biosynthetic pathway and on human erythroid disorders due to defective heme synthesis. The regulatory role of heme during erythroid differentiation is discussed as well as the heme-mediated regulatory mechanisms that allow the orchestration of the adaptive cell response to heme deficiency. Copyright© Ferrata Storti Foundation.

  7. Heme oxygenase-1: a metabolic nike.

    Wegiel, Barbara; Nemeth, Zsuzsanna; Correa-Costa, Matheus; Bulmer, Andrew C; Otterbein, Leo E

    2014-04-10

    Heme degradation, which was described more than 30 years ago, is still very actively explored with many novel discoveries on its role in various disease models every year. The heme oxygenases (HO) are metabolic enzymes that utilize NADPH and oxygen to break apart the heme moiety liberating biliverdin (BV), carbon monoxide (CO), and iron. Heme that is derived from hemoproteins can be toxic to the cells and if not removed immediately, it causes cell apoptosis and local inflammation. Elimination of heme from the milieu enables generation of three products that influences numerous metabolic changes in the cell. CO has profound effects on mitochondria and cellular respiration and other hemoproteins to which it can bind and affect their function, while BV and bilirubin (BR), the substrate and product of BV, reductase, respectively, are potent antioxidants. Sequestration of iron into ferritin and its recycling in the tissues is a part of the homeodynamic processes that control oxidation-reduction in cellular metabolism. Further, heme is an important component of a number of metabolic enzymes, and, therefore, HO-1 plays an important role in the modulation of cellular bioenergetics. In this review, we describe the cross-talk between heme oxygenase-1 (HO-1) and its products with other metabolic pathways. HO-1, which we have labeled Nike, the goddess who personified victory, dictates triumph over pathophysiologic conditions, including diabetes, ischemia, and cancer.

  8. Identification of the Mitochondrial Heme Metabolism Complex.

    Medlock, Amy E; Shiferaw, Mesafint T; Marcero, Jason R; Vashisht, Ajay A; Wohlschlegel, James A; Phillips, John D; Dailey, Harry A

    2015-01-01

    Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.

  9. Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

    Gardner, L.C.; Cox, T.M.

    1988-01-01

    Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular free heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation

  10. Mechanism governing heme synthesis reveals a GATA factor/heme circuit that controls differentiation.

    Tanimura, Nobuyuki; Miller, Eli; Igarashi, Kazuhiko; Yang, David; Burstyn, Judith N; Dewey, Colin N; Bresnick, Emery H

    2016-02-01

    Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governing heme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis elements strongly reduces GATA-1-induced Alas2 transcription, heme biosynthesis, and surprisingly, GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing ALAS-2 function in Alas2 cis element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescues heme biosynthesis and the GATA-1-dependent genetic network. Heme amplifies GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Through this dual mechanism, heme and a master regulator collaborate to orchestrate a cell type-specific transcriptional program that promotes cellular differentiation. © 2015 The Authors.

  11. Comparative analysis of the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a using Mössbauer spectroscopy with a high velocity resolution

    Alenkina, I. V.; Kumar, A.; Berkovsky, A. L.; Oshtrakh, M. I.

    2018-02-01

    A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using 57Fe Mössbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mössbauer hyperfine parameters. The Mössbauer spectra of tetrameric rabbit hemoglobin in both forms were fitted using two quadrupole doublets related to the 57Fe in ɑ- and β-subunits. In contrast, the Mössbauer spectra of monomeric soybean leghemoglobin a were fitted using: (i) two quadrupole doublets for the oxy-form related to two conformational states of the distal His E7 imidazole ring and different hydrogen bonding of oxygen molecule in the oxy-form and (ii) using three quadrupole doublets for deoxy-form related to three conformational states of the proximal His F8 imidazole ring. Small variations of Mössbauer hyperfine parameters related to small differences in the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a are discussed.

  12. Heme Sensor Proteins*

    Girvan, Hazel M.; Munro, Andrew W.

    2013-01-01

    Heme is a prosthetic group best known for roles in oxygen transport, oxidative catalysis, and respiratory electron transport. Recent years have seen the roles of heme extended to sensors of gases such as O2 and NO and cell redox state, and as mediators of cellular responses to changes in intracellular levels of these gases. The importance of heme is further evident from identification of proteins that bind heme reversibly, using it as a signal, e.g. to regulate gene expression in circadian rhythm pathways and control heme synthesis itself. In this minireview, we explore the current knowledge of the diverse roles of heme sensor proteins. PMID:23539616

  13. Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress

    Seguin, Alexandra; Bayot, Aurélien; Dancis, Andrew

    2009-01-01

    of 2muYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe-S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions......Friedreich's ataxia is generally associated with defects in [Fe-S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich's ataxia to study the physiological consequences of modulating...... for these independent roles are different....

  14. Moessbauer spectroscopic study of polymer-bound heme complexes

    Tsuchida, Eishun; Nishide, Hiroyuki; Yokoyama, Hiroyuki; Inoue, Hidenari; Shirai, Tsuneo.

    1984-01-01

    Moessbauer spectra were measured on the heme complexes of poly(1-vinyl- and 1-vinyl-2-methylimidazole)(PVI and PMI) and heme derivatives with covalently bound imidazoleligand (IH) and 2-methylimidazole-ligand (MIH) embedded in poly(1-vinyl-2-pyrrolidone) film. Quadrupole splitting (ΔE sub(Q)) for the carbon monoxide adduct of PMI-heme indicated large electronic field gradient at the iron nucleus, probably due to steric hindrance of the polymer chain, and this behavior agreed with its low affinity with carbon monoxide. PMI-heme formed an oxygen adduct and its isomer shift and ΔE sub(Q) values were obtained. (author)

  15. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

    Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multi...

  16. Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis

    Jacob E. Choby; Caroline M. Grunenwald; Arianna I. Celis; Svetlana Y. Gerdes; Jennifer L. DuBois; Eric P. Skaar; Kimberly A. Kline

    2018-01-01

    Staphylococcus aureus is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized de novo from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme hom...

  17. Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-05-01

    The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  18. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

  19. Hal Is a Bacillus anthracis Heme Acquisition Protein

    Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

    2012-01-01

    The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

  20. Kidney injury and heme oxygenase-1

    Hai-xing MAI

    2012-02-01

    Full Text Available     Heme oxygenase-1 (HO-1 is one of the main pathways to degrade heme in mammals, and the main degradation products are free iron (Fe2+, carbon monoxide (CO, and bilirubin. Heme plays an important role in promoting cell survival, circulation of intracellular substrates, and immune regulation. Previous studies suggest that HO-1 pathway is an important internal factor in determining the susceptibility and severity of acute kidney injury (AKI. The induction of HO-1 expression can attenuate the severity of renal ischemia-reperfusion injury (IRI, and the inhibition of HO-1 expression will aggravate IRI. The present article summarizes the latest advances in research abroad and at home on protective mechanism by which HO-1 prevents AKI to further deepen our understanding of the role of HO-1 in the treatment of AKI.   

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

    Strader, Michael Brad; Hicks, Wayne A; Kassa, Tigist; Singleton, Eileen; Soman, Jayashree; Olson, John S; Weiss, Mitchell J; Mollan, Todd L; Wilson, Michael T; Alayash, Abdu I

    2014-08-08

    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 β subunits of adult Hb (HbA) Bristol-Alesha (β67(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 β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Iron

    Iron is a mineral that our bodies need for many functions. For example, iron is part of hemoglobin, a protein which carries ... It helps our muscles store and use oxygen. Iron is also part of many other proteins and ...

  4. Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

    Majuri, R.

    1989-01-01

    Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of 35 S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with 35 S. The same two bands were observed if the cell surface proteins were labeled with 125 I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author)

  5. Heme acquisition mechanisms of Porphyromonas gingivalis - strategies used in a polymicrobial community in a heme-limited host environment.

    Smalley, J W; Olczak, T

    2017-02-01

    Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell-surface heme-containing pigment in the form of μ-oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB-dependent outer-membrane receptors (HmuR, HusB, IhtA), and hemophore-like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)-containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine- and lysine-specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Non-Heme Iron Catalysts with a Rigid Bis-Isoindoline Backbone and Their Use in Selective Aliphatic C−H Oxidation

    Chen, Jianming; Lutz, Martin; Milan, Michela; Costas, Miquel; Otte, Matthias; Klein Gebbink, Bert

    2017-01-01

    Iron complexes derived from a bis-isoindoline-bis-pyridine ligand platform based on the BPBP ligand (BPBP=N,N′-bis(2-picolyl)-2,2′-bis-pyrrolidine) have been synthesized and applied in selective aliphatic C−H oxidation with hydrogen peroxide under mild conditions. The introduction of benzene

  7. In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

    Mack, John; Vermeiren, Christie; Heinrichs, David E.; Stillman, Martin J.

    2004-01-01

    We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins

  8. Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems.

    Cavallaro, Gabriele; Decaria, Leonardo; Rosato, Antonio

    2008-11-01

    Heme is the prosthetic group of many proteins that carry out a variety of key biological functions. In addition, for many pathogenic organisms, heme (acquired from the host) may constitute a very important source of iron. Organisms can meet their heme demands by taking it up from external sources, by producing the cofactor through a dedicated biosynthetic pathway, or both. Here we analyzed the distribution of proteins specifically involved in the processes of heme biosynthesis and heme uptake in 474 prokaryotic organisms. These data allowed us to identify which organisms are capable of performing none, one, or both processes, based on the similarity to known systems. Some specific instances where one or more proteins along the pathways had unusual modifications were singled out. For two key protein domains involved in heme uptake, we could build a series of structural models, which suggested possible alternative modes of heme binding. Future directions for experimental work are given.

  9. Mimicking heme enzymes in the solid state: metal-organic materials with selectively encapsulated heme.

    Larsen, Randy W; Wojtas, Lukasz; Perman, Jason; Musselman, Ronald L; Zaworotko, Michael J; Vetromile, Carissa M

    2011-07-13

    To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal-organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a "ship-in-a-bottle" fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

  10. The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen

    Saederup, Kirstine Lindhardt; Stødkilde-Jørgensen, Kristian; Graversen, Jonas Heilskov

    2016-01-01

    Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd...

  11. Introduction of a covalent histidine-heme linkage in a hemoglobin: a promising tool for heme protein engineering.

    Rice, Selena L; Preimesberger, Matthew R; Johnson, Eric A; Lecomte, Juliette T J

    2014-12-01

    The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine-heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (>90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His-heme modification will be applicable to the design of proteins with a non-dissociable heme group. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.

    Hamza, Iqbal; Dailey, Harry A

    2012-09-01

    The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Increase on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro.

    Renata Stiebler

    Full Text Available BACKGROUND: Hemozoin (Hz is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH (the synthetic counterpart of Hz formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO and a series of polyethyleneglycols (PEGs. We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000 increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300 caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels.

  14. Electron Transport in a Dioxygenase-Ferredoxin Complex: Long Range Charge Coupling between the Rieske and Non-Heme Iron Center.

    Wayne K Dawson

    Full Text Available Dioxygenase (dOx utilizes stereospecific oxidation on aromatic molecules; consequently, dOx has potential applications in bioremediation and stereospecific oxidation synthesis. The reactive components of dOx comprise a Rieske structure Cys2[2Fe-2S]His2 and a non-heme reactive oxygen center (ROC. Between the Rieske structure and the ROC, a universally conserved Asp residue appears to bridge the two structures forming a Rieske-Asp-ROC triad, where the Asp is known to be essential for electron transfer processes. The Rieske and ROC share hydrogen bonds with Asp through their His ligands; suggesting an ideal network for electron transfer via the carboxyl side chain of Asp. Associated with the dOx is an itinerant charge carrying protein Ferredoxin (Fdx. Depending on the specific cognate, Fdx may also possess either the Rieske structure or a related structure known as 4-Cys-[2Fe-2S] (4-Cys. In this study, we extensively explore, at different levels of theory, the behavior of the individual components (Rieske and ROC and their interaction together via the Asp using a variety of density function methods, basis sets, and a method known as Generalized Ionic Fragment Approach (GIFA that permits setting up spin configurations manually. We also report results on the 4-Cys structure for comparison. The individual optimized structures are compared with observed spectroscopic data from the Rieske, 4-Cys and ROC structures (where information is available. The separate pieces are then combined together into a large Rieske-Asp-ROC (donor/bridge/acceptor complex to estimate the overall coupling between individual components, based on changes to the partial charges. The results suggest that the partial charges are significantly altered when Asp bridges the Rieske and the ROC; hence, long range coupling through hydrogen bonding effects via the intercalated Asp bridge can drastically affect the partial charge distributions compared to the individual isolated

  15. Coordinate expression of heme and globin is essential for effective erythropoiesis.

    Doty, Raymond T; Phelps, Susan R; Shadle, Christina; Sanchez-Bonilla, Marilyn; Keel, Siobán B; Abkowitz, Janis L

    2015-12-01

    Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.

  16. Isocyanides inhibit human heme oxygenases at the verdoheme stage.

    Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

    2009-09-22

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

  17. Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

    Evans, John P.; Kandel, Sylvie; Ortiz de Montellano, Paul R.

    2010-01-01

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides; isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides, and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 μM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

  18. Binding analysis of ferritin with heme using α-casein and biotinylated-hemin: detection of heme-binding capacity of Dpr derived from heme synthesis-deficient Streptococcus mutans.

    Mieno, Ayako; Yamamoto, Yuji; Yoshikawa, Yasunaga; Watanabe, Kiyotaka; Mukai, Takao; Orino, Koichi

    2013-01-01

    Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to an α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Escherichia coli, did not bind to an α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After preincubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin and that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.

  19. Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.

    Surdel, Matthew C; Dutter, Brendan F; Sulikowski, Gary A; Skaar, Eric P

    2016-08-12

    Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.

  20. Salivary proline-rich protein may reduce tannin-iron chelation: a systematic narrative review

    Delimont, Nicole M.; Rosenkranz, Sara K.; Haub, Mark D.; Lindshield, Brian L.

    2017-01-01

    Background Tannins are often cited for antinutritional effects, including chelation of non-heme iron. Despite this, studies exploring non-heme iron bioavailability inhibition with long-term consumption have reported mixed results. Salivary proline-rich proteins (PRPs) may mediate tannin-antinutritional effects on non-heme iron bioavailability. Aim To review evidence regarding biochemical binding mechanisms and affinity states between PRPs and tannins, as well as effects of PRPs on non-heme ir...

  1. DFT studies of the substituent effects of dimethylamino on non-heme active oxidizing species: iron(V)-oxo species or iron(IV)-oxo acetate aminopyridine cation radical species?

    Wang, Fang; Sun, Wei; Xia, Chungu; Wang, Yong

    2017-10-01

    Through the introduction of dimethylamino (Me 2 N) substituent at the pyridine ring of 2-((R)-2-[(R)-1-(pyridine-2-ylmethyl)pyrrolidin-2-yl]pyrrolidin-1-ylmethyl)pyridine (PDP) ligand, the non-heme Fe II ( Me2N PDP)/H 2 O 2 /AcOH catalyst system was found to exhibit significant higher catalytic activity and enantioselectivity than the non-substituent one in the asymmetric epoxidation experiments. The mechanistic origin of the remarkable substituent effects in these oxidation reactions has not been well established. To ascertain the potent oxidant and the related reaction mechanism, a detailed DFT calculation was performed. Interestingly, a novel Fe(IV)-oxo Me2N PDP cation radical species, [( Me2N PDP) + · Fe IV (O)(OAc)] 2+ ( Me2N 5), with about one spin spreading over the non-heme Me2N PDP ligand was formed via a carboxylic-acid-assisted O-O bond heterolysis, which is reminiscent of Compound I (an Fe(IV)(O)(porphyrin cation radical) species) in cytochrome P450 chemistry. Me2N 5 is energetically comparable with the cyclic ferric peracetate species Me2N 6, while in the pristine Fe(PDP) catalyst system, H 6 is more stable than H 5. Comparison of the activation energy for the ethylene epoxidation promoted by Me2N 5 and Me2N 6, Me2N 5 is supposed as the true oxidant triggering the epoxidation of olefins. In addition, a systematic research on the substituent effects varied from the electron-donating substituent (dMM, the substituents at sites 3, 4, and 5 of the pyridine ring: methyl, methoxyl, and methyl) to the electron-withdrawing one (CF 3 , 2,6-bis(trifluoromethyl)phenyl) on the electronic structure of the reaction intermediates has also been investigated. An alternative cyclic ferric peracetate complex is obtained, indicating that the substituents at the pyridine ring of PDP ligands have significant impacts on the electronic structure of the oxidants.

  2. Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

    Westberg, Johan A.; Jiang, Ji; Andersson, Leif C.

    2011-01-01

    Highlights: → Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. → Central iron atom of heme and cysteine-114 of STC1 are essential for binding. → STC1 binds Fe 2+ and Fe 3+ heme. → STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys 114 as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H 2 O 2 induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

  3. Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

    Westberg, Johan A., E-mail: johan.westberg@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Jiang, Ji, E-mail: ji.jiang@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Andersson, Leif C., E-mail: leif.andersson@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland)

    2011-06-03

    Highlights: {yields} Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. {yields} Central iron atom of heme and cysteine-114 of STC1 are essential for binding. {yields} STC1 binds Fe{sup 2+} and Fe{sup 3+} heme. {yields} STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys{sup 114} as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H{sub 2}O{sub 2} induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

  4. Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but not Disruption of the Distal Hydrogen Bonding Network†

    Wang, Jinling; Evans, John P.; Ogura, Hiroshi; La Mar, Gerd N.; Ortiz de Montellano, Paul R.

    2008-01-01

    Heme oxygenase regiospecifically oxidizes heme at the α-meso position to give biliverdin IXα, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry, but partially shifts the oxidation to the β/δ-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by ~90°, causes a slight loss of regiospecificity, but combined with the R183E and K18E mutations results primarily in β/δ-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network, impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane. PMID:16388581

  5. Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury.

    Sawicki, Konrad Teodor; Shang, Meng; Wu, Rongxue; Chang, Hsiang-Chun; Khechaduri, Arineh; Sato, Tatsuya; Kamide, Christine; Liu, Ting; Naga Prasad, Sathyamangla V; Ardehali, Hossein

    2015-07-31

    Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the

  6. Iron

    Hansen, Jakob Bondo; Moen, I W; Mandrup-Poulsen, T

    2014-01-01

    and discuss recent evidence, suggesting that iron is a key pathogenic factor in both type 1 and type 2 diabetes with a focus on inflammatory pathways. Pro-inflammatory cytokine-induced β-cell death is not fully understood, but may include iron-induced ROS formation resulting in dedifferentiation by activation...... of transcription factors, activation of the mitochondrial apoptotic machinery or of other cell death mechanisms. The pro-inflammatory cytokine IL-1β facilitates divalent metal transporter 1 (DMT1)-induced β-cell iron uptake and consequently ROS formation and apoptosis, and we propose that this mechanism provides...

  7. Iron

    ... Share: Search the ODS website Submit Search NIH Office of Dietary Supplements Consumer Datos en español Health ... eating a variety of foods, including the following: Lean meat, seafood, and poultry. Iron-fortified breakfast cereals ...

  8. Effect of lead on heme synthesis

    Neuberger, A.

    1975-01-01

    Recently, a fair amount of work has been done on the effect of lead on porphobilinogen dehydratase, which has been used as a sensitive indicator of lead poisoning. How far this is in itself harmful depends on the Michaelis constants of both the aminolaevulinic synthetase and of the dehydratase, and in addition on the relative activities of the two enzymes in a cell and also on the tissue concentration of glycine. Information on some of these points is still fragmentary, and a reliable judgement is at the present not very easy. Another step in the heme synthesis, which is sensitive to low concentrations of lead, is the incorporation of iron into protoporphyrin. Inhibition of this step may be important in accounting to a large extent for the anaemia found in individuals with lead poisoning. Reduction in the tissue concentration of heme or of heme-like compounds may also explain, through the mechanism of de-repression, the excretion of increased amounts of aminolaevulinic acid in the urine observed in cases of lead poisoning. A third step in heme synthesis, which might be sensitive to lead, is the oxidative decarboxylation of coproporphyrin to protoporphyrin, and this may explain why the former derivative is excreted in the urine. Recent work of the Harvard Medical School has indicated that greatly reduced levels of ALA dehydratase may be found in most cases of severe liver damage due to alcoholism. In most of these cases the level of lead in the blood is within normal limits, and there is no history of exposure to toxic amounts of lead. We therefore have to assume that a reduction in the blood level of this enzyme is not necessarily an indication of lead poisoning.

  9. Antibiotic suppression of intestinal microbiota reduces heme-induced lipoperoxidation associated with colon carcinogenesis in rats.

    Martin, O C B; Lin, C; Naud, N; Tache, S; Raymond-Letron, I; Corpet, D E; Pierre, F H

    2015-01-01

    Epidemiological studies show that heme iron from red meat is associated with increased colorectal cancer risk. In carcinogen-induced-rats, a heme iron-rich diet increases the number of precancerous lesions and raises associated fecal biomarkers. Heme-induced lipoperoxidation measured by fecal thiobarbituric acid reagents (TBARs) could explain the promotion of colon carcinogenesis by heme. Using a factorial design we studied if microbiota could be involved in heme-induced carcinogenesis, by modulating peroxidation. Rats treated or not with an antibiotic cocktail were given a control or a hemoglobin-diet. Fecal bacteria were counted on agar and TBARs concentration assayed in fecal water. The suppression of microbiota by antibiotics was associated with a reduction of crypt height and proliferation and with a cecum enlargement, which are characteristics of germ-free rats. Rats given hemoglobin diets had increased fecal TBARs, which were suppressed by the antibiotic treatment. A duplicate experiment in rats given dietary hemin yielded similar results. These data show that the intestinal microbiota is involved in enhancement of lipoperoxidation by heme iron. We thus suggest that microbiota could play a role in the heme-induced promotion of colorectal carcinogenesis.

  10. Polypeptide based hydrogels

    Hanay, Saltuk

    2018-01-01

    There is a need for biocompatible, biodegradable, 3-D printable and stable hydrogels especially in the areas of tissue engineering, drug delivery, bio-sensing technologies and antimicrobial coatings. The main aim of this Ph.D. work was to fabricate polypeptide based hydrogel which may find a potential application in those fields. Focusing on tyrosine or tryptophan-containing copolypeptides prepared by NCarboxyanhydride (NCA) polymerizations, three different crosslinking strategies have been t...

  11. Heme isomers substantially affect heme's electronic structure and function

    Kepp, Kasper Planeta

    2017-01-01

    Inspection of heme protein structures in the protein data bank reveals four isomers of heme characterized by different relative orientations of the vinyl side chains; remarkably, all these have been reported in multiple protein structures. Density functional theory computations explain this as du...

  12. Dietary Heme Induces Gut Dysbiosis, Aggravates Colitis, and Potentiates the Development of Adenomas in Mice

    Marco Constante

    2017-09-01

    Full Text Available Dietary heme can be used by colonic bacteria equipped with heme-uptake systems as a growth factor and thereby impact on the microbial community structure. The impact of heme on the gut microbiota composition may be particularly pertinent in chronic inflammation such as in inflammatory bowel disease (IBD, where a strong association with gut dysbiosis has been consistently reported. In this study we investigated the influence of dietary heme on the gut microbiota and inferred metagenomic composition, and on chemically induced colitis and colitis-associated adenoma development in mice. Using 16S rRNA gene sequencing, we found that mice fed a diet supplemented with heme significantly altered their microbiota composition, characterized by a decrease in α-diversity, a reduction of Firmicutes and an increase of Proteobacteria, particularly Enterobacteriaceae. These changes were similar to shifts seen in dextran sodium sulfate (DSS-treated mice to induce colitis. In addition, dietary heme, but not systemically delivered heme, contributed to the exacerbation of DSS-induced colitis and facilitated adenoma formation in the azoxymethane/DSS colorectal cancer (CRC mouse model. Using inferred metagenomics, we found that the microbiota alterations elicited by dietary heme resulted in non-beneficial functional shifts, which were also characteristic of DSS-induced colitis. Furthermore, a reduction in fecal butyrate levels was found in mice fed the heme supplemented diet compared to mice fed the control diet. Iron metabolism genes known to contribute to heme release from red blood cells, heme uptake, and heme exporter proteins, were significantly enriched, indicating a shift toward favoring the growth of bacteria able to uptake heme and protect against its toxicity. In conclusion, our data suggest that luminal heme, originating from dietary components or gastrointestinal bleeding in IBD and, to lesser extent in CRC, directly contributes to microbiota dysbiosis

  13. Enhancement of nitrite on heme-induced oxidative reactions: A potential toxicological implication.

    Lu, Naihao; Chen, Wei; Zhu, Jingjie; Peng, Yi-Yuan

    2012-02-01

    Evidence to support the role of heme as major inducers of oxidative damage is increasingly present. Nitrite (NO(2)(-)) is one of the major end products of NO metabolism. Although the biological significance of heme/NO(2)(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO(2)(-) on heme-dependent redox reaction have been greatly underestimated. In this study, we investigated the influence of NO(2)(-) on heme -dependent oxidative reactions. It was found that NO(2)(-) had the capacity to act as a reducing agent to remove high oxidation states of heme iron. In the reduction of ferryl heme to ferric heme, NO(2)(-) was oxidized to a nitrating agent NO(2), and subsequently, tyrosine residues in bovine serum albumin (BSA) were nitrated. However, the presence of NO(2)(-) surprisingly exerted pro-oxidant effect on heme-H(2)O(2)-induced formation of BSA carbonyls at lower concentrations and enhanced the loss of HepG2 cell viability dose-dependently, which was probably due to the ability of this inorganic compound to efficiently enhance the peroxidase activity and oxidative degradation of heme. These data provide novel evidence that the dietary intake and experimental use of NO(2)(-) in vivo and in vitro would possess the pro-oxidant activity through interfering in heme-dependent oxidative reactions. Besides the classic role in protein tyrosine nitration, the deleterious effects on heme redox reactions may provide new insights into the toxicological implications of NO(2)(-) with cellular heme proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.

    Babbitt, Shalon E; San Francisco, Brian; Bretsnyder, Eric C; Kranz, Robert G

    2014-08-19

    C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical "correction" supports the proposed role of heme binding for the corresponding residues.

  15. Mosaic HIV envelope immunogenic polypeptides

    Korber, Bette T. M.; Gnanakaran, S.; Perkins, Simon; Sodroski, Joseph; Haynes, Barton

    2018-01-02

    Disclosed herein are mosaic HIV envelope (Env) polypeptides that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). Also disclosed are sets of the disclosed mosaic Env polypeptides, which include two or more (for example, three) of the polypeptides. Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in a subject comprising administering to the subject at least one (such as two, three, or more) of the immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.

  16. Introduction of water into the heme distal side by Leu65 mutations of an oxygen sensor, YddV, generates verdoheme and carbon monoxide, exerting the heme oxygenase reaction.

    Stranava, Martin; Martínková, Markéta; Stiborová, Marie; Man, Petr; Kitanishi, Kenichi; Muchová, Lucie; Vítek, Libor; Martínek, Václav; Shimizu, Toru

    2014-11-01

    The globin-coupled oxygen sensor, YddV, is a heme-based oxygen sensor diguanylate cyclase. Oxygen binding to the heme Fe(II) complex in the N-terminal sensor domain of this enzyme substantially enhances its diguanylate cyclase activity which is conducted in the C-terminal functional domain. Leu65 is located on the heme distal side and is important for keeping the stability of the heme Fe(II)-O2 complex by preventing the entry of the water molecule to the heme complex. In the present study, it was found that (i) Escherichia coli-overexpressed and purified L65N mutant of the isolated heme-bound domain of YddV (YddV-heme) contained the verdoheme iron complex and other modified heme complexes as determined by optical absorption spectroscopy and mass spectrometry; (ii) CO was generated in the reconstituted system composed of heme-bound L65N and NADPH:cytochrome P450 reductase as confirmed by gas chromatography; (iii) CO generation of heme-bound L65N in the reconstituted system was inhibited by superoxide dismutase and catalase. In a concordance with the result, the reactive oxygen species increased the CO generation; (iv) the E. coli cells overexpressing the L65N protein of YddV-heme also formed significant amounts of CO compared to the cells overexpressing the wild type protein; (v) generation of verdoheme and CO was also observed for other mutants at Leu65 as well, but to a lesser extent. Since Leu65 mutations are assumed to introduce the water molecule into the heme distal side of YddV-heme, it is suggested that the water molecule would significantly contribute to facilitating heme oxygenase reactions for the Leu65 mutants. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Radiolysis of polypeptide

    Ogura, Isao; Nakamura, Katsuichi; Tanaka, Hiroshi; Takahashi, Katsuhiro; Ozaki, Makoto

    1981-01-01

    Almost the same results were obtained from the additional dipeptide, Gly-DL-Ala and DL-Ala-DL-Phe, by the γ-irradiation as previous report. Tri and tetrapeptide consisted of the same amino acid signified good stability than the others. Every polypeptide composed from sulfur contained amino acid exhaled the smell of hydrogen sulfide by the irradiation. It seemed that the stability by the difference of position of amino group in amino acid increased in order α, β, γ ... amino acid and that by the existence of hydroxyl group became smaller. (author)

  18. The heme-heme oxygenase system: a molecular switch in wound healing.

    Wagener, F.A.D.T.G.; Beurden, H.E. van; Hoff, J.W. Von den; Adema, G.J.; Figdor, C.G.

    2003-01-01

    When cells are injured they release their contents, resulting in a local accumulation of free heme proteins and heme. Here, we investigated the involvement of heme and its degrading enzyme heme oxygenase (HO) in the inflammatory process during wound healing. We observed that heme directly

  19. Heme oxygenase-1, oxidation, inflammation and atherosclerosis

    Jesus A Araujo

    2012-07-01

    Full Text Available Atherosclerosis is an inflammatory process of the vascular wall characterized by the infiltration of lipids and inflammatory cells. Oxidative modifications of infiltrating low density lipoproteins and induction of oxidative stress play a major role in lipid retention in the vascular wall, uptake by macrophages and generation of foam cells, a hallmark of this disorder. The vasculature has a plethora of protective resources against oxidation and inflammation, many of them regulated by the Nrf2 transcription factor. Heme oxygenase-1 (HO-1 is a Nrf2-regulated gene that plays a critical role in the prevention of vascular inflammation. It is the inducible isoform of heme oxygenase, responsible for the oxidative cleavage of heme groups leading to the generation of biliverdin, carbon monoxide and release of ferrous iron. HO-1 has important antioxidant, antiinflammatory, antiapoptotic, antiproliferative and immunomodulatory effects in vascular cells, most of which play a significant role in the protection against atherogenesis. HO-1 may also be an important feature in macrophage differentiation and polarization to certain subtypes. The biological effects of HO-1 are largely attributable to its enzymatic activity, which can be conceived as a system with three arms of action, corresponding to its three enzymatic byproducts. HO-1 mediated vascular protection may be due to a combination of systemic and vascular local effects. It is usually expressed at low levels but can be highly upregulated in the presence of several proatherogenic stimuli. The HO-1 system is amenable for use in the development of new therapies, some of them currently under experimental and clinical trials. Interestingly, in contrast to the HO-1 antiatherogenic actions, the expression of its transcriptional regulator Nrf2 leads to proatherogenic effects instead. This article reviews the evidence that supports the antiatherogenic role of HO-1, potential pathways and mechanisms mediating

  20. Human heme oxygenase oxidation of 5- and 15-phenylhemes.

    Wang, Jinling; Niemevz, Fernando; Lad, Latesh; Huang, Liusheng; Alvarez, Diego E; Buldain, Graciela; Poulos, Thomas L; de Montellano, Paul R Ortiz

    2004-10-08

    Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.

  1. Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

    Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

    2016-02-15

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

    Norifumi Muraki

    2016-05-01

    Full Text Available Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

  3. Heme-coordinated histidine residues form non-specific functional "ferritin-heme" peroxidase system: Possible and partial mechanistic relevance to oxidative stress-mediated pathology in neurodegenerative diseases.

    Esmaeili, Sajjad; Kooshk, Mohammad Reza Ashrafi; Asghari, Seyyed Mohsen; Khodarahmi, Reza

    2016-10-01

    Ferritin is a giant protein composed of 24 subunits which is able to sequester up to 4500 atoms of iron. We proposed two kinds of heme binding sites in mammalian ferritins and provided direct evidence for peroxidase activity of heme-ferritin, since there is the possibility that "ferritin-heme" systems display unexpected catalytic behavior like heme-containing enzymes. In the current study, peroxidase activity of heme-bound ferritin was studied using TMB(1), l-DOPA, serotonin, and dopamine, in the presence of H2O2, as oxidant substrate. The catalytic oxidation of TMB was consistent with first-order kinetics with respect to ferritin concentration. Perturbation of the binding affinity and catalytic behavior of heme-bound His-modified ferritin were also documented. We also discuss the importance of the peroxidase-/nitrative-mediated oxidation of vital molecules as well as ferritin-induced catalase inhibition using in vitro experimental system. Uncontrollable "heme-ferritin"-based enzyme activity as well as up-regulation of heme and ferritin may inspire that some oxidative stress-mediated cytotoxic effects in AD-affected cells could be correlated to ferritin-heme interaction and/or ferritin-induced catalase inhibition and describe its contribution as an important causative pathogenesis mechanism in some neurodegenerative disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2.

    Kathiresan, Meena; English, Ann M

    2017-02-01

    We recently reported that cytochrome c peroxidase (Ccp1) functions as a H 2 O 2 sensor protein when H 2 O 2 levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (Cyc II ), determines whether Ccp1 acts as a H 2 O 2 sensor or peroxidase. For H 2 O 2 to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H 2 O 2 in the absence of Cyc II to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H 2 O 2 at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H 2 O 2 shuts down heterolytic cleavage of H 2 O 2 at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H 2 O 2 signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O 2 reactivity.

  5. Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity.

    Padmamalini Baskaran

    Full Text Available Nitric oxide signals through activation of soluble guanylyl cyclase (sGC, a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain to the effector domain (catalytic domain, in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105 of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

  6. Current understanding of iron homeostasis.

    Anderson, Gregory J; Frazer, David M

    2017-12-01

    Iron is an essential trace element, but it is also toxic in excess, and thus mammals have developed elegant mechanisms for keeping both cellular and whole-body iron concentrations within the optimal physiologic range. In the diet, iron is either sequestered within heme or in various nonheme forms. Although the absorption of heme iron is poorly understood, nonheme iron is transported across the apical membrane of the intestinal enterocyte by divalent metal-ion transporter 1 (DMT1) and is exported into the circulation via ferroportin 1 (FPN1). Newly absorbed iron binds to plasma transferrin and is distributed around the body to sites of utilization with the erythroid marrow having particularly high iron requirements. Iron-loaded transferrin binds to transferrin receptor 1 on the surface of most body cells, and after endocytosis of the complex, iron enters the cytoplasm via DMT1 in the endosomal membrane. This iron can be used for metabolic functions, stored within cytosolic ferritin, or exported from the cell via FPN1. Cellular iron concentrations are modulated by the iron regulatory proteins (IRPs) IRP1 and IRP2. At the whole-body level, dietary iron absorption and iron export from the tissues into the plasma are regulated by the liver-derived peptide hepcidin. When tissue iron demands are high, hepcidin concentrations are low and vice versa. Too little or too much iron can have important clinical consequences. Most iron deficiency reflects an inadequate supply of iron in the diet, whereas iron excess is usually associated with hereditary disorders. These disorders include various forms of hemochromatosis, which are characterized by inadequate hepcidin production and, thus, increased dietary iron intake, and iron-loading anemias whereby both increased iron absorption and transfusion therapy contribute to the iron overload. Despite major recent advances, much remains to be learned about iron physiology and pathophysiology. © 2017 American Society for Nutrition.

  7. A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus.

    Laakso, Holly A; Marolda, Cristina L; Pinter, Tyler B; Stillman, Martin J; Heinrichs, David E

    2016-01-01

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

    Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

    2016-01-01

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

  9. Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

    Mouriño, Susana; Rodríguez-Ares, Isabel; Osorio, Carlos R.; Lemos, Manuel L.

    2005-01-01

    The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity. PMID:16332832

  10. Phenol degradation catalyzed by a peroxidase mimic constructed through the grafting of heme onto metal-organic frameworks.

    Jiang, Wei; Yang, Jiebing; Wang, Xinghuo; Han, Haobo; Yang, Yan; Tang, Jun; Li, Quanshun

    2018-01-01

    The aim of this work was to construct a peroxidase mimic for achieving the phenol degradation through Fenton reaction. The enzyme mimic was synthesized through the conjugation of heme with the amino group of 2-amino-1,4-benzene dicarboxylate in UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. Compared to free heme, the composite Heme-ZrMOF exhibited an obviously enhanced ability for phenol degradation with up to 97.3% of phenol removal after 2h. Meanwhile, it could achieve the easy separation of catalyst from the system and the elimination of iron residues in the process of phenol degradation. Finally, the catalyst Heme-ZrMOF was observed to possess good recyclability in the phenol degradation with still 76.2% of phenol removal after 4 cycles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Hemoglobin and heme scavenger receptors

    Nielsen, Marianne Jensby; Møller, Holger Jon; Moestrup, Søren Kragh

    2010-01-01

    Heme, the functional group of hemoglobin, myoglobin, and other hemoproteins, is a highly toxic substance when it appears in the extracellular milieu. To circumvent potential harmful effects of heme from hemoproteins released during physiological or pathological cell damage (such as hemolysis...... and rhabdomyolysis), specific high capacity scavenging systems have evolved in the mammalian organism. Two major systems, which essentially function in a similar way by means of a circulating latent plasma carrier protein that upon ligand binding is recognized by a receptor, are represented by a) the hemoglobin...

  12. Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis.

    Halina Wójtowicz

    2009-05-01

    Full Text Available Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-beta fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.

  13. Transmutation of a heme protein.

    Barker, P D; Ferrer, J C; Mylrajan, M; Loehr, T M; Feng, R; Konishi, Y; Funk, W D; MacGillivray, R T; Mauk, A G

    1993-01-01

    Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (E(m) = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the alpha carbon of the heme 4-vinyl group. Form BI has a covalently bound heme group coupled through a thioether linkage involving the beta carbon of the heme 4-vinyl group. Form BII is similar to BI except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (E(m) = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed. PMID:8341666

  14. Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

    Ramos-Santana, Brenda J.; López-Garriga, Juan

    2012-01-01

    Highlights: ► H-bonding network loop by PheB10Tyr mutation is proposed. ► The propionate group H-bonding network restricted the flexibility of the heme. ► The hydrogen bonding interaction modulates the electron density of the iron. ► Propionate H-bonding network loop explains the heme-ligand stabilization. -- Abstract: Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. 1 H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00 ppm, GlnE7 N ε1 H/N ε2 H at 10.66 ppm/−3.27 ppm, and PheE11 C δ H at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen

  15. Isoporphyrin Intermediate in Heme Oxygenase Catalysis

    Evans, John P.; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

    2008-01-01

    Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. PMID:18487208

  16. No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

    Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

    2017-07-20

    Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Identification of the heme acquisition system in Vibrio vulnificus M2799.

    Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Yamahata, Eri; Yamaguchi, Ryo; Miyaki, Yuta; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Tsujibo, Hiroshi

    2018-04-01

    Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Red meat and colon cancer : The cytotoxic and hyperproliferative effects of dietary heme

    Sesink, ALA; Termont, DSML; Kleibeuker, JH; Van der Meer, R

    1999-01-01

    The intake of a Western diet with a high amount of red meat is associated with a high risk for colon cancer. We hypothesize that heme, the iron carrier of red meat, is involved in diet-induced colonic epithelial damage, resulting in increased epithelial proliferation. Rats were fed purified control

  19. Heme oxygenase-1 deletion affects stress erythropoiesis.

    Yu-An Cao

    Full Text Available Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1 deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/- or hmox(+/+ bone marrow cells, we evaluated (i the erythrocyte parameters in the peripheral blood; (ii the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii the patterns of histological iron staining; and (iv the number of Mac-1(+-cells expressing TNF-α. In the spleens of mice that received hmox(+/- cells, we show (i decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii increases in the insoluble iron levels and decreases in the soluble iron levels; (iii increased numbers of Mac-1(+-cells expressing TNF-α; and (iv decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.

  20. Effective electrochemical method for investigation of hemoglobin unfolding based on the redox property of heme groups at glassy carbon electrodes.

    Li, Xianchan; Zheng, Wei; Zhang, Limin; Yu, Ping; Lin, Yuqing; Su, Lei; Mao, Lanqun

    2009-10-15

    This study demonstrates a facile and effective electrochemical method for investigation of hemoglobin (Hb) unfolding based on the electrochemical redox property of heme groups in Hb at bare glassy carbon (GC) electrodes. In the native state, the heme groups are deeply buried in the hydrophobic pockets of Hb with a five-coordinate high-spin complex and thus show a poor electrochemical property at bare GC electrodes. Upon the unfolding of Hb induced by the denaturant of guanidine hydrochloride (GdnHCl), the fifth coordinative bond between the heme groups and the residue of the polypeptides (His-F8) is broken, and as a result, the heme groups initially buried deeply in the hydrophobic pockets dissociate from the polypeptide chains and are reduced electrochemically at GC electrodes, which can be used to probe the unfolding of Hb. The results on the GdnHCl-induced Hb unfolding obtained with the electrochemical method described here well coincide with those studied with other methods, such as UV-vis spectroscopy, fluorescence, and circular dichroism. The application of the as-established electrochemical method is illustrated to study the kinetics of GdnHCl-induced Hb unfolding, the GdnHCl-induced unfolding of another kind of hemoprotein, catalase, and the pH-induced Hb unfolding/refolding.

  1. Methods for using polypeptides having cellobiohydrolase activity

    Morant, Marc D; Harris, Paul

    2016-08-23

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  3. Nitric oxide heme interactions in nitrophorin from Cimex lectularius

    Christmann, R.; Auerbach, H., E-mail: auerbach@physik.uni-kl.de [University of Kaiserslautern, Department of Physics (Germany); Berry, R. E.; Walker, F. A. [The University of Arizona, Department of Chemistry and Biochemistry (United States); Schünemann, V. [University of Kaiserslautern, Department of Physics (Germany)

    2016-12-15

    The nitrophorin from the bedbug Cimex lectularius (cNP) is a nitric oxide (NO) carrying protein. Like the nitrophorins (rNPs) from the kissing bug Rhodnius prolixus, cNP forms a stable heme Fe(III)-NO complex, where the NO can be stored reversibly for a long period of time. In both cases, the NPs are found in the salivary glands of blood-sucking bugs. The insects use the nitrophorins to transport the NO to the victim’s tissues, resulting in vasodilation and reduced blood coagulation. However, the structure of cNP is significantly different to those of the rNPs from Rhodnius prolixus. Furthermore, the cNP can bind a second NO molecule to the proximal heme cysteine when present at higher concentrations. High field Mössbauer spectroscopy on {sup 57}Fe enriched cNP complexed with NO shows reduction of the heme iron and formation of a ferrous nitric oxide (Fe(II)-NO) complex. Density functional theory calculations reproduce the experimental Mössbauer parameters and confirm this observation.

  4. Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

    Dailey, Harry A; Dailey, Tamara A; Gerdes, Svetlana; Jahn, Dieter; Jahn, Martina; O'Brian, Mark R; Warren, Martin J

    2017-03-01

    The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. Copyright © 2017 American Society for Microbiology.

  5. Interaction of nitric oxide with human heme oxygenase-1.

    Wang, Jinling; Lu, Shen; Moënne-Loccoz, Pierre; Ortiz de Montellano, Paul R

    2003-01-24

    NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

  6. Formation of nitrosyl non-heme iron-sulphur complexes of a mitrochondria electron-transport chain in a liver and kidneys under prolonged permanent action of radiation contamination in the Chernobyl region

    Sidorik, E.P.; Burlaka, A.P.; Druzhina, N.A.

    1995-01-01

    No-complexes with iron-sulfur protein of the N-type (EPR signal g=2.03 at 77 K) have been revealed in a mitochondria electron transport chain in a liver and kidneys of animals which were hold for 1.5 years in the Chernobyl area under action of low intensity ionizing radiation as a result of incorporated radionuclides. These alterations in protein give evidence of changes in oxidation and phosphorylation in tissues

  7. Heme Oxygenase-1 and breast cancer resistance protein protect against heme-induced toxicity

    Wagener, Frank A D T G; Dankers, Anita C A; van Summeren, Frank; Scharstuhl, Alwin; van den Heuvel, Jeroen J M W; Koenderink, Jan B; Pennings, Sebastiaan W C; Russel, Frans G M; Masereeuw, R.

    2013-01-01

    Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating

  8. A Heme Oxygenase-1 Transducer Model of Degenerative and Developmental Brain Disorders

    Hyman M. Schipper

    2015-03-01

    Full Text Available Heme oxygenase-1 (HO-1 is a 32 kDa protein which catalyzes the breakdown of heme to free iron, carbon monoxide and biliverdin. The Hmox1 promoter contains numerous consensus sequences that render the gene exquisitely sensitive to induction by diverse pro-oxidant and inflammatory stimuli. In “stressed” astroglia, HO-1 hyperactivity promotes mitochondrial iron sequestration and macroautophagy and may thereby contribute to the pathological iron deposition and bioenergetic failure documented in Alzheimer disease, Parkinson disease and certain neurodevelopmental conditions. Glial HO-1 expression may also impact neuroplasticity and cell survival by modulating brain sterol metabolism and the proteasomal degradation of neurotoxic proteins. The glial HO-1 response may represent a pivotal transducer of noxious environmental and endogenous stressors into patterns of neural damage and repair characteristic of many human degenerative and developmental CNS disorders.

  9. Host heme oxygenase-1: Friend or foe in tackling pathogens?

    Singh, Nisha; Ahmad, Zeeshan; Baid, Navin; Kumar, Ashwani

    2018-05-14

    Infectious diseases are a major challenge in management of human health worldwide. Recent literature suggests that host immune system could be modulated to ameliorate the pathogenesis of infectious disease. Heme oxygenase (HMOX1) is a key regulator of cellular signaling and it could be modulated using pharmacological reagents. HMOX1 is a cytoprotective enzyme that degrades heme to generate carbon monoxide (CO), biliverdin, and molecular iron. CO and biliverdin (or bilirubin derived from it) can restrict the growth of a few pathogens. Both of these also induce antioxidant pathways and anti-inflammatory pathways. On the other hand, molecular iron can induce proinflammatory pathway besides making the cellular environment oxidative in nature. Since microbial infections often induce oxidative stress in host cells/tissues, role of HMOX1 has been analyzed in the pathogenesis of number of infections. In this review, we have described the role of HMOX1 in pathogenesis of bacterial infections caused by Mycobacterium species, Salmonella and in microbial sepsis. We have also provided a succinct overview of the role of HMOX1 in parasitic infections such as malaria and leishmaniasis. In the end, we have also elaborated the role of HMOX1 in viral infections such as AIDS, hepatitis, dengue, and influenza. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

  10. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    Vogler, Malvina M.

    2018-01-01

    Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

  11. Histidine at Position 195 is Essential for Association of Heme- b in Lcp1VH2

    Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

    2018-05-01

    The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12 , 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly( cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme- b in the central region of Lcp1VH2.

  12. Clinically Important Features of Porphyrin and Heme Metabolism and the Porphyrias

    Siddesh Besur

    2014-11-01

    Full Text Available Heme, like chlorophyll, is a primordial molecule and is one of the fundamental pigments of life. Disorders of normal heme synthesis may cause human diseases, including certain anemias (X-linked sideroblastic anemias and porphyrias. Porphyrias are classified as hepatic and erythropoietic porphyrias based on the organ system in which heme precursors (5-aminolevulinic acid (ALA, porphobilinogen and porphyrins are chiefly overproduced. The hepatic porphyrias are further subdivided into acute porphyrias and chronic hepatic porphyrias. The acute porphyrias include acute intermittent, hereditary copro-, variegate and ALA dehydratase deficiency porphyria. Chronic hepatic porphyrias include porphyria cutanea tarda and hepatoerythropoietic porphyria. The erythropoietic porphyrias include congenital erythropoietic porphyria (Gűnther’s disease and erythropoietic protoporphyria. In this review, we summarize the key features of normal heme synthesis and its differing regulation in liver versus bone marrow. In both organs, principal regulation is exerted at the level of the first and rate-controlling enzyme, but by different molecules (heme in the liver and iron in the bone marrow. We also describe salient clinical, laboratory and genetic features of the eight types of porphyria.

  13. Radioisotope investigation of iron absorption in humans. Part of a WHO/IAEA coordinated programme on iron nutrition

    Bothwell, T.H.

    1975-08-01

    The gastrointestinal absorption of iron was investigated in a number of multiparous women living under low socio-economic conditions which are known to lead commonly to iron deficiency. The percentage absorption was deduced by comparing the concentration of 55 Fe and/or 59 Fe in blood with the activity of the corresponding isotope(s) administered orally about two weeks earlier. It was confirmed or established that: (1) food or supplemental iron, if available at all, tends to be absorbed from the intestines as if present there in one of two alternative pools: heme and non-heme, (2) vitamin C substantially increases absorption of food or supplemental non-heme iron, (3) it is technically possible to use common salt or sugar as a carrier for supplemental iron and vitamin C, but practical difficulties of using such systems in a public health prophylactic programme are severe, and (4) tea, presumably because of its tannin content, inhibits absorption of accompanying iron

  14. The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress.

    Mancini, Stefano; Imlay, James A

    2015-05-01

    Hydrogen peroxide pervades many natural environments, including the phagosomes that mediate cell-based immunity. Transcriptomic analysis showed that during protracted low-grade H(2)O(2) stress, Escherichia coli responds by activating both the OxyR defensive regulon and the Fur iron-starvation response. OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). Mutations that blocked either adaptation caused the accumulation of porphyrin intermediates, inadequate activation of heme enzymes, low catalase activity, defective clearance of H(2)O(2) and a failure to grow. Genetic analysis indicated that HemH induction is needed to compensate for iron sequestration by the mini-ferritin Dps. Dps activity protects DNA and proteins by limiting Fenton chemistry, but it interferes with the ability of HemH to acquire the iron that it needs to complete heme synthesis. HemF is a manganoprotein that displaces HemN, an iron-sulfur enzyme whose synthesis and/or stability is apparently problematic during H(2)O(2) stress. Thus, the primary responses to H(2)O(2), including the sequestration of iron, require compensatory adjustments in the mechanisms of iron-cofactor synthesis. The results support the growing evidence that oxidative stress is primarily an iron pathology. © 2015 John Wiley & Sons Ltd.

  15. Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake.

    Fiorito, Veronica; Geninatti Crich, Simonetta; Silengo, Lorenzo; Aime, Silvio; Altruda, Fiorella; Tolosano, Emanuela

    2013-01-01

    The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of (57)Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of (57)FeSO4 or of (57)Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis.

  16. Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

    Tsien, Roger Y; Wang, Lei

    2015-01-13

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  17. Improvement of bioavailability for iron from vegetarian meals by ascorbic acid

    Sritongkul, N.; Tuntawiroon, M.; Pleehachinda, R.; Suwanik, R.

    1996-01-01

    There are two kinds of iron in the diet with respect to the mechanism of absorption, heme-iron which is present as haemoglobin or myoglobin in meat and blood products, and, non-heme iron which is the main source of dietary iron. The bioavailability of the non-heme food iron is much lower than heme-iron. Vegetarian diets contain only non-heme iron. Iron intake from vegetarian meals are generally satisfied with the requirements, however, the bioavailabilities for non-heme iron is determined not only by iron content byt also the balance between different dietary factors enhancing and inhibiting iron absorption. The main enhancing factor in vegetarian meals is ascorbic acid in fruits and vegetables, inhibitors are phytate in cereals and grains, and tannins in some spices and vegetables. It has been reported that iron deficiency is one of the common micronutrient problems associated with unplanned vegetarian diets. In the present study the absorption of non-heme iron was measured from 2 vegetarian meals containing considerable amounts of phytate and tannin. The extrinsic tay method ( 59 Fe/ 55 Fe) was used to labelled the non-heme iron. The mean percentage absorption of non-heme iron from both meals was slightly different due to differences in their dietary contents. Their initial percentages iron absorption were apparent low (3.5% and 4.1%), however, the absorption progressively increased with increase in the level of ascorbic acid, 2-3 times with 100 mg and 4-5 times with 200 mg of ascorbic acid. The average amount of iron absorbed per 2000 kcal increased from 0.37 mg to 0.86 mg and 1.45 mg with the addition of 100 mg and 200 mg ascorbic acid respectively (p < 0.001). Considering the limited caloric intakes and the iron content in the meals, the amount of iron absorbed from vegetarian meals without ascorbic acid was not able to meet certain requirements for children, adolescents and menstruating women. The minimal requirement for dietary iron needed to be absorbed is

  18. A rapid, simple method for obtaining radiochemically pure hepatic heme

    Bonkowski, H.L.; Bement, W.J.; Erny, R.

    1978-01-01

    Radioactively-labelled heme has usually been isolated from liver to which unlabelled carrier has been added by long, laborious techniques involving organic solvent extraction followed by crystallization. A simpler, rapid method is devised for obtaining radiochemically-pure heme synthesized in vivo in rat liver from delta-amino[4- 14 C]levulinate. This method, in which the heme is extracted into ethyl acetate/glacial acetic acid and in which porphyrins are removed from the heme-containing organic phase with HCl washes, does not require addition of carrier heme. The new method gives better heme recoveries than and heme specific activities identical to, those obtained using the crystallization method. In this new method heme must be synthesized from delta-amino[4- 14 C]levulinate; it is not satisfactory to use [2- 14 C]glycine substrate because non-heme counts are isolated in the heme fraction. (Auth.)

  19. Plasma protein haptoglobin modulates renal iron loading

    Fagoonee, Sharmila; Gburek, Jakub; Hirsch, Emilio

    2005-01-01

    Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme...... distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting...... morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload....

  20. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

    Wojtowicz, Halina; Wojaczynski, Jacek; Olczak, Mariusz; Kroliczewski, Jaroslaw; Latos-Grazynski, Lechoslaw; Olczak, Teresa

    2009-01-01

    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and 1 H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  1. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

    Wojtowicz, Halina [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Wojaczynski, Jacek [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Mariusz [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Kroliczewski, Jaroslaw [Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, 50-148 Wroclaw (Poland); Latos-Grazynski, Lechoslaw [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Teresa [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland)

    2009-05-29

    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and {sup 1}H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  2. A Neisseria meningitidis fbpABC mutant is incapable of using nonheme iron for growth.

    Khun, H H; Kirby, S D; Lee, B C

    1998-05-01

    The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.

  3. Insights on Heme Synthesis in the Malaria Parasite.

    Nagaraj, Viswanathan A; Padmanaban, Govindarajan

    2017-08-01

    The malaria parasite has a functional heme-biosynthetic pathway, although it can access host hemoglobin-heme. The heme pathway is dispensable for blood stages, but essential in the mosquito stages which do not acquire hemoglobin-heme. We propose that the blood stage parasites maintain a dynamic heme pool through multiple back-up mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Polypeptides having catalase activity and polynucleotides encoding same

    Liu, Ye; Duan, Junxin; Zhang, Yu; Tang, Lan

    2017-05-02

    Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptides having xylanase activity and polynucleotides encoding same

    Spodsberg, Nikolaj

    2018-02-06

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

    Jiang, Yongying; Ortiz de Montellano, Paul R

    2008-05-05

    Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

  7. Mitochondrial Iron Transport and Homeostasis in Plants

    Anshika eJain

    2013-09-01

    Full Text Available Iron (Fe is an essential nutrient for plants and although the mechanisms controlling iron uptake from the soil are relatively well understood, comparatively little is known about subcellular trafficking of iron in plant cells. Mitochondria represent a significant iron sink within cells, as iron is required for the proper functioning of respiratory chain protein complexes. Mitochondria are a site of Fe-S cluster synthesis, and possibly heme synthesis as well. Here we review recent insights into the molecular mechanisms controlling mitochondrial iron transport and homeostasis. We focus on the recent identification of a mitochondrial iron uptake transporter in rice and a possible role for metalloreductases in iron uptake by mitochondria. In addition, we highlight recent advances in mitochondrial iron homeostasis with an emphasis on the roles of frataxin and ferritin in iron trafficking and storage within mitochondria.

  8. Glucose-dependent Insulinotropic Polypeptide

    Christensen, Mikkel B; Calanna, Salvatore; Holst, Jens Juul

    2014-01-01

    CONTEXT: Patients with type 2 diabetes mellitus (T2DM) have clinically relevant disturbances in the effects of the hormone glucose-dependent insulinotropic polypeptide (GIP). OBJECTIVE: We aimed to evaluate the importance of the prevailing plasma glucose levels for the effect of GIP on responses......: During fasting glycemia (plasma glucose ∼8 mmol/L), GIP elicited significant increments in both insulin and glucagon levels, resulting in neutral effects on plasma glucose. During insulin-induced hypoglycemia (plasma glucose ∼3 mmol/L), GIP elicited a minor early-phase insulin response and increased...... glucagon levels during the initial 30 minutes, resulting in less glucose needed to be infused to maintain the clamp (29 ± 8 vs 49 ± 12 mg × kg(-1), P glucose ∼12 mmol/L), GIP augmented insulin secretion throughout the clamp, with slightly less glucagon...

  9. Heme-containing enzymes and inhibitors for tryptophan metabolism.

    Yan, Daojing; Lin, Ying-Wu; Tan, Xiangshi

    2017-09-20

    Iron-containing enzymes such as heme enzymes play crucial roles in biological systems. Three distinct heme-containing dioxygenase enzymes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the initial and rate-limiting step of l-tryptophan catabolism through the kynurenine pathway in mammals. Overexpression of these enzymes causes depletion of tryptophan and the accumulation of metabolic products, which contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. In the past few decades, IDO1 has garnered the most attention as a therapeutic target with great potential in cancer immunotherapy. Many potential inhibitors of IDO1 have been designed, synthesized and evaluated, among which indoximod (d-1-MT), INCB024360, GDC-0919 (formerly NLG-919), and an IDO1 peptide-based vaccine have advanced to the clinical trial stage. However, recently, the roles of TDO and IDO2 have been elucidated in immune suppression. In this review, the current drug discovery landscape for targeting TDO, IDO1 and IDO2 is highlighted, with particular attention to the recent use of drugs in clinical trials. Moreover, the crystal structures of these enzymes, in complex with inhibitors, and the mechanisms of Trp catabolism in the first step, are summarized to provide information for facilitating the discovery of new enzyme inhibitors.

  10. Heme oxygenase and carbon monoxide protect from muscle dystrophy.

    Chan, Mun Chun; Ziegler, Olivia; Liu, Laura; Rowe, Glenn C; Das, Saumya; Otterbein, Leo E; Arany, Zoltan

    2016-11-28

    Duchenne muscle dystrophy (DMD) is one of the most common lethal genetic diseases of children worldwide and is 100% fatal. Steroids, the only therapy currently available, are marred by poor efficacy and a high side-effect profile. New therapeutic approaches are urgently needed. Here, we leverage PGC-1α, a powerful transcriptional coactivator known to protect against dystrophy in the mdx murine model of DMD, to search for novel mechanisms of protection against dystrophy. We identify heme oxygenase-1 (HO-1) as a potential novel target for the treatment of DMD. Expression of HO-1 is blunted in the muscles from the mdx murine model of DMD, and further reduction of HO-1 by genetic haploinsufficiency worsens muscle damage in mdx mice. Conversely, induction of HO-1 pharmacologically protects against muscle damage. Mechanistically, HO-1 degrades heme into biliverdin, releasing in the process ferrous iron and carbon monoxide (CO). We show that exposure to a safe low dose of CO protects against muscle damage in mdx mice, as does pharmacological treatment with CO-releasing molecules. These data identify HO-1 and CO as novel therapeutic agents for the treatment of DMD. Safety profiles and clinical testing of inhaled CO already exist, underscoring the translational potential of these observations.

  11. Measurement of Heme Synthesis Levels in Mammalian Cells.

    Hooda, Jagmohan; Alam, Maksudul; Zhang, Li

    2015-07-09

    Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-(14)C] 5-aminolevulinic acid ([(14)C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [(14)C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

  12. Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy.

    Kruglik, Sergei G; Jasaitis, Audrius; Hola, Klara; Yamashita, Taku; Liebl, Ursula; Martin, Jean-Louis; Vos, Marten H

    2007-05-01

    Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.

  13. Protein Hydrolysates as Promoters of Non-Haem Iron Absorption

    Li, Yanan; Jiang, Han; Huang, Guangrong

    2017-01-01

    Iron (Fe) is an essential micronutrient for human growth and health. Organic iron is an excellent iron supplement due to its bioavailability. Both amino acids and peptides improve iron bioavailability and absorption and are therefore valuable components of iron supplements. This review focuses on protein hydrolysates as potential promoters of iron absorption. The ability of protein hydrolysates to chelate iron is thought to be a key attribute for the promotion of iron absorption. Iron-chelatable protein hydrolysates are categorized by their absorption forms: amino acids, di- and tri-peptides and polypeptides. Their structural characteristics, including their size and amino acid sequence, as well as the presence of special amino acids, influence their iron chelation abilities and bioavailabilities. Protein hydrolysates promote iron absorption by keeping iron soluble, reducing ferric iron to ferrous iron, and promoting transport across cell membranes into the gut. We also discuss the use and relative merits of protein hydrolysates as iron supplements. PMID:28617327

  14. Protein Hydrolysates as Promoters of Non-Haem Iron Absorption

    Yanan Li

    2017-06-01

    Full Text Available Iron (Fe is an essential micronutrient for human growth and health. Organic iron is an excellent iron supplement due to its bioavailability. Both amino acids and peptides improve iron bioavailability and absorption and are therefore valuable components of iron supplements. This review focuses on protein hydrolysates as potential promoters of iron absorption. The ability of protein hydrolysates to chelate iron is thought to be a key attribute for the promotion of iron absorption. Iron-chelatable protein hydrolysates are categorized by their absorption forms: amino acids, di- and tri-peptides and polypeptides. Their structural characteristics, including their size and amino acid sequence, as well as the presence of special amino acids, influence their iron chelation abilities and bioavailabilities. Protein hydrolysates promote iron absorption by keeping iron soluble, reducing ferric iron to ferrous iron, and promoting transport across cell membranes into the gut. We also discuss the use and relative merits of protein hydrolysates as iron supplements.

  15. Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.

    S. de Weert; P.J. Punt; Christien Lokman; C.A. van den Hondel; A.C. Franken; A.F. Ram

    2011-01-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

  16. Heme biosynthesis and its regulation: Towards understanding and improvement of heme biosynthesis in filamentous fungi

    Franken, A.C.W.; Lokman, B.C.; Ram, A.F.J.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Weert, S. de

    2011-01-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

  17. Hemoglobin variants as models for investigation of dissociation of intact polypeptide chains by ESI tandem mass spectrometry

    Light, K.J.; Loo, J.A.; Edmonds, C.G.; Smith, R.D.

    1991-06-01

    Electrospray ionization mass spectroscopy (ESI-MS) is rapidly becoming a practical biochemical tool for peptide and protein sequence analysis. The utility of ESI-MS is through use of Collisionally Activated Dissociation (ESI-CAD-MS). Human hemoglobin (Hb, ∼62 kDa) consists of four polypeptide chains and a prosthetic heme group. There are over 400 Hb variants, characterized by amino acid substitutions in either the alpha or beta polypeptide chains. We investigated ESI-CAD-MS as a tool for rapidly analyzing amino acid substitutions, using eight Hb beta chain variants. The approximate location of the modification can be deduced from comparison of the CAD mass spectra and observance of the mass shifts of the fragment ion containing the substitution. Fragmentation occurs preferentially at the amino terminus of proline residues. For most substitutions, differences in CAD mass spectra were not seen. 2 figs

  18. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  19. Characterization of a Gene Family Encoding SEA (Sea-urchin Sperm Protein, Enterokinase and Agrin)-Domain Proteins with Lectin-Like and Heme-Binding Properties from Schistosoma japonicum

    Mbanefo, Evaristus Chibunna; Kikuchi, Mihoko; Huy, Nguyen Tien; Shuaibu, Mohammed Nasir; Cherif, Mahamoud Sama; Yu, Chuanxin; Wakao, Masahiro; Suda, Yasuo; Hirayama, Kenji

    2014-01-01

    Background We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Methodology/Principal Findings Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. Conclusions The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted. PMID:24416467

  20. Effect of irradiation and storage in the iron availability in lamb meat treated with different diets

    Souza, Adriana Regia Marques de; Arthur, Valter

    2008-01-01

    Irradiation is an efficient method to increase the microbiological safety and to maintain the nutrients such as iron in the meat. The best absorption form, heme iron, should be preserved in order to increase the nutritional quality of stored meat. The diet can alter the nutrients contents and form in the meat. The iron is provided from the diet and it is an essential element for the metabolic processes such as oxygen transport, oxidative metabolism, and cellular growth. Meat lamb samples treated with different diets (it controls, TAC1, TAC2 and sorghum) were wrapped to vacuous, and irradiated in the doses 0, 2 and 4 kGy and stored at 4 deg C during 15 days. The values of total iron and heme iron were measured at 0 and 15 days of storage. The storage reduced the content of total iron (18.36 for 14.28 mg.100 g -1 ) and heme iron (13.78 for 10.52 mg.100 g -1 ). The diets affected the levels of total and heme iron of the meat, and the sorghum diet was the one that presented the larger content. The dose of 2 kGy was the one that affected the iron the most independently of the storage time. It was verified that the amounts of total and heme iron varied according to the storage time, irradiation doses, and lamb diets. (author)

  1. Shigella Iron Acquisition Systems and their Regulation.

    Wei, Yahan; Murphy, Erin R

    2016-01-01

    Survival of Shigella within the host is strictly dependent on the ability of the pathogen to acquire essential nutrients, such as iron. As an innate immune defense against invading pathogens, the level of bio-available iron within the human host is maintained at exceeding low levels, by sequestration of the element within heme and other host iron-binding compounds. In response to sequestration mediated iron limitation, Shigella produce multiple iron-uptake systems that each function to facilitate the utilization of a specific host-associated source of nutrient iron. As a mechanism to balance the essential need for iron and the toxicity of the element when in excess, the production of bacterial iron acquisition systems is tightly regulated by a variety of molecular mechanisms. This review summarizes the current state of knowledge on the iron-uptake systems produced by Shigella species, their distribution within the genus, and the molecular mechanisms that regulate their production.

  2. Interaction between Mitochondrial Reactive Oxygen Species, Heme Oxygenase, and Nitric Oxide Synthase Stimulates Phagocytosis in Macrophages

    Andrea Müllebner

    2018-01-01

    Full Text Available BackgroundMacrophages are cells of the innate immune system that populate every organ. They are required not only for defense against invading pathogens and tissue repair but also for maintenance of tissue homeostasis and iron homeostasis.AimThe aim of this study is to understand whether heme oxygenase (HO and nitric oxide synthase (NOS contribute to the regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX activity and phagocytosis, two key components of macrophage function.MethodsThis study was carried out using resting J774A.1 macrophages treated with hemin or vehicle. Activity of NOS, HO, or NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS formation was determined by Amplex® red assay, and phagocytosis was measured using fluorescein isothiocyanate-labeled bacteria. In addition, we analyzed the fate of the intracellular heme by using electron spin resonance.ResultsWe show that both enzymes NOS and HO are essential for phagocytic activity of macrophages. NOS does not directly affect phagocytosis, but stimulates NOX activity via nitric oxide-triggered ROS production of mitochondria. Treatment of macrophages with hemin results in intracellular accumulation of ferrous heme and an inhibition of phagocytosis. In contrast to NOS, HO products, including carbon monoxide, neither clearly affect NOX activity nor clearly affect phagocytosis, but phagocytosis is accelerated by HO-mediated degradation of heme.ConclusionBoth enzymes contribute to the bactericidal activity of macrophages independently, by controlling different pathways.

  3. The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for Hemoglobin Scavenging

    Jens Haugbølle Thomsen

    2013-01-01

    Full Text Available The haptoglobin- (Hp- CD163-heme oxygenase-1 (HO-1 pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO, biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6, but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.

  4. Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

    Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

    2010-03-01

    The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

  5. Duodenal Cytochrome b (DCYTB in Iron Metabolism: An Update on Function and Regulation

    Darius J. R. Lane

    2015-03-01

    Full Text Available Iron and ascorbate are vital cellular constituents in mammalian systems. The bulk-requirement for iron is during erythropoiesis leading to the generation of hemoglobin-containing erythrocytes. Additionally; both iron and ascorbate are required as co-factors in numerous metabolic reactions. Iron homeostasis is controlled at the level of uptake; rather than excretion. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance non-heme iron absorption in the gut; ascorbate regulates iron homeostasis. The involvement of ascorbate in dietary iron absorption extends beyond the direct chemical reduction of non-heme iron by dietary ascorbate. Among other activities; intra-enterocyte ascorbate appears to be involved in the provision of electrons to a family of trans-membrane redox enzymes; namely those of the cytochrome b561 class. These hemoproteins oxidize a pool of ascorbate on one side of the membrane in order to reduce an electron acceptor (e.g., non-heme iron on the opposite side of the membrane. One member of this family; duodenal cytochrome b (DCYTB; may play an important role in ascorbate-dependent reduction of non-heme iron in the gut prior to uptake by ferrous-iron transporters. This review discusses the emerging relationship between cellular iron homeostasis; the emergent “IRP1-HIF2α axis”; DCYTB and ascorbate in relation to iron metabolism.

  6. Moessbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

    Harami, Taikan, E-mail: harami.taikan@jaea.go.jp [Japan Atomic Energy Agency (Japan); Kitao, Shinji; Kobayashi, Yasuhiro [Kyoto University, Research Reactor Institute (Japan); Mitsui, Takaya [Japan Atomic Energy Agency (Japan)

    2008-01-15

    The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Moessbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Moessbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.

  7. Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

    Du, Y.; Liu, G.; Yan, Y.; Huang, D.; Luo, W.; Martínková, M.; Man, Petr; Shimizu, T.

    2013-01-01

    Roč. 26, č. 5 (2013), s. 839-852 ISSN 0966-0844 Institutional support: RVO:61388971 Keywords : Heme oxygenase * Heme protein * Hydrogen sulfide Subject RIV: CE - Biochemistry Impact factor: 2.689, year: 2013

  8. Molecular hijacking of siroheme for the synthesis of heme and d1 heme.

    Bali, Shilpa; Lawrence, Andrew D; Lobo, Susana A; Saraiva, Lígia M; Golding, Bernard T; Palmer, David J; Howard, Mark J; Ferguson, Stuart J; Warren, Martin J

    2011-11-08

    Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

  9. Identification of two genes potentially associated in iron-heme ...

    2013-03-15

    Mar 15, 2013 ... include the degree of vessel stenosis, ulceration, inflamma- tory cell infiltration and ... ness, ulceration, hemorrhage, necrosis, calcification, in- flammation ... treatment. Clinical .... An alternative would be to use only tissue from ...

  10. Models for non-heme iron containing oxidation enzymes

    Roelfes, Johannes Gerhardus

    2000-01-01

    IJzer is een van de essentiël elementen voor alle levende wezens. Heel veel belangrijke functies in organismen worden vervuld door ijzer bevattende eiwitten. De bekendste voorbeelden hiervan zijn ongetwijfeld hemoglobine en myoglobine, die het transport van zuurstof van de longen naar de rest van

  11. Histidine at Position 195 is Essential for Association of Heme-b in Lcp1VH2

    Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

    2018-03-01

    The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12, 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly(cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme-b in the central region of Lcp1VH2.

  12. Cellular Studies with UVA Radiation: A Role for Iron (invited paper)

    Tyrrell, R.M.; Pourzand, C.A.; Brown, J.; Hejmadi, V.; Kvam, V.; Ryter, S.; Watkin, R.D.

    2000-01-01

    The UVA (320-380 nm) component of sunlight or sunbeds acts as an oxidising carcinogen and has been clearly implicated in skin cancer. Since UVA radiation interacts with cells by generating active oxygen species, the damaging effects of this radiation will be exacerbated by the presence of catalytically reactive iron in cells. It has now been shown by two independent techniques (dequenching of metal-quenched calcein fluorescence in cells and changes in the binding activity of the iron responsive protein IRPI) that UVA radiation causes an immediate release of 'free' iron in human skin fibroblasts via the proteolysis of ferritin (Ft). Within minutes of exposure to a range of doses of UVA at natural exposure levels, the binding activity of IRP-1, as well as Ft levels, decrease in a dose-dependent manner. It is proposed that the oxidative damage to lysosomes that leads to Ft degradation and the consequent release of potentially harmful 'free' iron to the cytosol might be a major factor in UVA-induced damage to the skin. UVA radiation also breaks down heme-containing proteins in the microsomal membrane to release free heme as an additional photosensitising component. This will provide another source of enhanced free iron in skin cells since constitutive heme oxygenase 2 (in keratinocytes) and UVA-inducible heme oxygenase-1 (in fibroblasts) are likely to break down any free heme to biliverdin and release iron and carbon monoxide in the process. It is postulated that, in skin fibroblasts, this free heme release and the enhanced free iron pools will lead to an adaptive response involving heme oxygenase (with a maximum about 10 h) and ferritin (in 24-48 h) that will scavenge the heme and iron released by subsequent oxidising (UVA) treatments. (author)

  13. Red meat and colon cancer : how dietary heme initiates hyperproliferation

    IJssennagger, N.

    2012-01-01

    Colorectal cancer is a leading cause of cancer deaths in Western countries. The risk to develop colorectal cancer is associated with the intake of red meat. Red meat contains the porphyrin pigment heme. Heme is an irritant for the colonic wall and it is previously shown that the addition of heme

  14. Dibromine radical anion reactions with heme enzymes

    Gebicka, L.; Gebicki, J.L.

    1996-01-01

    Reactions of Br 2 radical anion with heme enzymes, catalase horseradish peroxidase, have been studied by pulse radiolysis. It has been found that Br 2 - does not react with the heme centre of investigated enzymes. Dibromine radical anion reacts with tryptophan residues of catalase without any influence on the activity of catalase. It is suggested that in pulse radiolysis studies, where horseradish peroxidase is at about tenfold excess toward Br 2 - , the enzyme is modified rather by Br 2 , than by Br 2 - . (author). 26 refs., 3 figs

  15. Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

    Nitin Puri

    2017-01-01

    Full Text Available Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin-a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO is a stress response system which reduces oxidative stress. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in hepatic tissues from lean and obese mice. Obese mice exhibited hyperglycemia (p<0.05; increased levels of proinflammatory cytokines (MCP-1, IL-6, p<0.05; oxidative stress (p<0.05; and increased hepatic hepcidin levels (p<0.05. Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p<0.05. However, this effect is accompanied by a significant decline in ferritin expression. Additionally, there are reduced insulin receptor phosphorylation and attenuation of metabolic regulators pAMPK, pAKT, and pLKB1. Cobalt protoporphyrin- (CoPP- induced HO-1 upregulation in obese mice reversed these alterations (p<0.05, while attenuating hepatic hepcidin levels. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP (p<0.05. Our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin.

  16. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We inv...

  17. Coulomb repulsion in short polypeptides.

    Norouzy, Amir; Assaf, Khaleel I; Zhang, Shuai; Jacob, Maik H; Nau, Werner M

    2015-01-08

    Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge-charge repulsion, side chain functional group solvation, N-terminal and side chain charge-charge repulsion, and side chain-side chain as well as side chain-backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each

  18. Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis.

    Chang, Ling-Chu; Chiang, Shih-Kai; Chen, Shuen-Ei; Yu, Yung-Luen; Chou, Ruey-Hwang; Chang, Wei-Chao

    2018-03-01

    Ferroptosis is a form of oxidative cell death and has become a chemotherapeutic target for cancer treatment. BAY 11-7085 (BAY), which is a well-known IκBα inhibitor, suppressed viability in cancer cells via induction of ferroptotic death in an NF-κB-independent manner. Reactive oxygen species scavenging, relief of lipid peroxidation, replenishment of glutathione and thiol-containing agents, as well as iron chelation, rescued BAY-induced cell death. BAY upregulated a variety of Nrf2 target genes related to redox regulation, particularly heme oxygenase-1 (HO-1). Studies with specific inhibitors and shRNA interventions suggested that the hierarchy of induction is Nrf2-SLC7A11-HO-1. SLC7A11 inhibition by erastin, sulfasalazine, or shRNA interference sensitizes BAY-induced cell death. Overexperession of SLC7A11 attenuated BAY-inhibited cell viability. The ferroptotic process induced by hHO-1 overexpression further indicated that HO-1 is a key mediator of BAY-induced ferroptosis that operates through cellular redox regulation and iron accumulation. BAY causes compartmentalization of HO-1 into the nucleus and mitochondrion, and followed mitochondrial dysfunctions, leading to lysosome targeting for mitophagy. In this study, we first discovered that BAY induced ferroptosis via Nrf2-SLC7A11-HO-1 pathway and HO-1 is a key mediator by responding to the cellular redox status. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Impairment of heme synthesis in myelin as potential trigger of multiple sclerosis.

    Morelli, Alessandro; Ravera, Silvia; Calzia, Daniela; Panfoli, Isabella

    2012-06-01

    The pathogenesis of multiple sclerosis (MS), a disease characterized by demyelination and subsequent axonal degeneration, is as yet unknown. Also, the nature of the disease is as yet not established, since doubts have been cast on its autoimmune origin. Genetic and environmental factors have been implied in MS, leading to the idea of an overall multifactorial origin. An unexpected role in energizing the axon has been reported for myelin, supposed to be the site of consumption of most of oxygen in brain. Myelin would be able to perform oxidative phosphorylation to supply the axons with ATP, thanks to the expression therein of mitochondrial F(o)F(1)-ATP synthase, and respiratory chains. Interestingly, myelin expresses the pathway of heme synthesis, hence of cytochromes, that rely on heme group, in turn depending on Fe availability. Poisoning by these pollutants shares the common characteristic to bring about demyelination both in animal models and in man. Carbon monoxide (CO) and lead poisoning which cause functional imbalance of the heme group, as well as of heme synthesis, cause myelin damage. On the other hand, a lack of essential metals such as iron and copper, produces dramatic myelin decrease. Myelin is a primary target, of iron shortage, indicating that in myelin Fe-dependent processes are more active than in other tissues. The predominant spread of MS in industrialized countries where pollution by heavy metals, and CO poisoning is widespread, suggests a relationship among toxic action of metal pollutants and MS. According to the present hypothesis, MS can be primarily triggered by environmental factors acting on a genetic susceptibility, while the immune response may be a consequence of a primary oxidative damage due to reactive oxygen species produced consequently to an imbalance of cytochromes and respiratory chains in the sheath. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Staphylococcus aureus redirects central metabolism to increase iron availability.

    David B Friedman

    2006-08-01

    Full Text Available Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment or genetic (Deltafur alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB, a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.

  1. Heme pathway evolution in kinetoplastid protists

    Cenci, U.; Moog, D.; Curtis, B.A.; Tanifuji, G.; Eme, L.; Lukeš, Julius; Archibald, J.M.

    2016-01-01

    Roč. 16, MAY 18 (2016), č. článku 109. ISSN 1471-2148 Institutional support: RVO:60077344 Keywords : heme * kinetoplastea * Paramoeba pemaquidensis * Perkinsela * evolution * endosymbiosis * Prokinetoplastina * lateral gene transfer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.221, year: 2016

  2. An ethane-bridged porphyrin dimer as a model of di-heme proteins: inorganic and bioinorganic perspectives and consequences of heme-heme interactions.

    Sil, Debangsu; Rath, Sankar Prasad

    2015-10-07

    Interaction between heme centers has been cleverly implemented by Nature in order to regulate different properties of multiheme cytochromes, thereby allowing them to perform a wide variety of functions. Our broad interest lies in unmasking the roles played by heme-heme interactions in modulating different properties viz., metal spin state, redox potential etc., of the individual heme centers using an ethane-bridged porphyrin dimer as a synthetic model of dihemes. The large differences in the structure and properties of the diheme complexes, as compared to the monoheme analogs, provide unequivocal evidence of the role played by heme-heme interactions in the dihemes. This Perspective provides a brief account of our recent efforts to explore these interesting aspects and the subsequent outcomes.

  3. The Chemistry and Biochemistry of Heme c: Functional Bases for Covalent Attachment

    Bowman, Sarah E. J.; Bren, Kara L.

    2008-01-01

    A discussion of the literature concerning the synthesis, function, and activity of heme c-containing proteins is presented. Comparison of the properties of heme c, which is covalently bound to protein, is made to heme b, which is bound noncovalently. A question of interest is why nature uses biochemically expensive heme c in many proteins when its properties are expected to be similar to heme b. Considering the effects of covalent heme attachment on heme conformation and on the proximal histi...

  4. Iron, Oxidative Stress and Gestational Diabetes

    Taifeng Zhuang

    2014-09-01

    Full Text Available Both iron deficiency and hyperglycemia are highly prevalent globally for pregnant women. Iron supplementation is recommended during pregnancy to control iron deficiency. The purposes of the review are to assess the oxidative effects of iron supplementation and the potential relationship between iron nutrition and gestational diabetes. High doses of iron (~relative to 60 mg or more daily for adult humans can induce lipid peroxidation in vitro and in animal studies. Pharmaceutical doses of iron supplements (e.g., 10× RDA or more for oral supplements or direct iron supplementation via injection or addition to the cell culture medium for a short or long duration will induce DNA damage. Higher heme-iron intake or iron status measured by various biomarkers, especially serum ferritin, might contribute to greater risk of gestational diabetes, which may be mediated by iron oxidative stress though lipid oxidation and/or DNA damage. However, information is lacking about the effect of low dose iron supplementation (≤60 mg daily on lipid peroxidation, DNA damage and gestational diabetes. Randomized trials of low-dose iron supplementation (≤60 mg daily for pregnant women are warranted to test the relationship between iron oxidative stress and insulin resistance/gestational diabetes, especially for iron-replete women.

  5. Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption.

    Wang, Baowei; Cheng, Fansheng; Gao, Shun; Ge, Wenhua; Zhang, Mingai

    2017-02-15

    Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. GLYCOSYLATED YGHJ POLYPEPTIDES FROM ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

    2017-01-01

    The present invention relates to glycosylated YghJ polypeptides from or derived from enterotoxigenic Escherichia coli (ETEC) that are immunogenic. In particular, the present invention relates to compositions or vaccines comprising the polypeptides and their application in immunization, vaccination...

  7. Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia.

    Hascalovici, Jacob R; Song, Wei; Vaya, Jacob; Khatib, Soliman; Fuhrman, Bianca; Aviram, Michael; Schipper, Hyman M

    2009-01-01

    Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.

  8. Tuning Ice Nucleation with Supercharged Polypeptides

    Yang, Huige; Ma, Chao; Li, Kaiyong; Liu, Kai; Loznik, Mark; Teeuwen, Rosalie; van Hest, Jan C. M.; Zhou, Xin; Herrmann, Andreas; Wang, Jianjun

    2016-01-01

    Supercharged unfolded polypeptides (SUPs) are exploited for controlling ice nucleation via tuning the nature of charge and charge density of SUPs. The results show that positively charged SUPs facilitate ice nucleation, while negatively charged ones suppress it. Moreover, the charge density of the

  9. Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1.

    Otto, B R; Kusters, J G; Luirink, J; de Graaf, F K; Oudega, B

    1996-01-01

    An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open readi...

  10. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Wogulis, Mark; Sweeney, Matthew; Heu, Tia

    2017-06-14

    The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

  11. Polypeptides having xylanase activity and polynucleotides encoding same

    Spodsberg, Nikolaj; Shaghasi, Tarana

    2017-06-20

    The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

  12. Phase transitions in polypeptides: analysis of energy fluctuations

    Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

    2009-01-01

    The helix random coil transition in alanine, valine, and leucine polypeptides consisting of 30 amino acids is studied in vacuo using the Langevin molecular dynamics approach. The influence of side chain radicals on internal energy and heat capacity of the polypeptides is discussed. The heat...... of simulation time. This study provides a comparison of methods for the description of structural transitions in polypeptides....

  13. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    Harris, Paul; Golightly, Elizabeth

    2012-11-27

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  14. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    Morant, Marc D.; Harris, Paul

    2015-10-13

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2013-10-29

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polynucleotides encoding polypeptides having beta-glucosidase activity

    Harris, Paul; Golightly, Elizabeth

    2010-03-02

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  17. Human heme oxygenase-1 gene transfer lowers blood pressure and promotes growth in spontaneously hypertensive rats.

    Sabaawy, H E; Zhang, F; Nguyen, X; ElHosseiny, A; Nasjletti, A; Schwartzman, M; Dennery, P; Kappas, A; Abraham, N G

    2001-08-01

    Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, with release of free iron and carbon monoxide. Both heme and carbon monoxide have been implicated in the regulation of vascular tone. A retroviral vector containing human HO-1 cDNA (LSN-HHO-1) was constructed and subjected to purification and concentration of the viral particles to achieve 5x10(9) to 1x10(10) colony-forming units per milliliter. The ability of concentrated infectious viral particles to express human HO-1 (HHO-1) in vivo was tested. A single intracardiac injection of the concentrated infectious viral particles (expressing HHO-1) to 5-day-old spontaneously hypertensive rats resulted in functional expression of the HHO-1 gene and attenuation of the development of hypertension. Rats expressing HHO-1 showed a significant decrease in urinary excretion of a vasoconstrictor arachidonic acid metabolite and a reduction in myogenic responses to increased intraluminal pressure in isolated arterioles. Unexpectedly, HHO-1 chimeric rats showed a simultaneous significant proportionate increase in somatic growth. Thus, delivery of HHO-1 gene by retroviral vector attenuates the development of hypertension and promotes body growth in spontaneously hypertensive rats.

  18. Binding of Pseudomonas aeruginosa Apo-Bacterioferritin Associated Ferredoxin to Bacterioferritin B Promotes Heme Mediation of Electron Delivery and Mobilization of Core Mineral Iron†

    Weeratunga, Saroja K.; Gee, Casey E.; Lovell, Scott; Zeng, Yuhong; Woodin, Carrie L.; Rivera, Mario

    2009-01-01

    The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5–10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) Mol. Microbiol. 45, 1277–1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to

  19. Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

    Litwin, Christine M.; Byrne, Burke L.

    1998-01-01

    Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis. PMID:9632577

  20. Heme-based sensors in biological systems.

    Rodgers, K R

    1999-04-01

    The past several years have been witness to a staggering rate of advancement in the understanding of how organisms respond to changes in the availability of diatomic molecules that are toxic and/or crucial to survival. Heme-based sensors presently constitute the majority of the proteins known to sense NO, O2 and CO and to initiate the chemistry required to adapt to changes in their availabilities. Knowledge of the three characterized members of this class, soluble guanylate cyclase, FixL and CooA, has grown substantially during the past year. The major advances have resulted from a broad range of approaches to elucidation of both function and mechanism. They include growth in the understanding of the interplay between the heme and protein in soluble guanylate cyclase, as well as alternate means for its stimulation. Insight into the O2-induced structural changes in FixL has been supplied by the single crystal structure of the heme domain of Bradyrhizobium japonicum. Finally, the ligation environment and ligand interchange that facilitates CO sensing by CooA has been established by spectroscopic and mutagenesis techniques.

  1. Measles virus polypeptides in purified virions and in infected cells

    Vainionpaeae, R.; Ziola, B.; Salmi, A.

    1978-01-01

    A wild-type measles virus was radiolabeled during growth in VERO cells and purified by two successive potassium tartrate gradient centrifugations. The virion polypeptide composition was determined by SDS-polyacrylamide gel electrophoresis employing two different buffer systems. Six virus-specific polypeptides were consistently detected. The largest (L) had a molecular weight (MW) of greater than 150,000. The second largest polypeptide, G (MW 79,000), was the only glycoprotein found. The proteins designated polypeptide 2 (MW 66 to 70,000) and nucleocapsid protein or NP (MW 61,000) were phosphorylated. The remaining virus-coded proteins were polypeptide 5 (MW 40,000) and the matrix or M protein (MW 37,000). Measles virions also contained a polypeptide (MW 42,000) thought to be actin due to co-migration with this component of uninfected cells. Analysis of in vitro 3 H-acetic anhydride radiolabeled virions confirmed the presence of these seven polypeptides. Acetic anhydride also labeled a protein designated polypeptide 4 (MW 53,000) which was not consistently radiolabeled in vivo, as well as several other minor proteins believed to be cellular in origin. Synthesis of the six virus-specific structural polypeptides was detected in lysates of infected cells by SDS-polyacrylamide slab gel electrophoresis. Virus specificity of polypeptide 4 could not be confirmed due to the similar MW of several cellular polypeptides. Two non-virion, but virus-specified polypeptides, of MW 38,000 and 18,000 were also detected. Synthesis of the virus structural proteins was in the same proportions as the polypeptides found in virions except for under production of polypeptide G and over production of polypeptide 2. (author)

  2. Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

    Karina E. J. Tripodi

    2011-01-01

    Full Text Available Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi, leishmaniasis (Leishmania spp., and African trypanosomiasis (Trypanosoma brucei. Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents.

  3. Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

    Song, Linyang; Song, Wei; Schipper, Hyman M

    2007-08-01

    The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

  4. Association of dietary and supplemental iron and colorectal cancer in a population-based study.

    Ashmore, Joseph H; Lesko, Samuel M; Miller, Paige E; Cross, Amanda J; Muscat, Joshua E; Zhu, Junjia; Liao, Jason; Harper, Gregory; Lazarus, Philip; Hartman, Terryl J

    2013-11-01

    We evaluated the role of dietary iron, heme iron, and supplemental iron on colorectal cancer (CRC) risk in a population-based case-control study in Pennsylvania, including 1005 incident cases and 1062 controls. Diet was assessed through a modified food frequency questionnaire that included supplement use and a meat-specific module. Cases reported intakes for the year before diagnosis, whereas controls reported intakes for the year before interview. Heme iron intake was calculated using a new heme database developed by the US National Cancer Institute. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. After multivariate adjustment, there were no significant associations between heme iron or total iron intake and CRC incidence. Dietary iron intake was inversely associated with CRC among women (OR Q5 vs. Q1=0.45; 95% CI=0.22-0.92), but not among men. Supplemental iron intake of more than 18 mg/day versus none was positively associated with CRC incidence (OR=2.31; 95% CI=1.48-3.59; P-trendconsumption of more than 18 mg/day of supplemental iron may increase risk for CRC.

  5. Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer.

    Pintscher, Sebastian; Kuleta, Patryk; Cieluch, Ewelina; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2016-03-25

    In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemesb The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on hemebligand mutants of cytochromebc1in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functionalin vivo This reveals that cytochromebc1can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemesbin this cytochrome and in other membranous cytochromesbact as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Characterisation of Anopheles gambiae heme oxygenase and metalloporphyrin feeding suggests a potential role in reproduction.

    Spencer, Christopher S; Yunta, Cristina; de Lima, Glauber Pacelli Gomes; Hemmings, Kay; Lian, Lu-Yun; Lycett, Gareth; Paine, Mark J I

    2018-05-03

    The mosquito Anopheles gambiae is the principal vector for malaria in sub-Saharan Africa. The ability of A. gambiae to transmit malaria is strictly related to blood feeding and digestion, which releases nutrients for oogenesis, as well as substantial amounts of highly toxic free heme. Heme degradation by heme oxygenase (HO) is a common protective mechanism, and a gene for HO exists in the An. gambiae genome HO (AgHO), although it has yet to be functionally examined. Here, we have cloned and expressed An. gambiae HO (AgHO) in E. coli. Purified recombinant AgHO bound hemin stoichiometrically to form a hemin-enzyme complex similar to other HOs, with a K D of 3.9 ± 0.6 μM; comparable to mammalian and bacterial HOs, but 7-fold lower than that of Drosophila melanogaster HO. AgHO also degraded hemin to biliverdin and released CO and iron in the presence of NADPH cytochrome P450 oxidoreductase (CPR). Optimal AgHO activity was observed at 27.5 °C and pH 7.5. To investigate effects of AgHO inhibition, adult female A. gambiae were fed heme analogues Sn- and Zn-protoporphyrins (SnPP and ZnPP), known to inhibit HO. These led to a dose dependent decrease in oviposition. Cu-protoporphyrin (CuPP), which does not inhibit HO had no effect. These results demonstrate that AgHO is a catalytically active HO and that it may play a key role in egg production in mosquitoes. It also presents a potential target for the development of compounds aimed at sterilising mosquitoes for vector control. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Gas-phase spectroscopy of ferric heme-NO complexes

    Wyer, J.A.; Jørgensen, Anders; Pedersen, Bjarke

    2013-01-01

    and significantly blue-shifted compared to ferric heme nitrosyl proteins (maxima between 408 and 422 nm). This is in stark contrast to the Q-band absorption where the protein microenvironment is nearly innocent in perturbing the electronic structure of the porphyrin macrocycle. Photodissociation is primarily...... maxima of heme and its complexes with amino acids and NO. Not so innocent: Weakly bound complexes between ferric heme and NO were synthesised in the gas phase, and their absorption measured from photodissociation yields. Opposite absorption trends in the Soret-band are seen upon NO addition to heme ions...

  8. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.

    Kumar, Devesh; de Visser, Samuël P; Shaik, Sason

    2005-06-08

    The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem. The degradation is initiated by complexation of the heme, then formation of the iron-hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O-O bond homolysis of iron-hydroperoxide. The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H(3)O(+)(H(2)O)(n)(), n = 0-2. The effect of the acid strength is tested using the H(4)N(+)(H(2)O)(2) cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O-O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H(3)O(+)(H(2)O)(2) cluster. The calculated alpha-deuterium secondary kinetic isotope effect is inverse (0.95-0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism.

  10. Nanostructured complexes of polyelectrolytes and charged polypeptides

    Müller, M.; Ouyang, W.; Bohatá, Karolína; Kessler, B.

    2010-01-01

    Roč. 12, Sp. Iss. 9 (2010), B519-B528 ISSN 1438-1656. [Sino-German Symposium on Advanced Biomedical Nanostructures /1./. Jena, 26.10.2009-30.10.2009] Institutional research plan: CEZ:AV0Z40500505 Keywords : situ ATR-FTIR * alpha-helical polypeptides * multilayer films Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.746, year: 2010

  11. Synthetic heme/copper assemblies: toward an understanding of cytochrome c oxidase interactions with dioxygen and nitrogen oxides.

    Hematian, Shabnam; Garcia-Bosch, Isaac; Karlin, Kenneth D

    2015-08-18

    Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper or iron ions or both, those reacting with dioxygen (O2) or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2(-))) or both. As inspiration for this work, we turn to mitochondrial cytochrome c oxidase, which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis, and characterization of new O2 adducts whose further study will add insights into O2 reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO's function, which is intimately tied to cellular O2 balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-Fe(III)-O-Cu(II)(L) products; their properties are discussed. The O-atom is derived from dioxygen, and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a "naked" synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active sites. The other sector of research is focused on heme/Cu assemblies mediating the redox

  12. Relationship between natural and heme-mediated antibody polyreactivity

    Hadzhieva, Maya; Vassilev, Tchavdar [Stephan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113 (Bulgaria); Bayry, Jagadeesh; Kaveri, Srinivas; Lacroix-Desmazes, Sébastien [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France); Dimitrov, Jordan D., E-mail: jordan.dimitrov@crc.jussieu.fr [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France)

    2016-03-25

    Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.

  13. Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

    Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

    2014-01-01

    Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

  14. Iron accumulation with age, oxidative stress and functional decline.

    Jinze Xu

    2008-08-01

    Full Text Available Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength in male Fischer 344 X Brown Norway rats fed ad libitum (AL or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.

  15. Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia.

    Song, Wei; Su, Haixiang; Song, Sisi; Paudel, Hemant K; Schipper, Hyman M

    2006-03-01

    Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions. Copyright 2005 Wiley-Liss, Inc.

  16. Dietary heme-mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon.

    Noortje Ijssennagger

    Full Text Available Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARα target genes. The aim of this study was to investigate the role of PPARα in heme-induced hyperproliferation and hyperplasia. Male PPARα KO and WT mice received a purified diet with or without heme. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARα leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARα in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARα KO mice. We conclude that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.

  17. Role of heme in bromine-induced lung injury

    Lam, Adam; Vetal, Nilam; Matalon, Sadis; Aggarwal, Saurabh

    2016-01-01

    Bromine (Br2) gas inhalation poses an environmental and occupational hazard resulting in high morbidity and mortality. In this review, we underline the acute lung pathology (within 24 hours of exposure) and potential therapeutic interventions that may be utilized to mitigate Br2-induced human toxicity. We will discuss our latest published data, which suggests that an increase in heme-dependent tissue injury underlies the pathogenesis of Br2 toxicity. Our study was based on previous findings that demonstrated that Br2 upregulates the heme-degrading enzyme heme oxygenase-1 (HO-1), which converts toxic heme into billiverdin. Interestingly, following Br2 inhalation, heme levels were indeed elevated in bronchoalveolar lavage fluid, plasma, and whole lung tissue in C57BL/6 mice. High heme levels correlated with increased lung oxidative stress, lung inflammation, respiratory acidosis, lung edema, higher airway resistance, and mortality. However, therapeutic reduction of heme levels, by either scavenging with hemopexin or degradation by HO-1, improved lung function and survival. Therefore, heme attenuation may prove a useful adjuvant therapy to treat patients after Br2 exposure. PMID:27244263

  18. Heme: From quantum spin crossover to oxygen manager of life

    Kepp, Kasper Planeta

    2016-01-01

    The review discusses how the electronic structure of heme explains its central importance to oxygen-based life on Earth. Emphasis is on the chemical bonding of heme, its spin crossover, reversible O2 binding, and O-O bond activation, put in relation to its physiological functions. The review disc...

  19. SirR, a Novel Iron-Dependent Repressor in Staphylococcus epidermidis

    Hill, Philip J.; Cockayne, Alan; Landers, Patrick; Morrissey, Julie A.; Sims, Catriona M.; Williams, Paul

    1998-01-01

    In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation. To gain insights into the molecular basis of iron-dependent gene regulation in the staphylococci, we sequenced the DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to iron stress, for example, production of diphtheria toxin, siderophore, and a heme oxygenase. Upstream of the sitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a polypeptide of approximately 25 kDa with homology to the DtxR family of metal-dependent repressor proteins. This ORF has been designated SirR (staphylococcal iron regulator repressor). Within the sitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start of sitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR protein was overexpressed, purified, and used in DNA mobility shift assays; SirR retarded the migration of a synthetic oligonucleotide based on the Sir box in a metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus, S. epidermidis

  20. Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

    Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

    2014-01-01

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

  1. Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

    Babbitt, Shalon E; San Francisco, Brian; Mendez, Deanna L; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; Bretsnyder, Eric C; Kranz, Robert G

    2014-10-17

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Benzylic oxidation of gemfibrozil-1-O-beta-glucuronide by P450 2C8 leads to heme alkylation and irreversible inhibition.

    Baer, Brian R; DeLisle, Robert Kirk; Allen, Andrew

    2009-07-01

    Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.

  3. Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

    Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Di Rocco, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco

    2009-10-15

    The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

  4. Identification of the receptor scavenging hemopexin-heme complexes

    Hvidberg, Vibeke; Maniecki, Maciej B; Jacobsen, Christian

    2005-01-01

    and is suggested to facilitate cellular heme metabolism. Using a ligand-affinity approach, we purified the human hemopexin-heme receptor and identified it as the low-density lipoprotein receptor-related protein (LRP)/CD91, a receptor expressed in several cell types including macrophages, hepatocytes, neurons......, and syncytiotrophoblasts. Binding experiments, including Biacore analysis, showed that hemopexin-heme complex formation elicits the high receptor affinity. Uptake studies of radio-labeled hemopexin-heme complex in LRP/CD91-expressing COS cells and confocal microscopy of the cellular processing of fluorescent hemopexin......-heme complexes are removed by a receptor-mediated pathway showing striking similarities to the CD163-mediated haptoglobin-hemoglobin clearance in macrophages. Furthermore, the data indicate a hitherto unknown role of LRP/CD91 in inflammation....

  5. Iron(III) complexes of certain tetradentate phenolate ligands as ...

    non-heme iron enzymes, which catalyse the oxidative cleavage of catechols to cis, cis-muconic acids with the incorporation of ... nature of heterocyclic rings of the ligands and the methyl substituents on them regulate the electronic spectral features .... and simple substitution reactions.19,21 The complexes of [H2(L5)] and ...

  6. Differences and Comparisons of the Properties and Reactivities of Iron(III)–hydroperoxo Complexes with Saturated Coordination Sphere

    Faponle, Abayomi S; Quesne, Matthew G; Sastri, Chivukula V; Banse, Frédéric; de Visser, Sam P

    2015-01-01

    Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high-valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O–O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L52). Direct C–O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L52)FeIII(OOH)]2+ should be able to initiate an aromatic hydroxylation process, although a low-energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O–O bond, whereas a heterolytic O–O bond breaking in heme iron(III)–hydroperoxo is found. PMID:25399782

  7. [New drug developments of snake venom polypeptides and progress].

    Fu, Sihai; Feng, Mei; Xiong, Yan

    2017-11-28

    The value of snake venom polypeptides in clinical application has drawn extensive attention, and the development of snake polypeptides into new drugs with anti-tumor, anti-inflammatory, antithrombotic, analgesic or antihypertensive properties has become the recent research hotspot. With the rapid development of molecular biology and biotechnology, the mechanisms of snake venom polypeptides are also gradually clarified. Numerous studies have demonstrated that snake venom polypeptides exert their pharmacological effects by regulating ion channels, cell proliferation, apoptosis, intracellular signaling pathway, and expression of cytokine as well as binding to relevant active sites or receptors.

  8. Isotope aided studies of the bioavailability of iron from common diets from Peru

    Zavaleta, N.; Diaz, A.; Bedregal, P.; Montoya R, E.

    1993-01-01

    The nutritional iron absorption from a typical peruvian diet was studied by the Eakins and Brown method. the food were breakfast: coffee and bread with butter; lunch: vegetable soup rice with cow tripe stew and lemonade; dinner: vegetable soup and bread. The results show that despite low iron content in the meals, which is not enough to meet daily iron requirements absorption was good in lunch possibly by the action of the promoters ascorbic acid (lemonade) and heme iron (cow tripe). Iron absorption in lunch was good and different from dinner and breakfast. We cannot conclude if the low iron absorption from bread is affected by coffee. (authors). 11 refs., 3 tabs

  9. Monomeric Yeast Frataxin is an Iron-Binding Protein

    Cook, J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

    2006-01-01

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

  10. Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model.

    Millot, Sarah; Delaby, Constance; Moulouel, Boualem; Lefebvre, Thibaud; Pilard, Nathalie; Ducrot, Nicolas; Ged, Cécile; Lettéron, Philippe; de Franceschi, Lucia; Deybach, Jean Charles; Beaumont, Carole; Gouya, Laurent; De Verneuil, Hubert; Lyoumi, Saïd; Puy, Hervé; Karim, Zoubida

    2017-02-01

    Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis. Copyright© Ferrata Storti Foundation.

  11. Improvement of bioavailability for iron from vegetarian meals by ascorbic acid

    Sritongkul, N; Tuntawiroon, M; Pleehachinda, R; Suwanik, R [Siriraj Hospital Medical School, Bangkok (Thailand). Section of Nuclear Medicine

    1996-12-01

    There are two kinds of iron in the diet with respect to the mechanism of absorption, heme-iron which is present as haemoglobin or myoglobin in meat and blood products, and, non-heme iron which is the main source of dietary iron. The bioavailability of the non-heme food iron is much lower than heme-iron. Vegetarian diets contain only non-heme iron. Iron intake from vegetarian meals are generally satisfied with the requirements, however, the bioavailabilities for non-heme iron is determined not only by iron content byt also the balance between different dietary factors enhancing and inhibiting iron absorption. The main enhancing factor in vegetarian meals is ascorbic acid in fruits and vegetables, inhibitors are phytate in cereals and grains, and tannins in some spices and vegetables. It has been reported that iron deficiency is one of the common micronutrient problems associated with unplanned vegetarian diets. In the present study the absorption of non-heme iron was measured from 2 vegetarian meals containing considerable amounts of phytate and tannin. The extrinsic tay method ({sup 59}Fe/ {sup 55}Fe) was used to labelled the non-heme iron. The mean percentage absorption of non-heme iron from both meals was slightly different due to differences in their dietary contents. Their initial percentages iron absorption were apparent low (3.5% and 4.1%), however, the absorption progressively increased with increase in the level of ascorbic acid, 2-3 times with 100 mg and 4-5 times with 200 mg of ascorbic acid. The average amount of iron absorbed per 2000 kcal increased from 0.37 mg to 0.86 mg and 1.45 mg with the addition of 100 mg and 200 mg ascorbic acid respectively (p < 0.001). Considering the limited caloric intakes and the iron content in the meals, the amount of iron absorbed from vegetarian meals without ascorbic acid was not able to meet certain requirements for children, adolescents and menstruating women. The minimal requirement for dietary iron needed to be

  12. [Update on the biology of heme synthesis in erythroid cells].

    Fujiwara, Tohru; Harigae, Hideo

    2015-02-01

    Heme is a prosthetic group of hemoproteins playing important roles in oxygen transport, detoxification, circadian rhythm, microRNA processing, regulation of transcription, and translation. The majority of heme (-85%) is synthesized in red blood cells mainly for hemoglobin production, whereas hepatocytes account for most of the rest, functioning primarily in the synthesis of cytochrome P450 enzymes and mitochondrial respiratory enzymes. Thus, failure of heme biosynthesis causes severe inherited or acquired disorders in humans, including porphyria and sideroblastic anemia. The heme biosynthetic pathway is composed of eight enzymes that work in either mitochondria or the cytoplasm, which have been extensively researched and frequently reviewed. On the other hand, the mechanisms governing transport and intracellular trafficking of heme intermediates, as well as their potential links to human diseases, are poorly understood. Herein, we focus on recent understanding of the heme biosynthetic pathway and on human disorders due to defective heme synthesis in erythroid cells, such as X-linked sideroblastic anemia and erythropoietic protoporphyria.

  13. Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

    Lara, F.A.; Sant'Anna, C.; Lemos, D.; Laranja, G.A.T.; Coelho, M.G.P.; Reis Salles, I.; Michel, A.; Oliveira, P.L.; Cunha-e-Silva, N.; Salmon, D.; Paes, M.C.

    2007-01-01

    Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane

  14. Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

    Buisson, Christophe; Daou, Nadine; Kallassy, Mireille; Lereclus, Didier; Arosio, Paolo; Bou-Abdallah, Fadi; Nielsen Le Roux, Christina

    2014-01-01

    In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis. PMID:24550730

  15. Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization.

    Diego Segond

    2014-02-01

    Full Text Available In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.

  16. Effect of irradiation and storage in the iron availability in lamb meat treated with different diets; Efeito da irradiacao e do armazenamento na disponibilidade de ferro em carne de cordeiro tratado com diferentes dietas

    Souza, Adriana Regia Marques de; Arthur, Valter [Centro de Energia Nuclear na Agricultura (CENA/USP), Piracicaba, SP (Brazil). Lab. de Irradiacao de Alimentos e Radioentomologia]. E-mail: acornel@cena.usp.br; arthur@cena.usp.br; Canniatti-Brazaca, Solange Guidolin [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil). Dept. de Agroindustria, Alimentos e Nutricao]. E-mail: sgcbraza@esalq.usp.br; Couto, Meylene Aparecida Luzia [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil)]. E-mail: malcouto@esalq.usp.br

    2008-10-15

    Irradiation is an efficient method to increase the microbiological safety and to maintain the nutrients such as iron in the meat. The best absorption form, heme iron, should be preserved in order to increase the nutritional quality of stored meat. The diet can alter the nutrients contents and form in the meat. The iron is provided from the diet and it is an essential element for the metabolic processes such as oxygen transport, oxidative metabolism, and cellular growth. Meat lamb samples treated with different diets (it controls, TAC1, TAC2 and sorghum) were wrapped to vacuous, and irradiated in the doses 0, 2 and 4 kGy and stored at 4 deg C during 15 days. The values of total iron and heme iron were measured at 0 and 15 days of storage. The storage reduced the content of total iron (18.36 for 14.28 mg.100 g{sup -1}) and heme iron (13.78 for 10.52 mg.100 g{sup -1}). The diets affected the levels of total and heme iron of the meat, and the sorghum diet was the one that presented the larger content. The dose of 2 kGy was the one that affected the iron the most independently of the storage time. It was verified that the amounts of total and heme iron varied according to the storage time, irradiation doses, and lamb diets. (author)

  17. [Iron absorption of the habitual diet in a population of low socioeconomic level].

    Morón, C; Kremenchuzky, S; Passamai, M I; D'Andrea de Rivero, S; Pérez de Galíndez, G; Gerschcovich, C

    1985-06-01

    Iron absorption using the extrinsic double-tag method was determined in the habitual diet consumed by a group of 32 volunteers of both sexes, pertaining to the low socioeconomic strata. The diet was made up of bread, spaghetti, vegetables and meat, totalling 2,022 kcal, 65.0 g protein, 17.57 mg iron, and 28.75 mg ascorbic acid. According to our findings, men were found to be neither anemic nor iron-deficient. Among the women, however, 4.8% had anemia and 57.1% suffered from iron deficiency. The non-heme iron absorption was very low: 1.35% at breakfast, 3.29% at lunch, and 3.82% at dinner. Among those subjects found to be normal, the absorption was half the above figures, whereas among those with iron deficiency it was threefold, the differences being highly significant. The absorption of heme-iron for lunch and dinner was 17.53%. The iron deficient group had an absorption value four times greater than the normal group, the differences also being highly significant. The daily availability of non-heme, heme and total iron was 0.44, 1.13 and 1.57 mg, respectively. In the subjects who formed the normal group, total iron available was 1.14 mg, barely covering a man's daily requirements, but not those of a woman. In the iron-deficient group, it was 4.31 mg, that is, four times greater than in the normal group; while this value improves the balance, it does not prevent deficiency in women, with great blood losses. Bearing these results in mind, it is suggested that measures tending to improve dietary iron content and bio-availability, be enforced.

  18. Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

    Nagao, Satoshi; Hirai, Yueki; Kawano, Shin; Imai, Kiyohiro; Suzuki, Akihiro; Yamamoto, Yasuhiko

    2007-01-01

    A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12, 18-trimethyl-porphyrin-atoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using 19 F NMR and the O 2 binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in α- and β- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O 2 affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O 2 affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O 2 affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A

  19. Measles virus-specified polypeptides in infected cells

    Vainionpaepae, R.

    1979-01-01

    The synthesis of wild-type measles virus-specified polypeptides in Vero cells in pulse-chase experiments, in cells with synchronized protein synthesis by high salt concentration, and in the presence of proteolytic enzyme inhibitors was analyzed by polyacrylamide slab-gel electrophoresis. Six major (L, G, 2, NP, 5 and M) structural polypeptides were identified in infected cells. The results of pulse-chase experiments suggested that most of the structural polypeptides were synthesized at their final length. Polypeptide M was found to be sensitive to trypsin. In TLCK-treated cells its molecular weight was about 1000-2000 daltons higher than in untreated cells. A minor virus-specific polypeptide with a molecular weight of about 23,000 was found as a very faint and diffuse band. In addition, three nonstructural polypeptides with molecular weights of 65,000, 38,000 and 18,000 were also detected. The experiments with proteolytic enzyme inhibitors and with synchronized protein synthesis suggested that the polypeptide with a molecular weight of 65,000 might be a precursor of the structural polypeptide 5. (author)

  20. Caffeine-water-polypeptide interaction in aqueous solution

    Ghabi, Habib; Dhahbi, Mahmoud

    1999-04-01

    The interaction of caffeine monomer with the synthetic polypeptides polyasparagine (pAg) and polyaspartic acid (pAsp) was studied by UV spectrophotometry. The results show that different types of interactions are possible depending on the nature of polypeptide. The form of the complex was discussed.

  1. Cloning and characterization of a heme oxygenase-2 gene from alfalfa (Medicago sativa L.).

    Fu, Guang-Qing; Jin, Qi-Jiang; Lin, Yu-Ting; Feng, Jian-Fei; Nie, Li; Shen, Wen-Biao; Zheng, Tian-Qing

    2011-11-01

    Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H(2)O(2) (especially) treatment, respectively.

  2. Heme oxygenase-1: A new druggable target in the management of chronic and acute myeloid leukemia.

    Salerno, Loredana; Romeo, Giuseppe; Modica, Maria N; Amata, Emanuele; Sorrenti, Valeria; Barbagallo, Ignazio; Pittalà, Valeria

    2017-12-15

    Heme oxygenase-1 (HO-1) is the enzyme catalyzing the rate-limiting oxidative degradation of cellular heme into free iron, carbon monoxide (CO), and biliverdin, which is then rapidly converted into bilirubin. By means of these catabolic end-products and by removal of pro-oxidant heme, HO-1 exerts antioxidant, antiapoptotic, and immune-modulating effects, leading to overall cytoprotective and beneficial functions in mammalian cells. Therefore, HO-1 is considered a survival molecule in various stress-related conditions. By contrast, growing evidence suggests that HO-1 is a survival-enhancing molecule also in various solid and blood cancers, such as various types of leukemia, promoting carcinogenesis, tumor progression, and chemo-resistance. Among leukemias, chronic myeloid leukemia (CML) is currently therapeutically well treated with tyrosine kinase inhibitors (TKIs) such as Imatinib (IM) and its congeners; nevertheless, resistance to all kinds of current drugs persist in a number of patients. Moreover, treatment outcomes for acute myeloid leukemia (AML) remain unsatisfactory, despite progress in chemotherapy and hematopoietic stem cell transplantation. Therefore, identification of new eligible targets that may improve leukemias therapy is of general interest. Several recent papers prove that inhibition of HO-1 through HO-1 inhibitors as well as modulation of other pathways involving HO-1 by a number of different new or known molecules, are critical for leukemia treatment. This review summarizes the current understanding of the pro-tumorigenic role of HO-1 and its potential as a molecular target for the treatment of leukemias. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. A study of the mechanism of action of pyridoxal isonicotinoyl hydrazone at the cellular level using reticulocytes loaded with non-heme 59Fe

    Huang, A.R.; Ponka, P.; McGill Univ., Montreal, Quebec; Jewish General Hospital, Montreal, Quebec

    1983-01-01

    Pyridoxal isonicotinoyl hydrazone (PIH) has recently been identified as a new iron chelating agent with a high degree of iron mobilizing activity in vitro and in vivo which makes this compound a candidate drug in the treatment of iron overload. This study was undertaken to elucidate the mechanism of action of the iron mobilizing activity of PIH at the cellular level. An in vitro system of rabbit reticulocytes with a high level of non-heme 59 Fe was used as a model of iron overload. The effects of various biochemical and physiological manoeuvers on the mobilization of 59 Fe by PIH from the cells were studied. The fate of [ 14 C]-PIH in the in vitro system was also studied. Studies were also carried out using a crude mitochondrial fraction. (orig./AJ)

  4. Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.

    Ogura, Hiroshi; Evans, John P; de Montellano, Paul R Ortiz; La Mar, Gerd N

    2008-01-08

    The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.

  5. Rearrangement of the distal pocket accompanying E7 His → Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket

    Yu, L.P.; La Mar, G.N.; Mizukami, H.

    1990-01-01

    Two-dimensional 1 H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO 2 ). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO 2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The CζH proton of the Phe CD4 was found to move toward the iron of the heme by ∼4 angstrom relative to the position in sperm whale MbCO, requiring minimally a 3-angstrom movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His → Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties

  6. Chirality-selected phase behaviour in ionic polypeptide complexes

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

    2015-01-01

    Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation. PMID:25586861

  7. Iron and Zinc Complexes of Bulky Bis-Imidazole Ligands : Enzyme Mimicry and Ligand-Centered Redox Activity

    Folkertsma, E.

    2016-01-01

    The research described in this thesis is directed to the development of cheap and non-toxic iron-based homogeneous catalysts, using enzyme models and redox non-innocent ligands. Inspired by nature, the first approach focuses on the synthesis of structural models of the active site of non-heme iron

  8. Heme synthesis in the lead-intoxicated mouse embryo

    Gerber, G B; Maes, J

    1978-02-01

    Incorporation of /sup 55/Fe and of (/sup 14/C) glycine was studied in control embryos and mothers and in those which had received lead in the diet from day 7 of pregnancy. Incorporation of Fe into heme of embryonic liver which increases markedly for controls on day 17 of pregnancy was depressed greatly and showed no such increase in lead-intoxicated embryos. These embryos were retarded in growth but had normal heme concentrations in body and liver. Incorporation of glycine into embryonic heme and proteins was not affected. Data on incorporation in the mothers are also presented. It is thought that the impaired synthesis of heme in lead-intoxicated embryos limits their body growth during the late phase of pregnancy.

  9. Effect of a heme oxygenase-1 inducer on NADPH oxidase ...

    Effect of a heme oxygenase-1 inducer on NADPH oxidase expression in ... and immunohistochemistry of hepatic NOX1 and NOX4 were investigated in week 4. ... (HO-1 inhibitor) administration caused upregulation of NOX gene expression ...

  10. Heme and blood-feeding parasites: friends or foes?

    Glanfield Amber

    2010-11-01

    Full Text Available Abstract Hemoparasites, like malaria and schistosomes, are constantly faced with the challenges of storing and detoxifying large quantities of heme, released from their catabolism of host erythrocytes. Heme is an essential prosthetic group that forms the reactive core of numerous hemoproteins with diverse biological functions. However, due to its reactive nature, it is also a potentially toxic molecule. Thus, the acquisition and detoxification of heme is likely to be paramount for the survival and establishment of parasitism. Understanding the underlying mechanism involved in this interaction could possibly provide potential novel targets for drug and vaccine development, and disease treatment. However, there remains a wide gap in our understanding of these mechanisms. This review summarizes the biological importance of heme for hemoparasite, and the adaptations utilized in its sequestration and detoxification.

  11. Heme and blood-feeding parasites: friends or foes?

    2010-01-01

    Hemoparasites, like malaria and schistosomes, are constantly faced with the challenges of storing and detoxifying large quantities of heme, released from their catabolism of host erythrocytes. Heme is an essential prosthetic group that forms the reactive core of numerous hemoproteins with diverse biological functions. However, due to its reactive nature, it is also a potentially toxic molecule. Thus, the acquisition and detoxification of heme is likely to be paramount for the survival and establishment of parasitism. Understanding the underlying mechanism involved in this interaction could possibly provide potential novel targets for drug and vaccine development, and disease treatment. However, there remains a wide gap in our understanding of these mechanisms. This review summarizes the biological importance of heme for hemoparasite, and the adaptations utilized in its sequestration and detoxification. PMID:21087517

  12. Wiring of heme enzymes by methylene-blue labeled dendrimers

    Álvarez-Martos, Isabel; Shahdost-fard, Faezeh; Ferapontova, Elena

    2017-01-01

    Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme- and moli......Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme......- and molibdopterin-containing sulfite oxidase (SOx), wired to gold by the methylene blue (MB)-labeled polyamidoamine (PAMAM) dendrimers. The enzymes’ electrochemical transformation and bioelectrocatalytic function could be followed at both unlabeled and MB-labeled dendrimer-modified electrodes with the formal redox......, optimization of bioelectrocatalysis of complex intermembrane and, possibly, membrane enzymes....

  13. Immunolocalization of heme oxygenase-1 in periodontal diseases

    G Gayathri

    2014-01-01

    Conclusion: The results of our study is an increasing evidence of involvement of antioxidant enzymes like heme oxygenase-1 in periodontal inflammation and their implication for treatment of chronic periodontitis.

  14. Fibrillar dimer formation of islet amyloid polypeptides

    Chiu, Chi-cheng [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); de Pablo, Juan J. [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  15. Tissue polypeptide antigen activity in cerebrospinal fluid

    Bach, F; Söletormos, Georg; Dombernowsky, P

    1991-01-01

    Tissue polypeptide antigen (TPpA) in the cerebrospinal fluid (CSF) was measured in 59 consecutive breast cancer patients with suspected central nervous system (CNS) metastases. Subsequently, we determined that 13 patients had parenchymal brain metastases, 10 had leptomeningeal carcinomatosis......, and 36 had no CNS involvement. The concentration of TPpA, which is a nonspecific marker for cell proliferation, was significantly higher in patients with CNS metastases than in those without it (P less than .0001; Mann-Whitney test). A tentative cutoff value for CNS metastases was set at 95 U/L TPp...... metastases, no correlation was found between TPpA activity in corresponding CSF and blood samples (correlation coefficient, Spearman's rho = .4; P greater than .1). In three patients treated for leptomeningeal carcinomatosis, the measurements of CSF TPpA showed correlation between the presence of tumor cells...

  16. Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy.

    Loewen, Peter C; Villanueva, Jacylyn; Switala, Jacek; Donald, Lynda J; Ivancich, Anabella

    2015-05-01

    Heme-containing catalases and catalase-peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase-peroxidase led us to investigate the enzyme for comparison with other catalase-peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat  = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat  = 20 s(-1) ) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65-1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the Fe(III) high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. © 2015 Wiley Periodicals, Inc.

  17. Smart systems related to polypeptide sequences

    Lourdes Franco

    2016-03-01

    Full Text Available Increasing interest for the application of polypeptide-based smart systems in the biomedical field has developed due to the advantages given by the peptidic sequence. This is due to characteristics of these systems, which include: biocompatibility, potential control of degradation, capability to provide a rich repertoire of biologically specific interactions, feasibility to self-assemble, possibility to combine different functionalities, and capability to give an environmentally responsive behavior. Recently, applications concerning the development of these systems are receiving greater attention since a targeted and programmable release of drugs (e.g. anti-cancer agents can be achieved. Block copolymers are discussed due to their capability to render differently assembled architectures. Hybrid systems based on silica nanoparticles are also discussed. In both cases, the selected systems must be able to undergo fast changes in properties like solubility, shape, and dissociation or swelling capabilities. This review is structured in different chapters which explain the most recent advances on smart systems depending on the stimuli to which they are sensitive. Amphiphilic block copolymers based on polyanionic or polycationic peptides are, for example, typically employed for obtaining pH-responsive systems. Elastin-like polypeptides are usually used as thermoresponsive polymers, but performance can be increased by using techniques which utilize layer-by-layer electrostatic self-assembly. This approach offers a great potential to create multilayered systems, including nanocapsules, with different functionality. Recent strategies developed to get redox-, magnetic-, ultrasound-, enzyme-, light- and electric-responsive systems are extensively discussed. Finally, some indications concerning the possibilities of multi-responsive systems are discussed.

  18. Heme and menaquinone induced electron transport in lactic acid bacteria

    Brooijmans, Rob; Smit, Bart; Santos, Filipe; van Riel, Jan; de Vos, Willem M; Hugenholtz, Jeroen

    2009-01-01

    Abstract Background For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Results Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacill...

  19. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  20. Syntheses of carbon-13 labeled protoporphyrin-IX for spectroscopic studies of heme proteins

    Fujinari, E.M.

    1985-01-01

    The development of various methodologies for synthesis of selectively tailored protoporphyrin-IX dimethyl ester are presented. The iron(II) complex of protoporphyrin-IX is the heme, the prosthetic group for Hb, Mb, cytochromes and peroxidases. The significance of this research is to provide direct means to establish definitive carbon-13 NMR assignments of heme proteins in order to study not only the structure-function relationships, but also protein dynamics of these vital systems. Carbon-13 labeling at the beta-vinyl position was first achieved by ozonolysis of protoporphyrin-IX dimethyl ester. Column LC method were used to first isolate 2,4-diformyldeuteroporphyrin-IX dimethyl ester. Concomitantly, monofomyl-monovinyl porphyrins were obtained as a mixture of two isomers. This mixture was separated by MPLC or prep HPLC to afford the isomerically pure products, Spirographis porphyrin dimethyl ester and Iso-Spirographis porphyrin dimethyl ester. A Wittig reaction to each of these porphyrins with 13 C-methyltriphenylphosphonium iodide gave 2,4-bis[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, 2-[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, and the 4-[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, respectively

  1. TMEM14C is required for erythroid mitochondrial heme metabolism.

    Yien, Yvette Y; Robledo, Raymond F; Schultz, Iman J; Takahashi-Makise, Naoko; Gwynn, Babette; Bauer, Daniel E; Dass, Abhishek; Yi, Gloria; Li, Liangtao; Hildick-Smith, Gordon J; Cooney, Jeffrey D; Pierce, Eric L; Mohler, Kyla; Dailey, Tamara A; Miyata, Non; Kingsley, Paul D; Garone, Caterina; Hattangadi, Shilpa M; Huang, Hui; Chen, Wen; Keenan, Ellen M; Shah, Dhvanit I; Schlaeger, Thorsten M; DiMauro, Salvatore; Orkin, Stuart H; Cantor, Alan B; Palis, James; Koehler, Carla M; Lodish, Harvey F; Kaplan, Jerry; Ward, Diane M; Dailey, Harry A; Phillips, John D; Peters, Luanne L; Paw, Barry H

    2014-10-01

    The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

  2. Inflammation and ER Stress Downregulate BDH2 Expression and Dysregulate Intracellular Iron in Macrophages

    Susu M. Zughaier

    2014-01-01

    Full Text Available Macrophages play a very important role in host defense and in iron homeostasis by engulfing senescent red blood cells and recycling iron. Hepcidin is the master iron regulating hormone that limits dietary iron absorption from the gut and limits iron egress from macrophages. Upon infection macrophages retain iron to limit its bioavailability which limits bacterial growth. Recently, a short chain butyrate dehydrogenase type 2 (BDH2 protein was reported to contain an iron responsive element and to mediate cellular iron trafficking by catalyzing the synthesis of the mammalian siderophore that binds labile iron; therefore, BDH2 plays a crucial role in intracellular iron homeostasis. However, BDH2 expression and regulation in macrophages have not yet been described. Here we show that LPS-induced inflammation combined with ER stress led to massive BDH2 downregulation, increased the expression of ER stress markers, upregulated hepcidin expression, downregulated ferroportin expression, caused iron retention in macrophages, and dysregulated cytokine release from macrophages. We also show that ER stress combined with inflammation synergistically upregulated the expression of the iron carrier protein NGAL and the stress-inducible heme degrading enzyme heme oxygenase-1 (HO-1 leading to iron liberation. This is the first report to show that inflammation and ER stress downregulate the expression of BDH2 in human THP-1 macrophages.

  3. Heme-Induced Biomarkers Associated with Red Meat Promotion of colon Cancer Are Not Modulated by the Intake of Nitrite

    Chenni, Fatima Z; Taché, Sylviane; Naud, Nathalie; Guéraud, Françoise; Hobbs, Ditte A; Kunhle, Gunter G C; Pierre, Fabrice H; Corpet, Denis E

    2013-01-01

    Red and processed meat consumption is associated with the risk of colorectal cancer. Three hypotheses are proposed to explain this association, via heme/alcenal, heterocyclic amines or N-nitroso compounds. Rats have often been used to study these hypotheses, but the lack of enterosalivary cycle of nitrate in rats casts doubt on the relevance of this animal model to predict nitroso- and heme-associated human colon carcinogenesis. The present study was thus designed to clarify whether a nitrite intake that mimics the enterosalivary cycle can modulate heme-induced nitrosation and fat peroxidation. This study shows that, in contrast with the starting hypothesis, salivary nitrite did not change the effect of hemoglobin on biochemical markers linked to colon carcinogenesis, notably lipid peroxidation and cytotoxic activity in the colon of rat. However, ingested sodium nitrite increased fecal nitroso-compounds level, but their fecal concentration and their nature (iron-nitrosyl) would not be associated with an increased risk of cancer. The rat model could thus be relevant to study the effect of red meat on colon carcinogenesis in spite of the lack of nitrite recycling in rat’s saliva. PMID:23441609

  4. Comparison of the crystal structure and function to wild-type and His25Ala mutant human heme oxygenase-1.

    Zhou, Wen-Pu; Zhong, Wen-Wei; Zhang, Xue-Hong; Ding, Jian-Ping; Zhang, Zi-Li; Xia, Zhen-Wei

    2009-03-01

    Human heme oxygenase-1 (hHO-1) is a rate-limiting enzyme in heme metabolism. It regulates serum bilirubin level. Site-directed mutagenesis studies indicate that the proximal residue histidine 25 (His25) plays a key role in hHO-1 activity. A highly purified hHO-1 His25Ala mutant was generated and crystallized with a new expression system. The crystal structure of the mutant was determined by X-ray diffraction technology and molecular replacement at the resolution of 2.8 A, and the model of hHO-1 His25Ala mutant was refined. The final crystallographic and free R factors were 0.245 and 0.283, respectively. The standard bond length deviation was 0.007 A, and the standard bond angle deviation was 1.3 degrees . The mutation of His25 to Ala led to an empty pocket underneath the ferric ion in the heme, leading to loss of binding iron ligand. Although this did not cause an overall structural change, the enzymatic activity of the mutant hHO-1 was reduced by 90%. By supplementing imidazole, the HO-1 activity was restored approximately 90% to its normal level. These data suggest that Ala25 remains unchanged in the structure compared to His25, but the important catalytic function of hHO-1 is lost. Thus, it appears that His25 is a crucial residue for proper hHO-1 catalysis.

  5. Modelling Systemic Iron Regulation during Dietary Iron Overload and Acute Inflammation: Role of Hepcidin-Independent Mechanisms.

    Enculescu, Mihaela; Metzendorf, Christoph; Sparla, Richard; Hahnel, Maximilian; Bode, Johannes; Muckenthaler, Martina U; Legewie, Stefan

    2017-01-01

    Systemic iron levels must be maintained in physiological concentrations to prevent diseases associated with iron deficiency or iron overload. A key role in this process plays ferroportin, the only known mammalian transmembrane iron exporter, which releases iron from duodenal enterocytes, hepatocytes, or iron-recycling macrophages into the blood stream. Ferroportin expression is tightly controlled by transcriptional and post-transcriptional mechanisms in response to hypoxia, iron deficiency, heme iron and inflammatory cues by cell-autonomous and systemic mechanisms. At the systemic level, the iron-regulatory hormone hepcidin is released from the liver in response to these cues, binds to ferroportin and triggers its degradation. The relative importance of individual ferroportin control mechanisms and their interplay at the systemic level is incompletely understood. Here, we built a mathematical model of systemic iron regulation. It incorporates the dynamics of organ iron pools as well as regulation by the hepcidin/ferroportin system. We calibrated and validated the model with time-resolved measurements of iron responses in mice challenged with dietary iron overload and/or inflammation. The model demonstrates that inflammation mainly reduces the amount of iron in the blood stream by reducing intracellular ferroportin transcription, and not by hepcidin-dependent ferroportin protein destabilization. In contrast, ferroportin regulation by hepcidin is the predominant mechanism of iron homeostasis in response to changing iron diets for a big range of dietary iron contents. The model further reveals that additional homeostasis mechanisms must be taken into account at very high dietary iron levels, including the saturation of intestinal uptake of nutritional iron and the uptake of circulating, non-transferrin-bound iron, into liver. Taken together, our model quantitatively describes systemic iron metabolism and generated experimentally testable predictions for additional

  6. HemeBIND: a novel method for heme binding residue prediction by combining structural and sequence information

    Hu Jianjun

    2011-05-01

    Full Text Available Abstract Background Accurate prediction of binding residues involved in the interactions between proteins and small ligands is one of the major challenges in structural bioinformatics. Heme is an essential and commonly used ligand that plays critical roles in electron transfer, catalysis, signal transduction and gene expression. Although much effort has been devoted to the development of various generic algorithms for ligand binding site prediction over the last decade, no algorithm has been specifically designed to complement experimental techniques for identification of heme binding residues. Consequently, an urgent need is to develop a computational method for recognizing these important residues. Results Here we introduced an efficient algorithm HemeBIND for predicting heme binding residues by integrating structural and sequence information. We systematically investigated the characteristics of binding interfaces based on a non-redundant dataset of heme-protein complexes. It was found that several sequence and structural attributes such as evolutionary conservation, solvent accessibility, depth and protrusion clearly illustrate the differences between heme binding and non-binding residues. These features can then be separately used or combined to build the structure-based classifiers using support vector machine (SVM. The results showed that the information contained in these features is largely complementary and their combination achieved the best performance. To further improve the performance, an attempt has been made to develop a post-processing procedure to reduce the number of false positives. In addition, we built a sequence-based classifier based on SVM and sequence profile as an alternative when only sequence information can be used. Finally, we employed a voting method to combine the outputs of structure-based and sequence-based classifiers, which demonstrated remarkably better performance than the individual classifier alone

  7. Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

    Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

    2008-02-26

    Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

  8. Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin

    Benvenisti-Zarom Luna

    2004-09-01

    Full Text Available Abstract Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed

  9. Genetic/metabolic effect of iron metabolism and rare anemias

    Clara Camaschella

    2013-03-01

    Full Text Available Advances in iron metabolism have allowed a novel classification of iron disorders and to identify previously unknown diseases. These disorders include genetic iron overload (hemochromatosis and inherited iron-related anemias, in some cases accompanied by iron overload. Rare inherited anemias may affect the hepcidin pathway, iron absorption, transport, utilization and recycling. Among the genetic iron-related anemias the most common form is likely the iron-refractory iron-deficiency anemia (IRIDA, due to mutations of the hepcidin inhibitor TMPRSS6 encoding the serine protease matriptase-2. IRIDA is characterized by hepcidin up-regulation, decrease iron absorption and macrophage recycling and by microcytic- hypochromic anemia, unresponsive to oral iron. High serum hepcidin levels may suggest the diagnosis, which requires demonstrating the causal TMPRSS6 mutations by gene sequencing. Other rare microcytic hypochromic anemias associated with defects of iron transport-uptake are the rare hypotransferrinemia, and DMT1 and STEAP3 mutations. The degree of anemia is variable and accompanied by secondary iron overload even in the absence of blood transfusions. This is due to the iron-deficient or expanded erythropoiesis that inhibits hepcidin transcription, increases iron absorption, through the erythroid regulator, as in untransfused beta-thalassemia. Sideroblastic anemias are due to decreased mitochondrial iron utilization for heme or sulfur cluster synthesis. Their diagnosis requires demonstrating ringed sideroblasts by Perl’s staining of the bone marrow smears. The commonest X-linked form is due to deltaamino- levulinic-synthase-2-acid (ALAS2 mutations. The recessive, more severe form, affects SLC25A38, which encodes a potential mitochondrial importer of glycine, an amino acid essential for ALA synthesis and thus results in heme deficiency. Two disorders affect iron/sulfur cluster biogenesis: deficiency of the ATP-binding cassette B7 (ABCB7 causes X

  10. Iron Absorption from Two Milk Formulas Fortified with Iron Sulfate Stabilized with Maltodextrin and Citric Acid

    Fernando Pizarro

    2015-10-01

    Full Text Available Background: Fortification of milk formulas with iron is a strategy widely used, but the absorption of non-heme iron is low. The purpose of this study was to measure the bioavailability of two iron fortified milk formulas designed to cover toddlers´ nutritional needs. These milks were fortified with iron sulfate stabilized with maltodextrin and citric acid. Methods: 15 women (33–47 years old participated in study. They received on different days, after an overnight fast, 200 mL of Formula A; 200 mL of Formula B; 30 mL of a solution of iron and ascorbic acid as reference dose and 200 mL of full fat cow’s milk fortified with iron as ferrous sulfate. Milk formulas and reference dose were labeled with radioisotopes 59Fe or 55Fe, and the absorption of iron measured by erythrocyte incorporation of radioactive Fe. Results: The geometric mean iron absorption corrected to 40% of the reference dose was 20.6% for Formula A and 20.7% for Formula B, versus 7.5% of iron fortified cow’s milk (p < 0.001. The post hoc Sheffé indeed differences between the milk formulas and the cow’s milk (p < 0.001. Conclusion: Formulas A and B contain highly bioavailable iron, which contributes to covering toddlers´ requirements of this micronutrient.

  11. Pharmacological Induction of Heme Oxygenase-1 Impairs Nuclear Accumulation of Herpes Simplex Virus Capsids upon Infection

    Francisco J. Ibáñez

    2017-10-01

    Full Text Available Heme oxygenase-1 (HO-1 is an inducible enzyme that is expressed in response to physical and chemical stresses, such as ultraviolet radiation, hyperthermia, hypoxia, reactive oxygen species (ROS, as well as cytokines, among others. Its activity can be positively modulated by cobalt protoporphyrin (CoPP and negatively by tin protoporphirin (SnPP. Once induced, HO-1 degrades iron-containing heme into ferrous iron (Fe2+, carbon monoxide (CO and biliverdin. Importantly, numerous products of HO-1 are cytoprotective with anti-apoptotic, anti-oxidant, anti-inflammatory, and anti-cancer effects. The products of HO-1 also display antiviral properties against several viruses, such as the human immunodeficiency virus (HIV, influenza, hepatitis B, hepatitis C, and Ebola virus. Here, we sought to assess the effect of modulating HO-1 activity over herpes simplex virus type 2 (HSV-2 infection in epithelial cells and neurons. There are no vaccines against HSV-2 and treatment options are scarce in the immunosuppressed, in which drug-resistant variants emerge. By using HSV strains that encode structural and non-structural forms of the green fluorescent protein (GFP, we found that pharmacological induction of HO-1 activity with CoPP significantly decreases virus plaque formation and the expression of virus-encoded genes in epithelial cells as determined by flow cytometry and western blot assays. CoPP treatment did not affect virus binding to the cell surface or entry into the cytoplasm, but rather downstream events in the virus infection cycle. Furthermore, we observed that treating cells with a CO-releasing molecule (CORM-2 recapitulated some of the anti-HSV effects elicited by CoPP. Taken together, these findings indicate that HO-1 activity interferes with the replication cycle of HSV and that its antiviral effects can be recapitulated by CO.

  12. Considerations on the mechanism of action of artemisinin antimalarials: part 1--the 'carbon radical' and 'heme' hypotheses.

    Haynes, Richard K; Cheu, Kwan-Wing; N'Da, David; Coghi, Paolo; Monti, Diego

    2013-08-01

    The isolation of artemisinin from the traditional medicinal herb qīng hāo (Artemisia annua), its characterization as a peroxide and preparation of the derivatives dihydroartemisinin, artemether and artesunate in the 1970s and 1980s by Chinese scientists under the umbrella of Project 523 collectively represents one of the great events in medicine in the latter third of the 20(th) Century. Artemisinins have become the most important component of chemotherapy of malaria: although used initially in monotherapy, they are now used in combination therapies or ACTs with longer half-life quinolines or arylmethanols. Nevertheless, the recent emergence of artemisinin-tolerant strains of the malaria parasite as reflected in increased clearance times of parasitaemia in patients treated with ACTs represents the greatest threat to control of malaria since resistance to chloroquine was first reported over 55 years ago. Importantly, the event brings into sharp focus the realization that relatively little is precisely understood, as opposed to widely assumed, for the mechanism of drug action of artemisinins and their synthetic peroxide analogues. Thus, we review here their antimalarial activities, the use of artemisinins in combination therapies, drug-drug interactions with the quinolines and arylmethanols, and metabolism of the artemisinins and synthetic peroxides. The mechanism of action of quinolines and arylmethanols, in particular their ability to induce redistribution of heme into the parasite cytosol, is also highlighted. This collective information is then used as a counterpoint to screen the validity of two of the prevailing hypotheses of drug action of artemisinins and synthetic peroxides, namely i. 'the C-radical hypothesis' wherein the peroxide undergoes 'bioactivation' by ferrous iron to generate C-radicals that are held to be the cytotoxic agents and ii. the 'heme hypothesis' wherein ferrous heme may generate either the same type of 'cytotoxic' C-radical, or the

  13. The influence of high iron diet on rat lung manganese absorption

    Thompson, Khristy; Molina, Ramon; Donaghey, Thomas; Brain, Joseph D.; Wessling-Resnick, Marianne

    2006-01-01

    Individuals chronically exposed to manganese are at high risk for neurotoxic effects of this metal. A primary route of exposure is through respiration, although little is known about pulmonary uptake of metals or factors that modify this process. High dietary iron levels inversely affect intestinal uptake of manganese, and a major goal of this study was to determine if dietary iron loading could increase lung non-heme iron levels and alter manganese absorption. Rats were fed a high iron (1% carbonyl iron) or control diet for 4 weeks. Lung non-heme iron levels increased ∼2-fold in rats fed the high iron diet. To determine if iron-loading affected manganese uptake, 54 Mn was administered by intratracheal (it) instillation or intravenous (iv) injection for pharmacokinetic studies. 54 Mn absorption from the lungs to the blood was lower in it-instilled rats fed the 1% carbonyl iron diet. Pharmacokinetics of iv-injected 54 Mn revealed that the isotope was cleared more rapidly from the blood of iron-loaded rats. In situ analysis of divalent metal transporter-1 (DMT1) expression in lung detected mRNA in airway epithelium and bronchus-associated lymphatic tissue (BALT). Staining of the latter was significantly reduced in rats fed the high iron diet. In situ analysis of transferrin receptor (TfR) mRNA showed staining in BALT alone. These data demonstrate that manganese absorption from the lungs to the blood can be modified by iron status and the route of administration

  14. Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

    Nora Adriana Hernández-Cuevas

    Full Text Available Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron, low-iron medium (around 123 µM iron, iron-deficient medium (around 91 µM iron, and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.

  15. Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

    Hernández-Cuevas, Nora Adriana; Weber, Christian; Hon, Chung-Chau; Guillen, Nancy

    2014-01-01

    Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.

  16. The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder*

    Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N.; Torda, Andrew E.

    2014-01-01

    The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 μm) to aquo-cobalamin (H2OCbl+) but not to other cobalamins. Competition experiments with the H2OCbl+-coordinating ligand CN− and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl+ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl+. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins. PMID:25342754

  17. The Role of Heme Chirality in the Circular Dichroism of Heme Proteins

    Woody, Robert W.; Pescitelli, Gennaro

    2014-07-01

    The rotational strength (R) of the Soret transition in sperm-whale myoglobin (SW Mb), the hemoglobin from Chironomus thummi thummi (CTT Hb), and human hemoglobin (hHb) has been calculated using 20 high-resolution ( Raro > Rpep. For CTT Hb and hHB, the orders were, respectively, Rint > Rpep > Raro and Rint > Raro ≈ Rpep. Human Hb ɑ chains showed the same trend as CTT Hb. Only in the hHb β chains did Raro predominate, with the order Raro > Rint > Rpep. The total predicted Rtot for SW Mb, CTT Hb, and hHb averaged +0.77±0.10 (0.56 - 0.80), -0.37±0.12 (-0.5), and +0.31±0.17 DBM (0.23 - 0.50), respectively. (Values in parentheses are experimental values.) Thus, contrary to the currently accepted view, coupling with aromatic side-chain or peptide transitions is not the dominant factor in the Soret circular dichroism (CD) of these proteins. The Soret CD is dominated by intrinsic CD of the heme chromophore, of which vinyl torsion is the major determinant. This result suggests an explanation for the large effect of heme isomerism on the Soret CD of Mb and Hb. Rotation about the ɑ-γ axis may be associated with large changes in vinyl torsion and thus substantially alter the intrinsic CD, even reversing its sign.

  18. Target organs for avian pancreatic polypeptide

    Kimmel, J.R.; Pollock, H.G.

    1981-01-01

    The problem of the physiological function of pancreatic polypeptide (PP) has been approached by attempting to identify target organs. Avian PP (aPP) labeled with 125I at either the C-terminus (aPP-C) or the N-terminus (aPP-N) was injected into fasted chickens and allowed to circulate for 3-120 min. At the end of the equilibration period, the anesthetized bird was perfused first with saline, then with Buoin's solution. Samples of fixed tissue from various organs were collected, weighed, and counted. Control experiments consisted of coinjection of unlabeled aPP to compete for receptors. The rate of disappearance of aPP-N from plasma was greater than that of aPP-C. Binding of aPP-N by spleen, duodenum, ileum, pancreas, and bone marrow was markedly reduced by coinjection of unlabeled aPP. A similar but less marked reduction in binding was found in liver and proventriculus. aPP-C gave less conclusive results. The maximal competitive effect of unlabeled PP could be achieved in most cases with 30 microgram unlabeled aPP. It is concluded that pancreas, duodenum, ileum, spleen, and bone marrow, and probably liver and proventriculus, are target organs for aPP in the chicken and that the C-terminal region of aPP is involved in receptor binding

  19. Fibrillar dimer formation of islet amyloid polypeptides

    Chi-cheng Chiu

    2015-09-01

    Full Text Available Amyloid deposits of human islet amyloid polypeptide (hIAPP, a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  20. Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology

    Rehana Akter

    2016-01-01

    Full Text Available The hormone islet amyloid polypeptide (IAPP, or amylin plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

  1. Tunable drug loading and release from polypeptide multilayer nanofilms

    Jiang, Bingbing; Li, Bingyun

    2009-01-01

    Polypeptide multilayer nanofilms were prepared using electrostatic layer-by-layer self-assembly nanotechnology. Small charged drug molecules (eg, cefazolin, gentamicin, and methylene blue) were loaded in polypeptide multilayer nanofilms. Their loading and release were found to be pH-dependent and could also be controlled by changing the number of film layers and drug incubation time, and applying heat-treatment after film formation. Antibioticloaded polypeptide multilayer nanofilms showed controllable antibacterial properties against Staphylococcus aureus. The developed biodegradable polypeptide multilayer nanofilms are capable of loading both positively- and negatively-charged drug molecules and promise to serve as drug delivery systems on biomedical devices for preventing biomedical device-associated infection, which is a significant clinical complication for both civilian and military patients. PMID:19421369

  2. The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.

    Lill, Roland; Hoffmann, Bastian; Molik, Sabine; Pierik, Antonio J; Rietzschel, Nicole; Stehling, Oliver; Uzarska, Marta A; Webert, Holger; Wilbrecht, Claudia; Mühlenhoff, Ulrich

    2012-09-01

    Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides. Polypeptide vesicles by conformation-specific assembly. Ordered chiral macroporous hybrid silica-polypeptide composites

    Bellomo, Enrico Giuseppe

    2005-07-01

    Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides . The aqueous, lyotropic liquid-crystalline phase behavior of an alpha helical polypeptide, has been studied using optical microscopy and X-ray scattering. Solutions of optically pure polypeptide were found to form cholesteric liquid crystals at volume fractions that decreased with increasing average chain length. At very high volume fractions, the formation of a hexagonal mesophase was observed. The pitch of the cholesteric phase could be varied by a mixture of enantiomeric samples, where the pitch increased as the mixture approached equimolar. The cholesteric phases could be untwisted, using either magnetic field or shear flow, into nematic phases, which relaxed into cholesterics upon removal of field or shear. We have found that the phase diagram of this polypeptide in aqueous solution parallels that of poly(gamma-benzyl glutamate) in organic solvents, thus providing a useful system for liquid-crystal applications requiring water as solvent. Polypeptide vesicles by conformation-specific assembly. We have found that block copolymers composed of polypeptide segments provide significant advantages in controlling both the function and supramolecular structure of bioinspired self-assemblies. Incorporation of the stable chain conformations found in proteins into block copolymers was found to provide an additional element of control, beyond amphiphilicity and composition that defines self-assembled architecture. The abundance of functionality present in amino acids, and the ease by which they can be incorporated into these materials, also provides a powerful mechanism to impart block copolypeptides with function. This combination of structure and function work synergistically to enable significant advantages in the preparation of therapeutic agents as well as provide insight into design of self-assemblies beginning to approach the complexity of natural structures such as virus capsids. Ordered

  4. Ring-Opening Polymerization of N-Carboxyanhydrides for Preparation of Polypeptides and Polypeptide-Based Hybrid Materials with Various Molecular Architectures

    Pahovnik, David; Hadjichristidis, Nikolaos

    2015-01-01

    Different synthetic approaches utilizing ring-opening polymerization of N-carboxyanhydrides for preparation of polypeptide and polypeptide-based hybrid materials with various molecular architectures are described. An overview of polymerization

  5. Novel Insights in Mammalian Catalase Heme Maturation: Effect of NO and Thioredoxin-1

    Chakravarti, Ritu; Gupta, Karishma; Majors, Alana; Ruple, Lisa; Aronica, Mark; Stuehr, Dennis J.

    2015-01-01

    Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorpo...

  6. Iron absorption from adequate Filipino meals

    Trinidad, T.P.; Madriaga, J.R.; Valdez, D.H.; Cruz, E.M.; Mallillin, A.C.; Sison, C.C.; Kuizon, M.D.

    1991-01-01

    Iron absorption from adequate Filipino meals representing the three major island groups of the Philippines (Luzon, Visayas and Mindanao) was studied using double isotope extrinsic tag method. Mean iron absorption of the one-day meal for Metro Manila was 6.6 ± 1.26%, Central Visayas, 6.3 ± 1.15% and Southern Mindanao, 6.4 ± 1.19%. Comparison between meals (breakfast, lunch, dinner) for each region as well as one-day meal for the three regions showed no significant differences (P > .01). Correlation tests done between iron absorption and the following iron enhancers: ascorbic acid, amount of fish, meat or poultry and inhibitors: phytic acid and tannic acid did not give significant results. The overall bar x of 6.4 ± 1.20% may be used as the non-heme iron absorption level from an adequate Filipino meal. This value can be considered as one of the bases for arriving at recommended dietary allowances for iron among Filipinos instead of the 10% iron absorption assumed in 1976

  7. NO2-induced synthesis of nitrato-iron(III) porphyrin with diverse ...

    found serendipitously in the reaction of [Fe(4-Me-TPP)Cl] with nitrous acid, which ... Nitric oxide and its derivatives nitrite and nitrate ion ... oxide.2 Nitrate is produced in heme proteins from oxi- ... and nitrogen assimilation.4 Iron nitrate(III) porphyrins ... one-pot method.15 ... of the compound was determined based on the lack.

  8. Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

    Robert Staroń

    Full Text Available Among livestock, domestic pig (Sus scrofa is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

  9. Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin.

    Vu, B Christie; Nothnagel, Henry J; Vuletich, David A; Falzone, Christopher J; Lecomte, Juliette T J

    2004-10-05

    The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed

  10. Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

    Viswanathan Arun Nagaraj

    Full Text Available Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS, and the last enzyme, ferrochelatase (FC, in the heme-biosynthetic pathway of Plasmodium berghei (Pb. The wild-type and knockout (KO parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14C] aminolevulinic acid (ALA. We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

  11. Heme and menaquinone induced electron transport in lactic acid bacteria

    Brooijmans, R.J.W.; Smit, B.; Santos, dos F.; Riel, van J.; Vos, de W.M.; Hugenholtz, J.

    2009-01-01

    ABSTRACT: BACKGROUND: For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait.

  12. Cysteine-independent activation/inhibition of heme oxygenase-2

    Dragic Vukomanovic

    2016-01-01

    Full Text Available Reactive thiols of cysteine (cys residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2 isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2 and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

  13. Heme and HO-1 inhibition of HCV, HBV, and HIV

    Warren N Schmidt

    2012-10-01

    Full Text Available Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system are virucidal for all three viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections.

  14. Cysteine-independent activation/inhibition of heme oxygenase-2.

    Vukomanovic, Dragic; Rahman, Mona N; Maines, Mahin D; Ozolinš, Terence Rs; Szarek, Walter A; Jia, Zongchao; Nakatsu, Kanji

    2016-03-01

    Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

  15. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    An ELISA assay for heme oxygenase (HO-l ) Abstract A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  16. Isoporphyrin intermediate in heme oxygenase catalysis. Oxidation of alpha-meso-phenylheme.

    Evans, John P; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

    2008-07-11

    Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the alpha-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin pi-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of alpha-meso-phenylheme-IX, alpha-meso-(p-methylphenyl)-mesoheme-III, and alpha-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593-42604), only the alpha-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced alpha-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation.

  17. Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

    Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

    2018-05-15

    Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

  18. ATP-binding cassette B10 regulates early steps of heme synthesis.

    Bayeva, Marina; Khechaduri, Arineh; Wu, Rongxue; Burke, Michael A; Wasserstrom, J Andrew; Singh, Neha; Liesa, Marc; Shirihai, Orian S; Langer, Nathaniel B; Paw, Barry H; Ardehali, Hossein

    2013-07-19

    Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established. The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

  19. Iron-associated biology of Trypanosoma brucei.

    Basu, Somsuvro; Horáková, Eva; Lukeš, Julius

    2016-01-01

    Roč. 1860, č. 2 (2016), s. 363-370 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GA14-23986S; GA ČR GAP305/12/2261; GA MŠk(CZ) EE2.3.30.0032 EU Projects: European Commission(XE) COST Action CM1307; European Commission(XE) 316304 - MODBIOLIN Grant - others:AV ČR(CZ) M200961204 Institutional support: RVO:60077344 Keywords : iron * Fe/S cluster * heme * Trypanosoma * TAO Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.702, year: 2016

  20. Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

    Posayapisit, Navaporn; Songsungthong, Warangkhana; Koonyosying, Pongpisid; Falade, Mofolusho O; Uthaipibull, Chairat; Yuthavong, Yongyuth; Shaw, Philip J; Kamchonwongpaisan, Sumalee

    Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc 1 hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc 1 hl are both essential and thus may be antimalarial targets. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Isotope aided studies of the bioavailability of iron from human diets consumed in Peru

    Zavaleta, N.; Penny, M.; Berlanga, R.; Diaz, A.; Montoya, E.; Lonnerdal, B.

    1994-01-01

    Iron deficiency anaemia is an important health problem in Peru, which affects approximately 25% of the population. The most vulnerable groups are children below 5 years of age and pregnant women, of whom 64% and 53% respectively are anemic. The main reason for this deficiency is inadequate iron intake. Heme iron consumption is very low, and non-heme iron is virtually the only source of iron in the diet. Despite regional differences in food consumption, wheat, salt and sugar are widely consumed in all areas. Wheat is likely to be the most suitable food vehicle for iron fortification due to the processing required. Based on the recent food consumption surveys conducted in Lima by the IIN, we selected examples of typical main meals and measured iron bioavailability in the diet using an extrinsic tag method with 1.5 μCi of 59 Fe and 5 μCi of 55 Fe as markers. Coffee with bread and butter for breakfast, noodle soup with vegetables, rice with seasoned tripe (cow), bread and lemonade for lunch; and noodle soup with vegetables and bread for dinner were used to measure iron absorption. Thirteen adults in apparent good health, 5 male and 8 female, with normal hemoglobin levels participated in the study. The mean iron absorption from breakfast was 4.2% ± 4.1; from lunch 14.65% ± 10/95, and from dinner 5.1% ± 2.84. The presence of heme iron from tripe and ascorbic acid from lemonade improved iron absorption. (author). 17 refs, 3 tabs

  2. Computational modeling and analysis of iron release from macrophages.

    Alka A Potdar

    2014-07-01

    Full Text Available A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe2+ through ferroportin (FPN, the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp, oxidizes ferrous to ferric ion. Apo-transferrin (Tf, the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can

  3. Polypeptide profiles of human oocytes and preimplantation embryos.

    Capmany, G; Bolton, V N

    1993-11-01

    The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

  4. CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123

    Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

    2010-01-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biot...

  5. Dietary heme mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon

    IJssennagger, Noortje; Wit, de Nicole; Muller, Michael; Meer, van der Roelof

    2012-01-01

    Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome

  6. Dietary heme-mediated PPARa activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon

    IJssenagger, N.; Wit, de N.J.W.; Muller, M.R.; Meer, van der R.

    2012-01-01

    Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome

  7. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  8. Iron economy in Naegleria gruberi reflects its metabolic flexibility.

    Mach, Jan; Bíla, Jarmila; Ženíšková, Kateřina; Arbon, Dominik; Malych, Ronald; Glavanakovová, Marie; Nývltová, Eva; Sutak, Robert

    2018-05-05

    Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  9. Diagnosis and treatment of sideroblastic anemias: from defective heme synthesis to abnormal RNA splicing.

    Cazzola, Mario; Malcovati, Luca

    2015-01-01

    The sideroblastic anemias are a heterogeneous group of inherited and acquired disorders characterized by the presence of ring sideroblasts in the bone marrow. X-linked sideroblastic anemia (XLSA) is caused by germline mutations in ALAS2. Hemizygous males have a hypochromic microcytic anemia, which is generally mild to moderate and is caused by defective heme synthesis and ineffective erythropoiesis. XLSA is a typical iron-loading anemia; although most patients are responsive to pyridoxine, treatment of iron overload is also important in the management of these patients. Autosomal recessive sideroblastic anemia attributable to mutations in SLC25A38, a member of the mitochondrial carrier family, is a severe disease: patients present in infancy with microcytic anemia, which soon becomes transfusion dependent. Conservative therapy includes regular red cell transfusion and iron chelation, whereas allogenic stem cell transplantation represents the only curative treatment. Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome characterized mainly by anemia attributable to ineffective erythropoiesis. The clinical course of RARS is generally indolent, but there is a tendency to worsening of anemia over time, so that most patients become transfusion dependent in the long run. More than 90% of these patients carry somatic mutations in SF3B1, a gene encoding a core component of the RNA splicing machinery. These mutations cause misrecognition of 3' splice sites in downstream genes, resulting in truncated gene products and/or decreased expression attributable to nonsense-mediated RNA decay; this explains the multifactorial pathogenesis of RARS. Variants of RARS include refractory cytopenia with multilineage dysplasia and ring sideroblasts, and RARS associated with marked thrombocytosis; these variants involve additional genetic lesions. Inhibitors of molecules of the transforming growth factor-β superfamily have been shown recently to target ineffective

  10. Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

    Matuschewski, Kai; Haussig, Joana M.

    2016-01-01

    Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

  11. The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake.

    Marcelo L Merli

    2016-01-01

    Full Text Available Trypanosoma cruzi, the etiological agent of Chagas' disease, presents nutritional requirements for several metabolites. It requires heme for the biosynthesis of several heme-proteins involved in essential metabolic pathways like mitochondrial cytochromes and respiratory complexes, as well as enzymes involved in the biosynthesis of sterols and unsaturated fatty acids. However, this parasite lacks a complete route for its synthesis. In view of these facts, T. cruzi has to incorporate heme from the environment during its life cycle. In other words, their hosts must supply the heme for heme-protein synthesis. Although the acquisition of heme is a fundamental issue for the parasite's replication and survival, how this cofactor is imported and distributed is poorly understood. In this work, we used different fluorescent heme analogs to explore heme uptake along the different life-cycle stages of T. cruzi, showing that this parasite imports it during its replicative stages: the epimastigote in the insect vector and the intracellular amastigote in the mammalian host. Also, we identified and characterized a T. cruzi protein (TcHTE with 55% of sequence similarity to LHR1 (protein involved in L. amazonensis heme transport, which is located in the flagellar pocket, where the transport of nutrients proceeds in trypanosomatids. We postulate TcHTE as a protein involved in improving the efficiency of the heme uptake or trafficking in T. cruzi.

  12. Comparison between the availability of iron in the presence of vitamin a and β-carotene in foods and medications

    Fabiana Cristina Camargo Martini

    2011-06-01

    Full Text Available The objective of this work was to verify the availability of iron in the presence of vitamin A as components of foods and in combinations with medicines. The iron available was measured in the presence of vitamin A in foods - common bean (B, beef liver (Li and carrot (C - and medicines - Fer-In-Sol® (Fer (Mead Johnson, Arovit® (A (Roche and Neutrofer® (N (Sigma Pharma - as well as in combinations of both. β-carotene, vitamin A, total iron, heme and non heme iron, percentage of dialyzable iron and amount of dialyzable iron was determined. Vitamin A and β-carotene had a positive effect on the percentage of iron dialysis. Carrot and liver had a better percentage of dialyzable iron than their respective medicine at similar concentrations. Therefore, we can conclude that there has been an influence of vitamin A over the dialysis of iron, being the mixtures containing liver the ones which achieved the highest concentrations of dialyzable iron, and also that, according to the amounts needed to obtain the daily recommended intake of iron, they are good for consumption.

  13. Studies on the mechanism of pyrophosphate-mediated uptake of iron from transferrin by isolated rat-liver mitochondria

    Konopka, K.; Romslo, I.; Bergen Univ.

    1981-01-01

    1. Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1-1.5 nmol mg protein -1 h -1 , or approximately 60% of the amount of iron mobilized from transferrin. 2. The uptake declines if respiration is inhibited, substrate is depleted, or the experiments are run under anaerobic conditions. Substrate, depletion and respiratory inhibitors are less inhibitory under anaerobic conditions. 3. More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bathophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration-inhibited mitochondria under aerobic conditions is not available for heme synthesis. 4. With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate. 5. The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous irons thus formed is available for heme synthesis. (orig.) [de

  14. The FTO (fat mass and obesity associated gene codes for a novel member of the non-heme dioxygenase superfamily

    Andrade-Navarro Miguel A

    2007-11-01

    Full Text Available Abstract Background Genetic variants in the FTO (fat mass and obesity associated gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO. Results We performed a sequence similarity search using the human FTO protein as query and then generated a profile using the multiple sequence alignment of the homologous sequences. Profile-to-sequence and profile-to-profile based comparisons identified remote homologs of the non-heme dioxygenase family. Conclusion Our analysis suggests that human FTO is a member of the non-heme dioxygenase (Fe(II- and 2-oxoglutarate-dependent dioxygenases superfamily. Amino acid conservation patterns support this hypothesis and indicate that both 2-oxoglutarate and iron should be important for FTO function. This computational prediction of the function of FTO should suggest further steps for its experimental characterization and help to formulate hypothesis about the mechanisms by which it relates to obesity in humans.

  15. The Type VI Secretion System Engages a Redox-Regulated Dual-Functional Heme Transporter for Zinc Acquisition.

    Si, Meiru; Wang, Yao; Zhang, Bing; Zhao, Chao; Kang, Yiwen; Bai, Haonan; Wei, Dawei; Zhu, Lingfang; Zhang, Lei; Dong, Tao G; Shen, Xihui

    2017-07-25

    The type VI secretion system was recently reported to be involved in zinc acquisition, but the underlying mechanism remains unclear. Here, we report that Burkholderia thailandensis T6SS4 is involved in zinc acquisition via secretion of a zinc-scavenging protein, TseZ, that interacts with the outer membrane heme transporter HmuR. We find that HmuR is a redox-regulated dual-functional transporter that transports heme iron under normal conditions but zinc upon sensing extracellular oxidative stress, triggered by formation of an intramolecular disulfide bond. Acting as the first line of defense against oxidative stress, HmuR not only guarantees an immediate response to the changing environment but also provides a fine-tuned mechanism that allows a gradual response to perceived stress. The T6SS/HmuR-mediated active zinc transport system is also involved in bacterial virulence and contact-independent bacterial competition. We describe a sophisticated bacterial zinc acquisition mechanism affording insights into the role of metal ion transport systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: Implications for the enzyme mechanism‡

    Ogura, Hiroshi; Evans, John P.; Peng, Dungeng; Satterlee, James D.; de Montellano, Paul R. Ortiz; Mar, Gerd N. La

    2009-01-01

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase-1, (hHO) has been investigated by 1H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. 2D 1H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts, that places the lone iron π-spin in the dxz orbital, rather than the dyz orbital found in the cya...

  17. Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

    Brown, Kimberly; Harris, Paul

    2013-12-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    de Visser, Sam P.; Stillman, Martin J.

    2016-01-01

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties. PMID:27070578

  19. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    Sam P. de Visser

    2016-04-01

    Full Text Available In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1. This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties.

  20. Cyanide binding to human plasma heme-hemopexin: A comparative study

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Laboratorio Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Roma (Italy); Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Leboffe, Loris [Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Polticelli, Fabio [Dipartimento di Biologia, Universita Roma Tre, Roma (Italy)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  1. Functional imaging: monitoring heme oxygenase-1 gene expression in vivo

    Zhang, Weisheng; Reilly-Contag, Pamela; Stevenson, David K.; Contag, Christopher H.

    1999-07-01

    The regulation of genetic elements can be monitored in living animals using photoproteins as reporters. Heme oxygenase (HO) is the key catabolic enzyme in the heme degradation pathway. Here, HO expression serves as a model for in vivo functional imaging of transcriptional regulation of a clinically relevant gene. HO enzymatic activity is inhibited by heme analogs, metalloporphyrins, but many members of this family of compounds also activate transcription of the HO-1 promoter. The degree of transcriptional activation by twelve metalloporphyrins, differing at the central metal and porphyrin ring substituents, was evaluated in both NIH 3T3 stable lines and transgenic animals containing HO-1 promoter-luciferase gene fusions. In the correlative cell culture assays, the metalloporphyrins increased transcription form the full length HO promoter fusion to varying degrees, but none increased transcription from a truncated HO-1 promoter. These results suggested that one or both of the two distal enhancer elements located at -4 and -10 Kb upstream from transcriptional start are required for HO-1 induction by heme and its analogs. The full-length HO-1-luc fusion was then evaluated as a transgene in mice. It was possible to monitor the effects of the metalloporphyrins, SnMP and ZnPP, in living animals over time. This spatiotemporal analyses of gene expression in vivo implied that alterations in porphyrin ring substituents and the central metal may affect the extent of gene activation. These data further indicate that using photoprotein reporters, subtle differences in gene expression can be monitored in living animals.

  2. Vasoactive intestinal polypeptide and other preprovasoactive intestinal polypeptide-derived peptides in the female and male genital tract: localization, biosynthesis, and functional and clinical significance

    Ottesen, B; Fahrenkrug, J

    1995-01-01

    Vasoactive intestinal polypeptide, a neuropeptide with wide distribution in the central and peripheral nervous system, has a broad spectrum of biologic actions. The demonstration of vasoactive intestinal polypeptide containing nerve fibers within the female and male genital tract 17 years ago...... indicated a putative role for this peptide in the local nervous control of reproductive functions. The genes encoding the preprovasoactive intestinal polypeptide precursor molecule and the vasoactive intestinal polypeptide receptor have been identified. The gene expression has been studied by the use...... in the genital tracts (i.e., blood flow and nonvascular smooth muscle relaxation). In the ovary vasoactive intestinal polypeptide seems to play an important role as regulator and/or modulator of folliculogenesis and steroidogenesis. In the male genital tract vasoactive intestinal polypeptide seems to participate...

  3. Stability enhancement of cytochrome c through heme deprotonation and mutations.

    Sonoyama, Takafumi; Hasegawa, Jun; Uchiyama, Susumu; Nakamura, Shota; Kobayashi, Yuji; Sambongi, Yoshihiro

    2009-01-01

    The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.

  4. Effects of Metalloporphyrins on Heme Oxygenase-1 Transcription: Correlative Cell Culture Assays Guide in Vivo Imaging

    Monica Hajdena-Dawson; Weisheng Zhang; Pamela R. Contag; Ronald J. Wong; Hendrik J. Vreman; David K. Stevenson; Christopher H. Contag

    2003-01-01

    Heme oxygenase (HO) is the rate-limiting step in the heme degradation pathway and is a potential target for the control, or prevention, of pathologic jaundice in neonates. Metalloporphyrins (Mps), a diverse set of synthetic derivatives of heme, can competitively inhibit the HO enzymes. However, certain Mps are phototoxic and some increase transcription of HO-1, the inducible HO isozyme. Therefore, effective development of this class of compounds as therapeutics for treating pathologic jaundic...

  5. Nitrite to nitric oxide interconversion by heme FeII complex assisted by [CuI(tmpa)]+

    Turias, Francesc; Solà , Miquel; Falivene, Laura; Cavallo, Luigi; Poater, Albert

    2015-01-01

    The present computational study complements the recent experimental efforts by Karlin and coworkers to describe the interconversion of nitrite to nitric oxide by means of an iron porphyrin complex together with a Cu chemical system, i.e., the iron(II) complex (F8TPP)FeII [F8TPP = tetrakis(2,6-difluorophenyl)porphyrinate(2−)] and a preformed copper(II)–nitrito complex [(tmpa)CuII(NO2)][B(C6F5)4] [tmpa = tris(2-pyridylmethyl)amine], being the latter an oxidized species of [(tmpa)CuI(MeCN)]+. By DFT calculations, we unravel how the reduction of nitrite to nitric oxide takes place through a μ-oxo heme-FeIII–O–CuII complex, following a mimetic path as in the cytochrome c oxidase. Mayer bond order (MBO) and energy decomposition analyses are used to analyze the bonding strength of such nitro derivatives to either copper or iron. © 2015 Springer Science+Business Media New York

  6. Nitrite to nitric oxide interconversion by heme FeII complex assisted by [CuI(tmpa)]+

    Turias, Francesc

    2015-09-09

    The present computational study complements the recent experimental efforts by Karlin and coworkers to describe the interconversion of nitrite to nitric oxide by means of an iron porphyrin complex together with a Cu chemical system, i.e., the iron(II) complex (F8TPP)FeII [F8TPP = tetrakis(2,6-difluorophenyl)porphyrinate(2−)] and a preformed copper(II)–nitrito complex [(tmpa)CuII(NO2)][B(C6F5)4] [tmpa = tris(2-pyridylmethyl)amine], being the latter an oxidized species of [(tmpa)CuI(MeCN)]+. By DFT calculations, we unravel how the reduction of nitrite to nitric oxide takes place through a μ-oxo heme-FeIII–O–CuII complex, following a mimetic path as in the cytochrome c oxidase. Mayer bond order (MBO) and energy decomposition analyses are used to analyze the bonding strength of such nitro derivatives to either copper or iron. © 2015 Springer Science+Business Media New York

  7. Cast irons

    1996-01-01

    Cast iron offers the design engineer a low-cost, high-strength material that can be easily melted and poured into a wide variety of useful, and sometimes complex, shapes. This latest handbook from ASM covers the entire spectrum of one of the most widely used and versatile of all engineered materials. The reader will find the basic, but vital, information on metallurgy, solidification characteristics, and properties. Extensive reviews are presented on the low-alloy gray, ductile, compacted graphite, and malleable irons. New and expanded material has been added covering high-alloy white irons used for abrasion resistance and high-alloy graphitic irons for heat and corrosion resistance. Also discussed are melting furnaces and foundry practices such as melting, inoculation, alloying, pouring, gating and rising, and molding. Heat treating practices including stress relieving, annealing, normalizing, hardening and tempering, autempering (of ductile irons), and surface-hardening treatments are covered, too. ASM Spec...

  8. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  9. Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase.

    Quang M Tran

    Full Text Available The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.

  10. In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

    Coelho, Pedro S; Brustad, Eric M; Arnold, Frances H; Wang, Zhan; Lewis, Jared C

    2015-03-31

    The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

  11. [Dinitrosyl iron complexes are endogenous signaling agents in animal and human cells and tissues (a hypothesis)].

    Vanin, A F

    2004-01-01

    The hypothesis was advanced that dinitrosyl iron complexes generated in animal and human cells and tissues producing nitric oxide can function as endogenous universal regulators of biochemical and physiological processes. This function is realized by the ability of dinitrosyl iron complexes to act as donors of free nitric oxide molecules interacting with the heme groups of proteins, nitrosonium ions, or Fe+(NO+)2 interacting with the thiol groups of proteins. The effect of dinitrosyl iron complexes on the activity of some enzymes and the expression of the genome at the translation and transcription levels was considered.

  12. Iron-Deficiency Anemia

    Full Text Available ... risk for iron-deficiency anemia, including: Vegetarian or vegan eating patterns. Not eating enough iron-rich foods, ... iron-fortified foods that have iron added. Vegetarian diets can provide enough iron if you choose nonmeat ...

  13. PfsR is a key regulator of iron homeostasis in Synechocystis PCC 6803.

    Dan Cheng

    Full Text Available Iron is an essential cofactor in numerous cellular processes. The iron deficiency in the oceans affects the primary productivity of phytoplankton including cyanobacteria. In this study, we examined the function of PfsR, a TetR family transcriptional regulator, in iron homeostasis of the cyanobacterium Synechocystis PCC 6803. Compared with the wild type, the pfsR deletion mutant displayed stronger tolerance to iron limitation and accumulated significantly more chlorophyll a, carotenoid, and phycocyanin under iron-limiting conditions. The mutant also maintained more photosystem I and photosystem II complexes than the wild type after iron deprivation. In addition, the activities of photosystem I and photosystem II were much higher in pfsR deletion mutant than in wild-type cells under iron-limiting conditions. The transcripts of pfsR were enhanced by iron limitation and inactivation of the gene affected pronouncedly expression of fut genes (encoding a ferric iron transporter, feoB (encoding a ferrous iron transporter, bfr genes (encoding bacterioferritins, ho genes (encoding heme oxygenases, isiA (encoding a chlorophyll-binding protein, and furA (encoding a ferric uptake regulator. The iron quota in pfsR deletion mutant cells was higher than in wild-type cells both before and after exposure to iron limitation. Electrophoretic mobility shift assays showed that PfsR bound to its own promoter and thereby auto-regulated its own expression. These data suggest that PfsR is a critical regulator of iron homeostasis.

  14. Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes.

    Neritza Campo Beltrán

    Full Text Available Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

  15. Mutations in iron-sulfur cluster proteins that improve xylose utilization

    Froehlich, Allan; Henningsen, Brooks; Covalla, Sean; Zelle, Rintze M.

    2018-03-20

    There is provided an engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use thereof.

  16. Photochemical organic oxidations and dechlorinations with a mu-oxo bridged heme/non-heme diiron complex.

    Wasser, Ian M; Fry, H Christopher; Hoertz, Paul G; Meyer, Gerald J; Karlin, Kenneth D

    2004-12-27

    Steady state and laser flash photolysis studies of the heme/non-heme mu-oxo diiron complex [((6)L)Fe(III)-O-Fe(III)-Cl](+) (1) have been undertaken. The anaerobic photolysis of benzene solutions of 1 did not result in the buildup of any photoproduct. However, the addition of excess triphenylphosphine resulted in the quantitative photoreduction of 1 to [((6)L)Fe(II)...Fe(II)-Cl](+) (2), with concomitant production by oxo-transfer of 1 equiv of triphenylphosphine oxide. Under aerobic conditions and excess triphenylphosphine, the reaction produces multiple turnovers (approximately 28) before the diiron complex is degraded. The anaerobic photolysis of tetrahydrofuran (THF) or toluene solutions of 1 likewise results in the buildup of 2. The oxidation products from these reactions included gamma-butyrolactone (approximately 15%) for the reaction in THF and benzaldehyde (approximately 23%) from the reaction in toluene. In either case, the O-atom which is incorporated into the carbonyl product is derived from dioxygen present under workup or under aerobic photolysis conditions. Transient absorption measurements of low-temperature THF solutions of 1 revealed the presence of an (P)Fe(II)-like [P = tetraaryl porphyrinate dianion] species suggesting that the reactive species is a formal (heme)Fe(II)/Fe(IV)=O(non-heme) pair. The non-heme Fe(IV)=O is thus most likely responsible for C-H bond cleavage and subsequent radical chemistry. The photolysis of 1 in chlorobenzene or 1,2-dichlorobenzene resulted in C-Cl cleavage reactions and the formation of [[((6)L)Fe(III)-Cl...Fe(III)-Cl](2)O](2+) (3), with chloride ligands that are derived from solvent dehalogenation chemistry. The resulting organic products are biphenyl trichlorides or biphenyl monochlorides, derived from dichlorobenzene and chlorobenzene, respectively. Similarly, product 3 is obtained by the photolysis of benzene-benzyl chloride solutions of 1; the organic product is benzaldehyde (approximately 70%). A brief

  17. O2-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-01-01

    Research highlights: → Human serum heme-albumin displays globin-like properties. → O 2 -mediated oxidation of ferrous nitrosylated human serum heme-albumin. → Allosteric modulation of human serum heme-albumin reactivity. → Rifampicin is an allosteric effector of human serum heme-albumin. → Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O 2 -mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O 2 -mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10 -5 and 8.3 x 10 -4 s -1 , and h = 1.3 x 10 -4 and 8.5 x 10 -4 s -1 , in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 o C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O 2 -mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O 2 does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O 2 -mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  18. Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics

    Walker, Aisha L.; Lancaster, Cynthia S.; Finkelstein, David; Ware, Russell E.; Sparreboom, Alex

    2013-01-01

    Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assess...

  19. Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins

    Deming, TJ

    2017-01-01

    Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-cont...

  20. Tea fungus fermentation on a substrate with iron(ii-ions

    Malbaša Radomir V.

    2002-01-01

    Full Text Available Iron is essential element for human metabolism and it is a constituent of both heme- containing and nonheme proteins. Its deficiency can cause serious diseases, i.e. iron-deficiency anemia, with some fatal consequences. Tea fungus beverage has high nutritional value and some pharmaceutical effects. It is widely consumed allover the world and its benefits were proved a number of times. The aim of this paper was to investigate tea fungus fermentation on a substrate containing iron(II-ions and the possibility of obtaining a beverage enriched with iron. We monitored pH, iron content and also the production of L-ascorbic acid, which is very important for iron absorption in humans.

  1. Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

    Walker, Candace Rae; Pushpavanam, Karthik; Nair, Divya Geetha; Potta, Thrimoorthy; Sutiyoso, Caesario; Kodibagkar, Vikram D; Sapareto, Stephen; Chang, John; Rege, Kaushal

    2013-08-13

    Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

  2. Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

    Eichmann, Cédric; Preissler, Steffen; Riek, Roland; Deuerling, Elke

    2010-05-18

    The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.

  3. Effect of dietary iron loading on recognition memory in growing rats.

    Murui Han

    Full Text Available While nutritional and neurobehavioral problems are associated with both iron deficiency during growth and overload in the elderly, the effect of iron loading in growing ages on neurobehavioral performance has not been fully explored. To characterize the role of dietary iron loading in memory function in the young, weanling rats were fed iron-loading diet (10,000 mg iron/kg diet or iron-adequate control diet (50 mg/kg for one month, during which a battery of behavioral tests were conducted. Iron-loaded rats displayed elevated non-heme iron levels in serum and liver, indicating a condition of systemic iron overload. In the brain, non-heme iron was elevated in the prefrontal cortex of iron-loaded rats compared with controls, whereas there was no difference in iron content in other brain regions between the two diet groups. While iron loading did not alter motor coordination or anxiety-like behavior, iron-loaded rats exhibited a better recognition memory, as represented by an increased novel object recognition index (22% increase from the reference value than control rats (12% increase; P=0.047. Western blot analysis showed an up-regulation of dopamine receptor 1 in the prefrontal cortex from iron-loaded rats (142% increase; P=0.002. Furthermore, levels of glutamate receptors (both NMDA and AMPA and nicotinic acetylcholine receptor (nAChR were significantly elevated in the prefrontal cortex of iron-loaded rats (62% increase in NR1; 70% increase in Glu1A; 115% increase in nAChR. Dietary iron loading also increased the expression of NMDA receptors and nAChR in the hippocampus. These results support the idea that iron is essential for learning and memory and further reveal that iron supplementation during developmental and rapidly growing periods of life improves memory performance. Our investigation also demonstrates that both cholinergic and glutamatergic neurotransmission pathways are regulated by dietary iron and provides a molecular basis for the

  4. Potent heme-degrading action of antimony and antimony-containing parasiticidal agents.

    Drummond, G S; Kappas, A

    1981-02-01

    The ability of antimony and antimony-containing parasiticidal agents to enhance the rate of heme degradation in liver and kidney was investigated. Trivalent antimony was shown to be an extremely potent inducer of heme oxygenase, the initial and rate-limiting enzyme in heme degradation, in both organs, whereas the pentavalent form was a weak inducer of this enzyme. The ability of antimony to induce heme oxygenase was dose-dependent, independent of the salt used, and not a result of a direct activation of the enzyme in vitro. Concomitant with heme oxygenase induction by antimony, microsomal heme and cytochrome P-450 contents decreased, the cyto-chrome P-450-dependent mixed function oxidase system was impaired, and delta-ami-nolevulinate synthase (ALAS), the rate-limiting enzyme of heme synthesis, underwent the sequential changes-initial inhibition followed by rebound induction-usually associated with the administration of transition elements such as cobalt. Antimony induction of heme oxygenase however, unlike the enzyme induction elicited by cobalt, was not prevented either by cysteine administered orally or as a cysteine metal complex, or by simultaneous zinc administration. Desferoxamine also did not block heme oxygenase induction by antimony, but this chelator did prevent the rebound increase in ALAS activity associated with antimony or cobalt treatment. Antimony-containing parasiticidal drugs were also potent inducers of heme oxygenase in liver and kidney. The heme degradative action of these drugs may be related in part to the jaundice commonly associated with the prolonged therapeutic use of these agents. The heme-oxygenase-inducing action of antimony-containing parasiticidal drugs is a newly defined biological property of these compounds. The relation between the parasiticidal and the heme-oxygenase-inducing actions of such drugs is unknown. However, certain parasites contain hemoproteins or require heme compounds during their life cycle. It may therefore be

  5. Chemical proteomics approach reveals the direct targets and the heme-dependent activation mechanism of artemisinin in Plasmodium falciparum using an activity-based artemisinin probe

    Jigang Wang

    2016-04-01

    Full Text Available Artemisinin and its analogues are currently the most effective anti-malarial drugs. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources. Once activated, artemisinins attack macromolecules through alkylation and propagate a series of damages, leading to parasite death. Even though several parasite proteins have been reported as artemisinin targets, the exact mechanism of action (MOA of artemisinin is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted for. Recently, we have developed an unbiased chemical proteomics approach to directly probe the MOA of artemisinin in P. falciparum. We synthesized an activity-based artemisinin probe with an alkyne tag, which can be coupled with biotin through click chemistry. This enabled selective purification and identification of 124 protein targets of artemisinin. Many of these targets are critical for the parasite survival. In vitro assays confirmed the specific artemisinin binding and inhibition of selected targets. We thus postulated that artemisinin kills the parasite through disrupting its biochemical landscape. In addition, we showed that artemisinin activation requires heme, rather than free ferrous iron, by monitoring the extent of protein binding using a fluorescent dye coupled with the alkyne-tagged artemisinin. The extremely high level of heme released from the hemoglobin digestion by the parasite makes artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages compared to parasites at early ring stage, which have low level of heme, possibly derived from endogenous synthesis. Such a unique activation mechanism also confers artemisinin with extremely high specificity against the parasites, while the healthy red blood cells are unaffected. Our results provide a sound explanation of the MOA of artemisinin and its specificity against malaria

  6. The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

    Mezö, G; Hudecz, F; Kajtár, J; Szókán, G; Szekerke, M

    1989-10-01

    New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n

  7. Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal.

    Correia, Maria Almira; Sinclair, Peter R; De Matteis, Francesco

    2011-02-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers, with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein.

  8. IRON DOME

    6 Israeli Navy 'First Arm of the Sea: The Successful Interception of the Iron Dome Rocket .... sky to destroy them whilst in flight to minimise civilian casualties. ..... Including The Moon and Celestial Bodies.53 Demeyere further emphasises the.

  9. Iron overdose

    ... tracing) X-ray to detect and track iron tablets through the stomach and intestines Treatment may include: ... BF, St. Geme JW, Schor NF, eds. Nelson Textbook of Pediatrics . 20th ed. Philadelphia, PA: Elsevier; 2016: ...

  10. Pilot-scale tests of HEME and HEPA dissolution process

    Qureshi, Z.H.; Strege, D.K.

    1994-06-01

    A series of pilot-scale demonstration tests for the dissolution of High Efficiency Mist Eliminators (HEME`s) and High Efficiency Particulate Airfilters (HEPA) were performed on a 1/5th linear scale. These fiberglass filters are to be used in the Defense Waste Processing Facility (DWPF) to decontaminate the effluents from the off-gases generated during the feed preparation process and vitrification. When removed, these filters will be dissolved in the Decontamination Waste Treatment Tank (DWTT) using 5 wt% NaOH solution. The contaminated fiberglass is converted to an aqueous stream which will be transferred to the waste tanks. The filter metal structure will be rinsed with process water before its disposal as low-level solid waste. The pilot-scale study reported here successfully demonstrated a simple one step process using 5 wt% NaOH solution. The proposed process requires the installation of a new water spray ring with 30 nozzles. In addition to the reduced waste generated, the total process time is reduced to 48 hours only (66% saving in time). The pilot-scale tests clearly demonstrated that the dissolution process of HEMEs has two stages - chemical digestion of the filter and mechanical erosion of the digested filter. The digestion is achieved by a boiling 5 wt% caustic solutions, whereas the mechanical break down of the digested filter is successfully achieved by spraying process water on the digested filter. An alternate method of breaking down the digested filter by increased air sparging of the solution was found to be marginally successful are best. The pilot-scale tests also demonstrated that the products of dissolution are easily pumpable by a centrifugal pump.

  11. Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer.

    Sunamura, Makoto; Duda, Dan G; Ghattas, Maivel H; Lozonschi, Lucian; Motoi, Fuyuhiko; Yamauchi, Jun-Ichiro; Matsuno, Seiki; Shibahara, Shigeki; Abraham, Nader G

    2003-01-01

    Angiogenesis is necessary for the continued growth of solid tumors, invasion and metastasis. Several studies clearly showed that heme oxygenase-1 (HO-1) plays an important role in angiogenesis. In this study, we used the vital microscope system, transparent skinfold model, lung colonization model and transduced pancreatic cancer cell line (Panc-1)/human heme oxygenase-1 (hHO-1) cells, to precisely analyze, for the first time, the effect of hHO-1 gene on tumor growth, angiogenesis and metastasis. Our results revealed that HO-1 stimulates angiogenesis of pancreatic carcinoma in severe combined immune deficient mice. Overexpression of human hHO-1 after its retroviral transfer into Panc-1 cells did not interfere with tumor growth in vitro. While in vivo the development of tumors was accelerated upon transfection with hHO-1. On the other hand, inhibition of heme oxygenase (HO) activity by stannous mesoporphyrin was able transiently to delay tumor growth in a dose dependent manner. Tumor angiogenesis was markedly increased in Panc-1/hHO-1 compared to mock transfected and wild type. Lectin staining and Ki-67 proliferation index confirmed these results. In addition hHO-1 stimulated in vitro tumor angiogenesis and increased endothelial cell survival. In a lung colonization model, overexpression of hHO-1 increased the occurrence of metastasis, while inhibition of HO activity by stannous mesoporphyrin completely inhibited the occurrence of metastasis. In conclusion, overexpression of HO-1 genes potentiates pancreatic cancer aggressiveness, by increasing tumor growth, angiogenesis and metastasis and that the inhibition of the HO system may be of useful benefit for the future treatment of the disease.

  12. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1982-01-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X

  13. Heme metabolism in stress regulation and protein production: from Cinderella to a key player

    Martinez Ruiz, José Luis; Petranovic, D.; Nielsen, Jens

    2016-01-01

    Heme biosynthesis is a highly conserved pathway which is present in all kingdoms, from Archaea to higher organisms such as plants and mammals. The heme molecule acts as a prosthetic group for different proteins and enzymes involved in energy metabolism and reactions involved in electron transfer....

  14. Electrochemical and spectroscopic investigations of immobilized de novo designed heme proteins on metal electrodes

    Albrecht, Tim; Li, WW; Ulstrup, Jens

    2005-01-01

    On the basis of rational design principles, template-assisted four-helix-bundle proteins that include two histidines for coordinative binding of a heme were synthesized. Spectroscopic and thermodynamic characterization of the proteins in solution reveals the expected bis-histidine coordinated heme...

  15. Unsaturated Glycerophospholipids Mediate Heme Crystallization: Biological Implications for Hemozoin Formation in the Kissing Bug Rhodnius prolixus

    Stiebler, R.; Majerowicz, David; Knudsen, Jens

    2014-01-01

    Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membrane...

  16. The haptoglobin-CD163-heme oxygenase-1 pathway for hemoglobin scavenging

    Thomsen, Jens Haugbølle; Etzerodt, Anders; Svendsen, Pia

    2013-01-01

    The haptoglobin- (Hp-) CD163-heme oxygenase-1 (HO-1) pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex...

  17. Directing a Non-Heme Iron(III)-Hydroperoxide Species on a Trifurcated Reactivity Pathway

    Wegeberg, Christina; Lauritsen, Frants R.; Frandsen, Cathrine

    2018-01-01

    The reactivity of [FeIII(tpena)]2+ (tpena=N,N,N'-tris(2-pyridylmethyl)ethylenediamine-N'-acetate) as a catalyst for oxidation reactions depends on its ratio to the terminal oxidant H2O2 and presence or absence of sacrificial substrates. The outcome can be switched between: 1)catalysed H2O2...

  18. Spray nozzle pattern test for the DWPF HEME Task QA Plan

    Lee, L.

    1991-01-01

    The DWPF melter off-gas systems have two High Efficiency Mist Eliminators (HEME) upstream of the High-Efficiency Particulates Air filters (HEPA) to remove fine mists and particulates from the off-gas. To have an acceptable filter life and an efficient operation, an air atomized water is spray on the HEME. The water spray keeps the HEME wet and dissolves the soluble particulates and enhances and HEME efficiency. DWPF Technical asked SRL to determine the conditions which will give satisfactory atomization and distribution of water so that the HEME will operate efficiently. The purpose of this document is to identify, QA controls to be applied in the pursuit of this task (WSRC-RP-91-1151)

  19. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  20. Hemoglobin fructation promotes heme degradation through the generation of endogenous reactive oxygen species

    Goodarzi, M.; Moosavi-Movahedi, A. A.; Habibi-Rezaei, M.; Shourian, M.; Ghourchian, H.; Ahmad, F.; Farhadi, M.; Saboury, A. A.; Sheibani, N.

    2014-09-01

    Protein glycation is a cascade of nonenzymatic reactions between reducing sugars and amino groups of proteins. It is referred to as fructation when the reducing monosaccharide is fructose. Some potential mechanisms have been suggested for the generation of reactive oxygen species (ROS) by protein glycation reactions in the presence of glucose. In this state, glucose autoxidation, ketoamine, and oxidative advance glycation end products (AGEs) formation are considered as major sources of ROS and perhaps heme degradation during hemoglobin glycation. However, whether fructose mediated glycation produces ROS and heme degradation is unknown. Here we report that ROS (H2O2) production occurred during hemoglobin fructation in vitro using chemiluminescence methods. The enhanced heme exposure and degradation were determined using UV-Vis and fluorescence spectrophotometry. Following accumulation of ROS, heme degradation products were accumulated reaching a plateau along with the detected ROS. Thus, fructose may make a significant contribution to the production of ROS, glycation of proteins, and heme degradation during diabetes.

  1. Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury

    Aggarwal, Saurabh; Lam, Adam; Bolisetty, Subhashini; Carlisle, Matthew A.; Traylor, Amie; Agarwal, Anupam

    2016-01-01

    Abstract Aims: Exposure to irritant gases, such as bromine (Br2), poses an environmental and occupational hazard that results in severe lung and systemic injury. However, the mechanism(s) of Br2 toxicity and the therapeutic responses required to mitigate lung damage are not known. Previously, it was demonstrated that Br2 upregulates the heme degrading enzyme, heme oxygenase-1 (HO-1). Since heme is a major inducer of HO-1, we determined whether an increase in heme and heme-dependent oxidative injury underlies the pathogenesis of Br2 toxicity. Results: C57BL/6 mice were exposed to Br2 gas (600 ppm, 30 min) and returned to room air. Thirty minutes postexposure, mice were injected intraperitoneally with a single dose of the heme scavenging protein, hemopexin (Hx) (3 μg/gm body weight), or saline. Twenty-four hours postexposure, saline-treated mice had elevated total heme in bronchoalveolar lavage fluid (BALF) and plasma and acute lung injury (ALI) culminating in 80% mortality after 10 days. Hx treatment significantly lowered heme, decreased evidence of ALI (lower protein and inflammatory cells in BALF, lower lung wet-to-dry weight ratios, and decreased airway hyperreactivity to methacholine), and reduced mortality. In addition, Br2 caused more severe ALI and mortality in mice with HO-1 gene deletion (HO-1−/−) compared to wild-type controls, while transgenic mice overexpressing the human HO-1 gene (hHO-1) showed significant protection. Innovation: This is the first study delineating the role of heme in ALI caused by Br2. Conclusion: The data suggest that attenuating heme may prove to be a useful adjuvant therapy to treat patients with ALI. Antioxid. Redox Signal. 24, 99–112. PMID:26376667

  2. Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury.

    Aggarwal, Saurabh; Lam, Adam; Bolisetty, Subhashini; Carlisle, Matthew A; Traylor, Amie; Agarwal, Anupam; Matalon, Sadis

    2016-01-10

    Exposure to irritant gases, such as bromine (Br2), poses an environmental and occupational hazard that results in severe lung and systemic injury. However, the mechanism(s) of Br2 toxicity and the therapeutic responses required to mitigate lung damage are not known. Previously, it was demonstrated that Br2 upregulates the heme degrading enzyme, heme oxygenase-1 (HO-1). Since heme is a major inducer of HO-1, we determined whether an increase in heme and heme-dependent oxidative injury underlies the pathogenesis of Br2 toxicity. C57BL/6 mice were exposed to Br2 gas (600 ppm, 30 min) and returned to room air. Thirty minutes postexposure, mice were injected intraperitoneally with a single dose of the heme scavenging protein, hemopexin (Hx) (3 μg/gm body weight), or saline. Twenty-four hours postexposure, saline-treated mice had elevated total heme in bronchoalveolar lavage fluid (BALF) and plasma and acute lung injury (ALI) culminating in 80% mortality after 10 days. Hx treatment significantly lowered heme, decreased evidence of ALI (lower protein and inflammatory cells in BALF, lower lung wet-to-dry weight ratios, and decreased airway hyperreactivity to methacholine), and reduced mortality. In addition, Br2 caused more severe ALI and mortality in mice with HO-1 gene deletion (HO-1-/-) compared to wild-type controls, while transgenic mice overexpressing the human HO-1 gene (hHO-1) showed significant protection. This is the first study delineating the role of heme in ALI caused by Br2. The data suggest that attenuating heme may prove to be a useful adjuvant therapy to treat patients with ALI.

  3. Enhanced Heme Function and Mitochondrial Respiration Promote the Progression of Lung Cancer Cells

    Alam, Md Maksudul; Shah, Ajit; Cao, Thai M.; Sullivan, Laura A.; Brekken, Rolf; Zhang, Li

    2013-01-01

    Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer

  4. Basal serum pancreatic polypeptide is dependent on age and gender in an adult population

    Brimnes Damholt, M; Rasmussen, B K; Hilsted, L

    1997-01-01

    This study is the first epidemiologically based study of basal levels of serum pancreatic polypeptide (s-PP). The basal level of serum PP has become a field of interest mainly due to the role of PP as an endocrine tumour marker, and as a marker of pancreatic neuroendocrine function after pancreas...... a monospecific radioimmunoassay. Fasting serum pancreatic polypeptide depended on age and gender. The results demonstrated that fasting pancreatic polypeptide levels increase exponentially with age. Fitted separately for each sex, basal serum pancreatic polypeptide was found to increase by approximately 3% per...... reports on the fasting levels of serum pancreatic polypeptide are most likely due to lack of adjustment for age and gender. Thus, variation due to age and gender should be considered in evaluating fasting levels of serum pancreatic polypeptide. Whether similar considerations are important when evaluating...

  5. Heme and menaquinone induced electron transport in lactic acid bacteria

    Santos Filipe

    2009-05-01

    Full Text Available Abstract Background For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Results Heme- (and menaquinone stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. Conclusion We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species.

  6. The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication.

    Hill-Batorski, Lindsay; Halfmann, Peter; Neumann, Gabriele; Kawaoka, Yoshihiro

    2013-12-01

    Ebola virus (EBOV) is the causative agent of a severe hemorrhagic fever in humans with reported case fatality rates as high as 90%. There are currently no licensed vaccines or antiviral therapeutics to combat EBOV infections. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting step in heme degradation, has antioxidative properties and protects cells from various stresses. Activated HO-1 was recently shown to have antiviral activity, potently inhibiting the replication of viruses such as hepatitis C virus and human immunodeficiency virus. However, the effect of HO-1 activation on EBOV replication remains unknown. To determine whether the upregulation of HO-1 attenuates EBOV replication, we treated cells with cobalt protoporphyrin (CoPP), a selective HO-1 inducer, and assessed its effects on EBOV replication. We found that CoPP treatment, pre- and postinfection, significantly suppressed EBOV replication in a manner dependent upon HO-1 upregulation and activity. In addition, stable overexpression of HO-1 significantly attenuated EBOV growth. Although the exact mechanism behind the antiviral properties of HO-1 remains to be elucidated, our data show that HO-1 upregulation does not attenuate EBOV entry or budding but specifically targets EBOV transcription/replication. Therefore, modulation of the cellular enzyme HO-1 may represent a novel therapeutic strategy against EBOV infection.

  7. Heme and menaquinone induced electron transport in lactic acid bacteria.

    Brooijmans, Rob; Smit, Bart; Santos, Filipe; van Riel, Jan; de Vos, Willem M; Hugenholtz, Jeroen

    2009-05-29

    For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species.

  8. Energy transfer at the active sites of heme proteins

    Dlott, D.D.; Hill, J.R.

    1995-01-01

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

  9. Heme oxygenase-1 comes back to endoplasmic reticulum

    Kim, Hong Pyo [School of Biological Sciences, Ulsan University (Korea, Republic of); Pae, Hyun-Ock [Department of Immunology, Wonkwang University School of Medicine (Korea, Republic of); Back, Sung Hun; Chung, Su Wol [School of Biological Sciences, Ulsan University (Korea, Republic of); Woo, Je Moon [Department of Opthalmology, Ulasn University Hospital (Korea, Republic of); Son, Yong [Department of Anesthesiology and Pain Medicine, Wonkwang University School of Medicine (Korea, Republic of); Chung, Hun-Taeg, E-mail: chung@ulsan.ac.kr [School of Biological Sciences, Ulsan University (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

  10. Biochemical map of polypeptides specified by foot-and-mouth disease virus.

    Grubman, M J; Robertson, B H; Morgan, D O; Moore, D M; Dowbenko, D

    1984-01-01

    Pulse-chase labeling of foot-and-mouth disease virus-infected bovine kidney cells revealed stable and unstable viral-specific polypeptides. To identify precursor-product relationships among these polypeptides, antisera against a number of structural and nonstructural viral-specific polypeptides were used. Cell-free translations programmed with foot-and-mouth disease virion RNA or foot-and-mouth disease virus-infected bovine kidney cell lysates, which were shown to contain almost identical pol...

  11. Primary structure, gene organization and polypeptide expression of poliovirus RNA

    Kitamura, N. (State Univ. of New York, Stony Brook); Semler, B.L.; Rothberg, P.G.

    1981-06-18

    The primary structure of the poliovirus genome has been determined. The RNA molecule is 7433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

  12. Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

    Garrison, W.M.

    1985-01-01

    The purpose of this review is to bring together and to correlate the wide variety of experimental studies that provide information on the reaction products and reaction mechanisms involved in the radiolysis of peptides, polypeptides and proteins (including chromosomal proteins) in both aqueous and solid-state systems. The comparative radiation chemistry of these systems is developed in terms of specific reactions of the peptide main-chain and the aliphatic, aromatic-unsaturated and sulfur-containing side-chains. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis and ESR spectroscopy is included. 147 refs.

  13. Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

    Garrison, W.M.

    1985-01-01

    The purpose of this review is to bring together and to correlate the wide variety of experimental studies that provide information on the reaction products and reaction mechanisms involved in the radiolysis of peptides, polypeptides and proteins (including chromosomal proteins) in both aqueous and solid-state systems. The comparative radiation chemistry of these systems is developed in terms of specific reactions of the peptide main-chain and the aliphatic, aromatic-unsaturated and sulfur-containing side-chains. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis and ESR spectroscopy is included. 147 refs

  14. NMR study of the cooperative behavior of thermotropic model polypeptides

    Kurková, Dana; Kříž, Jaroslav; Rodríguez-Cabello, J. C.; Arias, F. J.

    2007-01-01

    Roč. 56, č. 2 (2007), s. 186-194 ISSN 0959-8103 R&D Projects: GA AV ČR IAA400500604 Grant - others:Spanish Ministry of Science and Culture(ES) A002/02; MAT2000-1764-C02; MAT2001-1853-C02-01; MAT2003- Institutional research plan: CEZ:AV0Z40500505 Keywords : thermotropic polymers * cooperativity * synthetic polypeptides Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.557, year: 2007

  15. Well-defined (co)polypeptides bearing pendant alkyne groups

    Zhao, Wei

    2016-03-18

    A novel metal-free strategy, using hydrogen-bonding catalytic ring opening polymerization of acetylene-functionalized N-carboxy anhydrites of α-amino acids, was developed for the synthesis of well-defined polypeptides bearing pendant alkyne groups. This method provides an efficient way to synthesize novel alkyne-functionalized homopolypeptides (A) and copolypeptides, such as AB diblock (B: non-functionalized), ABA triblock and star-AB diblock, as well as linear and star random copolypeptides, precursors of a plethora complex macromolecular architectures by click chemistry.

  16. Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins.

    Deming, Timothy J

    2017-03-15

    Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

  17. Well-defined (co)polypeptides bearing pendant alkyne groups

    Zhao, Wei; Gnanou, Yves; Hadjichristidis, Nikolaos

    2016-01-01

    A novel metal-free strategy, using hydrogen-bonding catalytic ring opening polymerization of acetylene-functionalized N-carboxy anhydrites of α-amino acids, was developed for the synthesis of well-defined polypeptides bearing pendant alkyne groups. This method provides an efficient way to synthesize novel alkyne-functionalized homopolypeptides (A) and copolypeptides, such as AB diblock (B: non-functionalized), ABA triblock and star-AB diblock, as well as linear and star random copolypeptides, precursors of a plethora complex macromolecular architectures by click chemistry.

  18. Iron-Deficiency Anemia

    Full Text Available ... iron-rich foods, especially during certain stages of life when more iron is needed, such as childhood and pregnancy. Good sources of iron are meat, poultry, fish, and iron- ...

  19. Iron-Deficiency Anemia

    Full Text Available ... that are good sources of iron include dried beans, dried fruits, eggs, lean red meat, salmon, iron- ... of iron, including iron-fortified breads and cereals, beans, tofu, dried fruits, and spinach and other dark ...

  20. Iron in diet

    ... Reasonable amounts of iron are also found in lamb, pork, and shellfish. Iron from vegetables, fruits, grains, ... strawberries, tomatoes, and potatoes) also increase iron absorption. Cooking foods in a cast-iron skillet can also ...

  1. Iron-Deficiency Anemia

    Full Text Available ... from developing iron-deficiency anemia. Foods that are good sources of iron include dried beans, dried fruits, ... iron is needed, such as childhood and pregnancy. Good sources of iron are meat, poultry, fish, and ...

  2. Iron-Deficiency Anemia

    Full Text Available ... you are diagnosed with iron-deficiency anemia. Risk Factors You may have an increased risk for iron- ... iron-deficiency anemia if you have certain risk factors , including pregnancy. To prevent iron-deficiency anemia, your ...

  3. Iron-Deficiency Anemia

    Full Text Available ... for your body to absorb iron from the gastrointestinal tract (GI tract). Blood loss When you lose blood, ... iron deficiency. Endurance athletes lose iron through their gastrointestinal tracts. They also lose iron through the breakdown of ...

  4. Iron-Deficiency Anemia

    Full Text Available ... iron in your body is low. For this reason, other iron tests are also done. Ferritin measure ... iron is needed, such as childhood and pregnancy. Good sources of iron are meat, poultry, fish, and ...

  5. Iron-Deficiency Anemia

    Full Text Available ... develop new therapies for conditions that affect the balance of iron in the body and lead to ... Disease Control and Prevention) Iron - Health Professional Fact Sheet (NIH) Iron Dietary Supplement Fact Sheet (NIH) Iron- ...

  6. Iron-Deficiency Anemia

    Full Text Available ... to moderate iron-deficiency anemia, or red blood cell transfusion for severe iron-deficiency anemia. You may ... body needs iron to make healthy red blood cells. Iron-deficiency anemia usually develops over time because ...

  7. Iron-Deficiency Anemia

    Full Text Available ... enough iron-rich foods, such as meat and fish, may result in you getting less than the ... pregnancy. Good sources of iron are meat, poultry, fish, and iron-fortified foods that have iron added. ...

  8. Iron Dextran Injection

    Iron dextran injection is used to treat iron-deficiency anemia (a lower than normal number of red blood cells ... treated with iron supplements taken by mouth. Iron dextran injection is in a class of medications called ...

  9. Iron-Deficiency Anemia

    Full Text Available ... and severity. Treatments may include iron supplements, procedures, surgery, and dietary ... iron supplements, also called iron pills or oral iron, by mouth once or several times a ...

  10. Iron-Deficiency Anemia

    Full Text Available ... developing iron-deficiency anemia. Foods that are good sources of iron include dried beans, dried fruits, eggs, ... is needed, such as childhood and pregnancy. Good sources of iron are meat, poultry, fish, and iron- ...

  11. Iron deficiency

    Schou, Morten; Bosselmann, Helle; Gaborit, Freja

    2015-01-01

    BACKGROUND: Both iron deficiency (ID) and cardiovascular biomarkers are associated with a poor outcome in heart failure (HF). The relationship between different cardiovascular biomarkers and ID is unknown, and the true prevalence of ID in an outpatient HF clinic is probably overlooked. OBJECTIVES.......043). CONCLUSION: ID is frequent in an outpatient HF clinic. ID is not associated with cardiovascular biomarkers after adjustment for traditional confounders. Inflammation, but not neurohormonal activation is associated with ID in systolic HF. Further studies are needed to understand iron metabolism in elderly HF...

  12. Regulation of human heme oxygenase-1 gene expression under thermal stress.

    Okinaga, S; Takahashi, K; Takeda, K; Yoshizawa, M; Fujita, H; Sasaki, H; Shibahara, S

    1996-06-15

    Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.

  13. A Heme-Sensing Mechanism in the Translational Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

    Soto, Iliana C.; Fontanesi, Flavia; Myers, Richard S.; Hamel, Patrice; Barrientos, Antoni

    2012-01-01

    Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, is a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N-terminus. Using a combination of in vitro and in vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production. PMID:23217259

  14. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-01-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and 14 C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of 14 C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose

  15. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    Christopher M Brennan

    Full Text Available The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA, the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  16. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-01-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  17. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    Souza, C.F. [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Carneiro, A.B.; Silveira, A.B. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); Laranja, G.A.T. [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); Silva-Neto, M.A.C. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); INCT, Entomologia Molecular (Brazil); Costa, S.C. Goncalves da [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Paes, M.C., E-mail: mcpaes@uerj.br [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); INCT, Entomologia Molecular (Brazil)

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  18. Relationship between Antimalarial Activity and Heme Alkylation for Spiro- and Dispiro-1,2,4-Trioxolane Antimalarials▿

    Creek, Darren J.; Charman, William N.; Chiu, Francis C. K.; Prankerd, Richard J.; Dong, Yuxiang; Vennerstrom, Jonathan L.; Charman, Susan A.

    2008-01-01

    The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study. PMID:18268087

  19. Heme as a danger molecule in pathogen recognition.

    Wegiel, Barbara; Hauser, Carl J; Otterbein, Leo E

    2015-12-01

    Appropriate control of redox mechanisms are critical for and effective innate immune response, which employs multiple cell types, receptors and molecules that recognize danger signals when they reach the host. Recognition of pathogen-associated pattern molecules (PAMPs) is a fundamental host survival mechanism for efficient elimination of invading pathogens and resolution of the infection and inflammation. In addition to PAMPs, eukaryotic cells contain a plethora of intracellular molecules that are normally secured within the confines of the plasma membrane, but if liberated and encountered in the extracellular milieu can provoke rapid cell activation. These are known as Alarmins or Danger-Associated Molecular Patterns (DAMPs) and can be released actively by cells or passively as a result of sterile cellular injury after trauma, ischemia, or toxin-induced cell rupture. Both PAMPs and DAMPs are recognized by a series of cognate receptors that increase the generation of free radicals and activate specific signaling pathways that result in regulation of a variety of stress response, redox sensitive genes. Multiple mediators released, as cells die include, but are not limited to ATP, hydrogen peroxide, heme, formyl peptides, DNA or mitochondria provide the second signal to amplify immune responses. In this review, we will focus on how sterile and infective stimuli activate the stress response gene heme oxygenase-1 (Hmox1, HO-1), a master gene critical to an appropriate host response that is now recognized as one with enormous therapeutic potential. HO-1 gene expression is regulated in large part by redox-sensitive proteins including but not limited to nrf2. Both PAMPs and DAMPs increase the activation of nrf2 and HO-1. Heme is a powerful pro-oxidant and as such should be qualified as a DAMP. With its degradation by HO-1a molecule of carbon monoxide (CO) is generated that in turn serves as a bioactive signaling molecule. PAMPs such as bacterial endotoxin activate HO-1

  20. CDNA encoding a polypeptide including a hevein sequence

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  1. cDNA encoding a polypeptide including a hevein sequence

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  2. Light Scattering Characterization of Elastin-Like Polypeptide Trimer Micelles

    Tsuper, Ilona; Terrano, Daniel; Maraschky, Adam; Holland, Nolan; Streletzky, Kiril

    The elastin-like polypeptides (ELP) nanoparticles are composed of three-armed star polypeptides connected by a negatively charged foldon. Each of the three arms extending from the foldon domain includes 20 repeats of the (GVGVP) amino acid sequence. The ELP polymer chains are soluble at room temperature and become insoluble at the transition temperature (close to 50 ° C), forming micelles. The size and shape of the micelle are dependent on the temperature and the pH of the solution, and on the concentration of the phosphate buffered saline (PBS). The depolarized dynamic light scattering (DDLS) was employed to study the structure and dynamics of micelles at 62 ° C. The solution was maintained at an approximate pH level of 7.3 - 7.5, while varying PBS concentration. At low salt concentrations (60 mM) displayed an apparent elongation of the micelles evident by a significant VH signal, along with a surge in the apparent Rh. A model of micelle growth (and potential elongation) with increase in salt concentration is considered.

  3. cDNA encoding a polypeptide including a hevein sequence

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

  4. cDNA encoding a polypeptide including a hevein sequence

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  5. cDNA encoding a polypeptide including a hevein sequence

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

  6. Iron-Deficiency Anemia

    Full Text Available ... Research Home / < Back To Health Topics / Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer en español Iron-deficiency ... iron-deficiency anemia. Blood tests to screen for iron-deficiency anemia To screen for iron-deficiency anemia, your doctor ...

  7. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

    Kathiresan, Meena; Martins, Dorival; English, Ann M.

    2014-01-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

  8. Allocation of Heme is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells

    Nino Asuela Espinas

    2016-08-01

    Full Text Available Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1 and null (fc1-2 mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1 and null (fc2-2 mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.

  9. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

    Kathiresan, Meena; Martins, Dorival; English, Ann M

    2014-12-09

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

  10. Iron and iron derived radicals

    Borg, D.C.; Schaich, K.M.

    1987-04-01

    We have discussed some reactions of iron and iron-derived oxygen radicals that may be important in the production or treatment of tissue injury. Our conclusions challenge, to some extent, the usual lines of thought in this field of research. Insofar as they are born out by subsequent developments, the lessons they teach are two: Think fast! Think small! In other words, think of the many fast reactions that can rapidly alter the production and fate of highly reactive intermediates, and when considering the impact of competitive reactions on such species, think how they affect the microenvironment (on the molecular scale) ''seen'' by each reactive molecule. 21 refs., 3 figs., 1 tab

  11. Iron – a key nexus in the virulence of Aspergillus fumigatus

    Hubertus eHaas

    2012-02-01

    Full Text Available Iron is an essential but in excess toxic nutrient. Therefore, fungi evolved fine-tuned mechanisms for uptake and storage of iron, such as the production of siderophores (low-molecular mass iron-specific chelators. In Aspergillus fumigatus, iron starvation causes extensive transcriptional remodeling involving two central transcription factors, which are interconnected in a negative transcriptional feed-back loop: the GATA-factor SreA and the bZip-factor HapX. During iron sufficiency SreA represses iron uptake, including reductive iron assimilation and siderophore-mediated iron uptake, to avoid toxic effects. During iron starvation HapX represses iron-consuming pathways, including heme biosynthesis and respiration, to spare iron and activates synthesis of ribotoxin AspF1 and siderophores, the latter partly by ensuring supply of the precursor ornithine. In agreement with the expression pattern and mode of action, detrimental effects of inactivation of SreA and HapX are confined to growth during iron sufficiency and iron starvation, respectively. Deficiency in HapX, but not SreA, attenuates virulence of A. fumigatus in a murine model of aspergillosis, which underlines the crucial role of adaptation to iron limitation in virulence. Consistently, production of both extra- and intracellular siderophores is crucial for virulence of A. fumigatus. Recently, the sterol-regulatory element-binding protein SrbA was found to be essential for adaptation to iron starvation, thereby linking regulation of iron metabolism, ergosterol biosynthesis, azole drug resistance and hypoxia adaptation.

  12. Effects of 1,2-dibromo-3-chloropropane on hepatic heme synthesis

    Moody, D.E.; Clawson, G.A.; Piper, W.N.; Smuckler, E.A.

    1984-01-01

    Previous studies showed that 1,2-dibromo-3-chloropropane (DBCP) caused a decrease in hepatic microsomal cytochrome P-450 suggesting that hepatic heme metabolism may be affected by DBCP treatment. Various parameters of hepatic heme synthesis were measured at intervals ranging from 0 to 72 hr in male Sprague-Dawley rats given a single oral dose (200 mg/kg) of DBCP. Incorporation of the radiolabeled heme precursor [delta-14C]aminolevulinic acid (14C-ALA) into liver, protein, extracted heme, and subcellular fractions of liver homogenates was significantly decreased to 75, 58, and 81% of controls, respectively, at 24 hr. At 48 and 72 hr after DBCP treatment, the accumulation of 14C-ALA label after 4 hr in liver homogenates and subcellular fractions was significantly increased in comparison to controls. These changes in 14C-ALA uptake were accompanied by decreases in total liver and microsomal heme, but not mitochondrial heme. Decreases were found in the spectral content of two heme proteins, cytochromes P-450 and b5, and the activity of another heme protein, catalase. Heme oxygenase activity increased to 130, 151, 209, and 186% of control values at 12, 24, 48, and 72 hr after DBCP, respectively. A slight, but significant, increase in ALA-synthetase to 112% of controls occurred at 24 hr, and slight, but significant, decreases in ALA-dehydratase to 90 and 80% of control occurred at 12 and 24 hr, respectively. No significant changes in uroporphyrinogen-1-synthetase or ferrochelatase at the time points tested was noted. The porphyrin content of liver was increased to 130% of control, while the serum and urine porphyrin levels were decreased to 30% of the control values at 24 hr. Liver ALA content was not significantly altered through the time period studied, but serum and urine levels were increased at 24 hr to 176 and 130% of the control values, respectively. In conclusion, the decreases in liver heme proteins following a single oral dose of DBCP are accompanied by

  13. Selective activation of heme oxygenase-2 by menadione.

    Vukomanovic, Dragic; McLaughlin, Brian E; Rahman, Mona N; Szarek, Walter A; Brien, James F; Jia, Zongchao; Nakatsu, Kanji

    2011-11-01

    While substantial progress has been made in elucidating the roles of heme oxygenases-1 (HO-1) and -2 (HO-2) in mammals, our understanding of the functions of these enzymes in health and disease is still incomplete. A significant amount of our knowledge has been garnered through the use of nonselective inhibitors of HOs, and our laboratory has recently described more selective inhibitors for HO-1. In addition, our appreciation of HO-1 has benefitted from the availability of tools for increasing its activity through enzyme induction. By comparison, there is a paucity of information about HO-2 activation, with only a few reports appearing in the literature. This communication describes our observations of the up to 30-fold increase in the in-vitro activation of HO-2 by menadione. This activation was due to an increase in Vmax and was selective, in that menadione did not increase HO-1 activity.

  14. Coordination Chemistry of Linear Oligopyrrolic Fragments Inspired by Heme Metabolites

    Gautam, Ritika

    Linear oligopyrroles are degradation products of heme, which is converted in the presence of heme oxygenase to bile pigments, such as biliverdin and bilirubin. These tetrapyrrolic oligopyrroles are ubiquitously present in biological systems and find applications in the fields of catalysis and sensing. These linear tetrapyrrolic scaffolds are further degraded into linear tripyrrolic and dipyrrolic fragments. Although these lower oligopyrroles are abundantly present, their coordination chemistry requires further characterization. This dissertation focuses mainly on two classes of bioinspired linear oligopyrroles, propentdyopent and tripyrrindione, and their transition metal complexes, which present a rich ligand-based redox chemistry. Chapter 1 offers an overview of heme degradation to different classes of linear oligopyrroles and properties of their transition metal complexes. Chapter 2 is focused on the tripyrrin-1,14-dione scaffold of the urinary pigment uroerythrin, which coordinates divalent transition metals palladium and copper with square planar geometry. Specifically, the tripyrrin-1, 14-dione ligand binds Cu(II) and Pd(II) as a dianionic organic radical under ambient conditions. The electrochemical study confirms the presence of ligand based redox chemistry, and one electron oxidation or reduction reactions do not alter the planar geometry around the metal center. The X-Ray analysis and the electron paramagnetic resonance (EPR) studies of the complexes in the solid and solution phase reveals intermolecular interactions between the ligand based unpaired electrons and therefore formation of neutral pi-pi dimers. In Chapter 3, the antioxidant activity and the fluorescence sensor properties of the tripyrrin-1,14-dione ligand in the presence of superoxide are described. We found that the tripyrrindione ligand undergoes one-electron reduction in the presence of the superoxide radical anion (O2•- ) to form highly fluorescent H3TD1•- radical anion, which emits

  15. Pilot-scale tests of HEME and HEPA dissolution process

    Qureshi, Z.H.; Strege, D.K.

    1994-06-01

    A series of pilot-scale demonstration tests for the dissolution of High Efficiency Mist Eliminators (HEME's) and High Efficiency Particulate Airfilters (HEPA) were performed on a 1/5th linear scale. These fiberglass filters are to be used in the Defense Waste Processing Facility (DWPF) to decontaminate the effluents from the off-gases generated during the feed preparation process and vitrification. When removed, these filters will be dissolved in the Decontamination Waste Treatment Tank (DWTT) using 5 wt% NaOH solution. The contaminated fiberglass is converted to an aqueous stream which will be transferred to the waste tanks. The filter metal structure will be rinsed with process water before its disposal as low-level solid waste. The pilot-scale study reported here successfully demonstrated a simple one step process using 5 wt% NaOH solution. The proposed process requires the installation of a new water spray ring with 30 nozzles. In addition to the reduced waste generated, the total process time is reduced to 48 hours only (66% saving in time). The pilot-scale tests clearly demonstrated that the dissolution process of HEMEs has two stages - chemical digestion of the filter and mechanical erosion of the digested filter. The digestion is achieved by a boiling 5 wt% caustic solutions, whereas the mechanical break down of the digested filter is successfully achieved by spraying process water on the digested filter. An alternate method of breaking down the digested filter by increased air sparging of the solution was found to be marginally successful are best. The pilot-scale tests also demonstrated that the products of dissolution are easily pumpable by a centrifugal pump

  16. Peroxide-Dependent Analyte Conversion by the Heme Prosthetic Group, the Heme Peptide “Microperoxidase-11” and Cytochrome c on Chitosan Capped Gold Nanoparticles Modified Electrodes

    Frieder W. Scheller

    2012-05-01

    Full Text Available In view of the role ascribed to the peroxidatic activity of degradation products of cytochrome c (cyt c in the processes of apoptosis, we investigate the catalytic potential of heme and of the cyt c derived heme peptide MP-11 to catalyse the cathodic reduction of hydrogen peroxide and to oxidize aromatic compounds. In order to check whether cyt c has an enzymatic activity in the native state where the protein matrix should suppress the inherent peroxidatic activity of its heme prosthetic group, we applied a biocompatible immobilization matrix and very low concentrations of the co-substrate H2O2. The biocatalysts were entrapped on the surface of a glassy carbon electrode in a biocompatible chitosan layer which contained gold nanoparticles. The electrochemical signal for the peroxide reduction is generated by the redox conversion of the heme group, whilst a reaction product of the substrate oxidation is cathodically reduced in the substrate indication. The catalytic efficiency of microperoxidase-11 is sufficient for sensors indicating HRP substrates, e.g., p-aminophenol, paracetamol and catechol, but also the hydroxylation of aniline and dehalogenation of 4-fluoroaniline. The lower limit of detection for p-aminophenol is comparable to previously published papers with different enzyme systems. The peroxidatic activity of cyt c immobilized in the chitosan layer for catechol was found to be below 1 per mill and for p-aminophenol about 3% as compared with that of heme or MP-11.

  17. Polypeptide synthesis in alphavirus-infected aedes albopictus cells during the establishment of persistent infection

    Richardson, M.A.; Boulton, R.W.; Raghow, R.S.; Dalgarno, L.

    1980-01-01

    Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cells, RRV reached peak titres at 34-48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was not shutdown of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s).The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9-11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity. (author)

  18. Anti-inflammatory effect of transduced PEP-1-heme oxygenase-1 in Raw 264.7 cells and a mouse edema model

    Kwon, Soon Won; Sohn, Eun Jeong; Kim, Dae Won; Jeong, Hoon Jae; Kim, Mi Jin; Ahn, Eun Hee; Kim, Young Nam; Dutta, Suman; Kim, Duk-Soo; Park, Jinseu; Eum, Won Sik; Hwang, Hyun Sook; Choi, Soo Young

    2011-01-01

    Highlights: → Recombinant PEP-1 heme oxygenase-1 expression vector was constructed and overexpressed. → We investigated transduction efficiency of PEP-1-HO-1 protein in Raw 264.7 cells. → PEP-1-HO-1 was efficiently transduced into Raw 264.7 cells in a dose and time dependent manner. → PEP-1-HO-1 exerted anti-inflammatory activity in Raw 264.7 cells and in a mice edema model. → PEP-1-HO-1 could be used as a therapeutic drug against inflammatory diseases. -- Abstract: Heme oxygenase-1 (HO-1), which catalyzes the degradation of free heme to biliverdin, carbon monoxide (CO), and free iron (Fe 2+ ), is up-regulated by several cellular stress and cell injuries, including inflammation, ischemia and hypoxia. In this study, we examined whether fusion of HO-1 with PEP-1, a protein transduction domain that is able to deliver exogenous molecules to living cells or tissues, would facilitate HO-1 delivery to target cells and tissues, and thereby effectively exert a therapeutically useful response against inflammation. Western blot analysis demonstrated that PEP-1-HO-1 fusion proteins were transduced into Raw 264.7 cells in time- and dose-dependent manners, and were stably maintained in the cells for about 60 h. In addition, fluorescence analysis revealed that only PEP-1-HO-1 fusion proteins were significantly transduced into the cytoplasm of cells, while HO-1 proteins failed to be transduced. In lipopolysaccharide (LPS)-stimulated Raw 264.7 cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse edema model, transduced PEP-1-HO-1 fusion proteins effectively inhibited the overexpression of pro-inflammatory mediators and cytokines. Also, histological analysis demonstrated that PEP-1-HO-1 remarkably suppressed ear edema. The results suggest that the PEP-1-HO-1 fusion protein can be used as a therapeutic molecule against reactive oxygen species-related inflammatory diseases.

  19. Ring-Opening Polymerization of N-Carboxyanhydrides for Preparation of Polypeptides and Polypeptide-Based Hybrid Materials with Various Molecular Architectures

    Pahovnik, David

    2015-09-01

    Different synthetic approaches utilizing ring-opening polymerization of N-carboxyanhydrides for preparation of polypeptide and polypeptide-based hybrid materials with various molecular architectures are described. An overview of polymerization mechanisms using conventional (various amines) as well as some recently developed initiators (hexamethyldisilazane, N-heterocyclic persistent carbenes, etc.) is presented, and their benefits and drawbacks for preparation of polypeptides with well-defined chain lengths and chain-end functionality are discussed. Recent examples from literature are used to illustrate different possibilities for synthesis of pure polypeptide materials with different molecular architectures bearing various functional groups, which are introduced either by modification of amino acids, before they are transformed into corresponding Ncarboxyanhydrides, or by post-polymerization modifications using protective groups and/or orthogonal functional groups. Different approaches for preparation of polypeptide-based hybrid materials are discussed as well using examples from recent literature. Syntheses of simple block copolymers or copolymers with more complex molecular architectures (graft and star copolymers) as well as modifications of nanoparticles and other surfaces with polypeptides are described.

  20. Intersections of pathways involving biotin and iron relative to therapeutic mechanisms for progressive multiple sclerosis.

    Heidker, Rebecca M; Emerson, Mitchell R; LeVine, Steven M

    2016-12-01

    While there are a variety of therapies for relapsing remitting multiple sclerosis (MS), there is a lack of treatments for progressive MS. An early study indicated that high dose biotin therapy has beneficial effects in approximately 12-15% of patients with progressive MS. The mechanisms behind the putative improvements seen with biotin therapy are not well understood, but have been postulated to include: 1) improving mitochondrial function which is impaired in MS, 2) increasing synthesis of lipids and cholesterol to facilitate remyelination, and 3) affecting gene expression. We suggest one reason that a greater percentage of patients with MS didn't respond to biotin therapy is the inaccessibility or lack of other nutrients, such as iron. In addition to biotin, iron (or heme) is necessary for energy production, biosynthesis of cholesterol and lipids, and for some protective mechanisms. Both biotin and iron are required for myelination during development, and by inference, remyelination. However, iron can also play a role in the pathology of MS. Increased deposition of iron can occur in some CNS structures possibly promoting oxidative damage while low iron levels can occur in other areas. Thus, the potential, detrimental effects of iron need to be considered together with the need for iron to support metabolic demands associated with repair and/or protective processes. We propose the optimal utilization of iron may be necessary to maximize the beneficial effects of biotin. This review will examine the interactions between biotin and iron in pathways that may have therapeutic or pathogenic implications for MS.

  1. Density functional theory calculations of the lowest energy quintet and triplet states of model hemes: role of functional, basis set, and zero-point energy corrections.

    Khvostichenko, Daria; Choi, Andrew; Boulatov, Roman

    2008-04-24

    We investigated the effect of several computational variables, including the choice of the basis set, application of symmetry constraints, and zero-point energy (ZPE) corrections, on the structural parameters and predicted ground electronic state of model 5-coordinate hemes (iron(II) porphines axially coordinated by a single imidazole or 2-methylimidazole). We studied the performance of B3LYP and B3PW91 with eight Pople-style basis sets (up to 6-311+G*) and B97-1, OLYP, and TPSS functionals with 6-31G and 6-31G* basis sets. Only hybrid functionals B3LYP, B3PW91, and B97-1 reproduced the quintet ground state of the model hemes. With a given functional, the choice of the basis set caused up to 2.7 kcal/mol variation of the quintet-triplet electronic energy gap (DeltaEel), in several cases, resulting in the inversion of the sign of DeltaEel. Single-point energy calculations with triple-zeta basis sets of the Pople (up to 6-311G++(2d,2p)), Ahlrichs (TZVP and TZVPP), and Dunning (cc-pVTZ) families showed the same trend. The zero-point energy of the quintet state was approximately 1 kcal/mol lower than that of the triplet, and accounting for ZPE corrections was crucial for establishing the ground state if the electronic energy of the triplet state was approximately 1 kcal/mol less than that of the quintet. Within a given model chemistry, effects of symmetry constraints and of a "tense" structure of the iron porphine fragment coordinated to 2-methylimidazole on DeltaEel were limited to 0.3 kcal/mol. For both model hemes the best agreement with crystallographic structural data was achieved with small 6-31G and 6-31G* basis sets. Deviation of the computed frequency of the Fe-Im stretching mode from the experimental value with the basis set decreased in the order: nonaugmented basis sets, basis sets with polarization functions, and basis sets with polarization and diffuse functions. Contraction of Pople-style basis sets (double-zeta or triple-zeta) affected the results

  2. Iron and stony-iron meteorites

    Ruzicka, Alex M.; Haack, Henning; Chabot, Nancy L.

    2017-01-01

    By far most of the melted and differentiated planetesimals that have been sampled as meteorites are metal-rich iron meteorites or stony iron meteorites. The parent asteroids of these meteorites accreted early and differentiated shortly after the solar system formed, producing some of the oldest...... and interpretations for iron and stony iron meteorites (Plate 13.1). Such meteorites provide important constraints on the nature of metal-silicate separation and mixing in planetesimals undergoing partial to complete differentiation. They include iron meteorites that formed by the solidification of cores...... (fractionally crystallized irons), irons in which partly molten metal and silicates of diverse types were mixed together (silicate-bearing irons), stony irons in which partly molten metal and olivine from cores and mantles were mixed together (pallasites), and stony irons in which partly molten metal...

  3. Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38

    Amin, Faisal Mohammad; Hougaard, Anders; Schytz, Henrik W

    2014-01-01

    Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide are structurally and functionally closely related but show differences in migraine-inducing properties. Mechanisms responsible for the difference in migraine induction are unknown. Here, for the ...

  4. Identification and phylogenetic analysis of heme synthesis genes in trypanosomatids and their bacterial endosymbionts.

    João M P Alves

    Full Text Available It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellate's cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.

  5. The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi

    Marcia Cristina Paes

    2011-01-01

    Full Text Available Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

  6. Analysis of the electrochemistry of hemes with Ems spanning 800 mV

    Zheng, Zhong; Gunner, M. R.

    2009-01-01

    The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of Ems with a single type of redox cofactor. Proteins containing 141 unique hemes of a-, b-, and c-type, with bis-His, His-Met, and aquo-His ligation were calculated using Multi-Conformation Continuum Electrostatics (MCCE). The experimental Ems range over 800 mV from −350 mV in cytochrome c3 to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the protein backbone, and sidechains. MCCE calculated Ems are in good agreement with measured values. Using no free parameters the slope of the line comparing calculated and experimental Ems is 0.73 (R2 = 0.90), showing the method accounts for 73% of the observed Em range. Adding a +160 mV correction to the His-Met c-type hemes yields a slope of 0.97 (R2 = 0.93). With the correction 65% of the hemes have an absolute error smaller than 60 mV and 92% are within 120 mV. The overview of heme proteins with known structures and Ems shows both the lowest and highest potential hemes are c-type, whereas the b-type hemes are found in the middle Em range. In solution, bis-His ligation lowers the Em by ≈205 mV relative to hemes with His-Met ligands. The bis-His, aquo-His, and His-Met ligated b-type hemes all cluster about Ems which are ≈200 mV more positive in protein than in water. In contrast, the low potential bis-His c-type hemes are shifted little from in solution, whereas the high potential His-Met c-type hemes are raised by ≈300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. For example, the loss of solvation (reaction field) energy, which raises the Em, has been suggested to be a major factor in

  7. Synthesis and Evaluation of Amyloid β Derived and Amyloid β Independent Enhancers of the Peroxidase-like Activity of Heme.

    Wißbrock, Amelie; Kühl, Toni; Silbermann, Katja; Becker, Albert J; Ohlenschläger, Oliver; Imhof, Diana

    2017-01-12

    Labile heme has been suggested to have an impact in several severe diseases. In the context of Alzheimer's disease (AD), however, decreased levels of free heme have been reported. Therefore, we were looking for an assay system that can be used for heme concentration determination. From a biochemical point of view the peroxidase activity of the Aβ-heme complex seemed quite attractive to pursue this goal. As a consequence, a peptide that is able to increase the readout even in the case of a low heme concentration is favorable. The examination of Aβ- and non-Aβ-derived peptides in complex with heme revealed that the peroxidase-like activity significantly depends on the peptide sequence and length. A 23mer His-based peptide derived from human fatty acyl-CoA reductase 1 in complex with heme exhibited a significantly higher peroxidase activity than Aβ(40)-heme. Structural modeling of both complexes demonstrated that heme binding via a histidine can be supported by hydrogen bond interactions of a basic residue near the propionate carboxyl function of protoporphyrin IX. Furthermore, the interplay of Aβ-heme and the lipoprotein LDL as a potential physiological effector of Aβ was examined.

  8. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    Lee, Seung-Min [Department of Biological Sciences, University of Columbia, NY (United States); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Attieh, Zouhair K. [Department of Laboratory Science and Technology, American University of Science and Technology, Ashrafieh (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Son, Hee Sook [Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University (Korea, Republic of); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Chen, Huijun [Medical School, Nanjing University, Nanjing 210008, Jiangsu Province (China); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Bacouri-Haidar, Mhenia [Department of Biology, Faculty of Sciences (I), Lebanese University, Hadath (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Vulpe, Chris D., E-mail: vulpe@berkeley.edu [Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  9. NMR and rotational angles in solution conformation of polypeptides

    Bystrov, V. F.

    1985-01-01

    Professor San-Ichiro Mizushima and Professor Yonezo Morino's classical contributions provided unique means and firm basis for understanding of conformational states and internal rotation in polypeptide molecules. Now the NMR spectroscopy is the best choice to study molecular conformation, mechanism of action and structure-functional relationships of peptide and proteins in solution under conditions approaching those of their physiological environments. Crucial details of spatial structure and interactions of these molecules in solution are revealed by using proton-proton and carbon-proton vicinal coupling constants, proton nuclear Overhauser effect and spectral perturbation techniques. The results of NMR conformational analysis are presented for valinomycin "bracelet", gramicidin A double helices, honey-bee neurotoxin apamin, scorpion insectotoxins and snake neurotoxins of long and short types.

  10. A radioimmunoassay of gastric inhibitory polypeptide in human plasma

    Sarson, D.L.; Bryant, M.G.; Bloom, S.R.

    1980-01-01

    A sensitive radioimmunoassay for the measurement of human gastric inhibitory polypeptide (GIP), using pure porcine GIP, has been developed. Cross-reactivity of the antiserum with all available mammalian gut peptide preparations was negligible with the exception of glucagon when it was approximately 1%. Two major molecular forms of GIP were detectable in plasma and tissue extracts, one of large molecular size and the other corresponding to the elution coefficient of pure porcine standard. Concentrations of GIP in plasma from 50 normal subjects after overnight fasting were 9+-1.0(S.E.M.) pmol/1 rising to a peak of 34+-2.8 pmol/1 following the ingestion of a small mixed test meal. Ingestion of glucose or fat resulted in a similar rise of plasma GIP, whereas no change was observed after the ingestion of protein. (author)

  11. Gastric inhibitory polypeptide does not inhibit gastric emptying in humans

    Meier, Juris J; Goetze, Oliver; Anstipp, Jens

    2004-01-01

    ) = 0.15, P = 0.15 for intact GIP; r(2) = 0.21, P = 0.086 for total GIP). We conclude that gastric emptying does not appear to be influenced by GIP. The secretion of GIP after meal ingestion is not suppressed by its exogenous administration. The lack of effect of GIP on gastric emptying underlines......The insulinotropic gut hormone gastric inhibitory polypeptide (GIP) has been demonstrated to inhibit gastric acid secretion and was proposed to possess "enterogastrone" activity. GIP effects on gastric emptying have not yet been studied. Fifteen healthy male volunteers (23.9 +/- 3.3 yr, body mass....... Gastric emptying was calculated from the (13)CO(2) exhalation rates in breath samples collected over 360 min. Venous blood was drawn in 30-min intervals for the determination of glucose, insulin, C-peptide, and GIP (total and intact). Statistical calculations were made by use of repeated-measures ANOVA...

  12. Vasoactive intestinal polypeptide (VIP) in the pig pancreas

    Poulsen, Steen Seier

    1984-01-01

    Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion...... of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas...... with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas....

  13. Biosynthesis of human sialophorins and analysis of the polypeptide core

    Remold-O'Donnell, E.; Kenney, D.; Rosen, F.S.

    1987-01-01

    Biosynthesis was examined of sialophorin (formerly called gpL115) which is altered in the inherited immunodeficiency Wiskott-Aldrich syndrome. Sialophorin is greater than 50% carbohydrate, primarily O-linked units of sialic acid, galactose, and galactosamine. Pulse-labeling with [ 35 S]methionine and chase incubation established that sialophorin is synthesized in CEM lymphoblastoid cells as an Mr 62,000 precursor which is converted within 45 min to mature glycosylated sialophorin, a long-lived molecule. Experiments with tunicamycin and endoglycosidase H demonstrated that sialophorin contains N-linked carbohydrate (approximately two units per molecule) and is therefore an N,O-glycoprotein. Pulse-labeling of tunicamycin-treated CEM cells together with immunoprecipitation provided the means to isolate the [ 35 S]-methionine-labeled polypeptide core of sialophorin and determine its molecular weight (58,000). This datum allowed us to express the previously established composition on a per molecule basis and determine that sialophorin molecules contain approximately 520 amino acid residues and greater than or equal to 100 O-linked carbohydrate units. A recent study showed that various blood cells express sialophorin and that there are two molecular forms: lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin. Biosynthesis of the two forms was compared by using sialophorin of CEM cells and sialophorin of MOLT-4 cells (another lymphoblastoid line) as models for lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin, respectively. The time course of biosynthesis and the content of N units were found to be identical for the two sialophorin species. [ 35 S]Methionine-labeled polypeptide cores of CEM sialophorin and MOLT sialophorin were isolated and compared by electrophoresis, isoelectrofocusing, and a newly developed peptide mapping technique

  14. Hemopexin and haptoglobin: allies against heme toxicity from hemoglobin not contenders.

    Ann eSmith

    2015-06-01

    Full Text Available The goal here is to describe our current understanding of heme metabolism and the deleterious effects of free heme on immunological processes, endothelial function, systemic inflammation, and various end-organ tissues (e.g. kidney, lung, liver, etc., with particular attention paid to the role of hemopexin (HPX. Because heme toxicity is the impetus for much of the pathology in sepsis, sickle cell disease, and other hemolytic conditions, the biological importance and clinical relevance of HPX, the predominant heme binding protein, is reinforced. A perspective on the function of HPX and haptoglobin (Hp is presented, updating how these two proteins and their respective receptors act simultaneously to protect the body in clinical conditions that entail hemolysis and/or systemic intravascular inflammation. Evidence from longitudinal studies in patients supports that HPX plays a Hp-independent role in genetic and non-genetic hemolytic diseases without the need for global Hp depletion. Evidence also supports that HPX has an important role in the prognosis of complex illnesses characterized predominantly by the presence of hemolysis, such as sickle cell disease, sepsis, hemolytic-uremic syndrome, and conditions involving intravascular and extravascular hemolysis, such as that generated by extracorporeal circulation during cardiopulmonary bypass and from blood transfusions. We propose that quantitating the amounts of plasma heme, HPX, Hb-Hp, heme-HPX and heme-albumin levels in various disease states may aid in the diagnosis and treatment of the above-mentioned conditions, which is crucial to developing targeted plasma protein supplementation (i.e. replenishment therapies for patients with heme toxicity due to HPX depletion.

  15. Effects of Zinc Deuteroporphyrin Bis Glycol on Newborn Mice After Heme-Loading

    He, Cynthia X.; Campbell, Claire M.; Zhao, Hui; Kalish, Flora S.; Schulz, Stephanie; Vreman, Hendrik J.; Wong, Ronald J.; Stevenson, David K.

    2011-01-01

    Infants with hemolytic diseases frequently develop hyperbilirubinemia, but standard phototherapy only eliminates bilirubin after its production. A better strategy might be to directly inhibit heme oxygenase (HO), the rate-limiting enzyme in bilirubin production. Metalloporphyrins (Mps) are heme analogs that competitively inhibit HO activity in vitro and in vivo and suppress plasma bilirubin levels in vivo. A promising Mp, zinc deuteroporphyrin bis glycol (ZnBG), is orally absorbed and effecti...

  16. In vitro Activation of heme oxygenase-2 by menadione and its analogs.

    Vukomanovic, Dragic; Rahman, Mona N; Bilokin, Yaroslav; Golub, Andriy G; Brien, James F; Szarek, Walter A; Jia, Zongchao; Nakatsu, Kanji

    2014-02-18

    Previously, we reported that menadione activated rat, native heme oxygenase-2 (HO-2) and human recombinant heme oxygenase-2 selectively; it did not activate spleen, microsomal heme oxygenase-1. The purpose of this study was to explore some structure-activity relationships of this activation and the idea that redox properties may be an important aspect of menadione efficacy. Heme oxygenase activity was determined in vitro using rat spleen and brain microsomes as the sources of heme oxygenase-1 and -2, respectively, as well as recombinant, human heme oxygenase-2. Menadione analogs with bulky aliphatic groups at position-3, namely vitamins K1 and K2, were not able to activate HO-2. In contrast, several compounds with similar bulky but less lipophilic moieties at position-2 (and -3) were able to activate HO-2 many fold; these compounds included polar, rigid, furan-containing naphthoquinones, furan-benzoxazine naphthoquinones, 2-(aminophenylphenyl)-3-piperidin-1-yl naphthoquinones. To explore the idea that redox properties might be involved in menadione efficacy, we tested analogs such as 1,4-dimethoxy-2-methylnaphthalene, pentafluoromenadione, monohalogenated naphthoquinones, α-tetralone and 1,4-naphthoquinone. All of these compounds were inactive except for 1,4-naphthoquinone. Menadione activated full-length recombinant human heme oxygenase-2 (FL-hHO-2) as effectively as rat brain enzyme, but it did not activate rat spleen heme oxygenase. These observations are consistent with the idea that naphthoquinones such as menadione bind to a receptor in HO-2 and activate the enzyme through a mechanism that may involve redox properties.

  17. Heme A synthesis and CcO activity are essential for Trypanosoma cruzi infectivity and replication.

    Merli, Marcelo L; Cirulli, Brenda A; Menéndez-Bravo, Simón M; Cricco, Julia A

    2017-06-27

    Trypanosoma cruzi , the causative agent of Chagas disease, presents a complex life cycle and adapts its metabolism to nutrients' availability. Although T. cruzi is an aerobic organism, it does not produce heme. This cofactor is acquired from the host and is distributed and inserted into different heme-proteins such as respiratory complexes in the parasite's mitochondrion. It has been proposed that T. cruzi's energy metabolism relies on a branched respiratory chain with a cytochrome c oxidase-type aa 3 (C c O) as the main terminal oxidase. Heme A, the cofactor for all eukaryotic C c O, is synthesized via two sequential enzymatic reactions catalyzed by heme O synthase (HOS) and heme A synthase (HAS). Previously, TcCox10 and TcCox15 ( Trypanosoma cruzi Cox10 and Cox15 proteins) were identified in T. cruzi They presented HOS and HAS activity, respectively, when they were expressed in yeast. Here, we present the first characterization of TcCox15 in T. cruzi , confirming its role as HAS. It was differentially detected in the different T. cruzi stages, being more abundant in the replicative forms. This regulation could reflect the necessity of more heme A synthesis, and therefore more C c O activity at the replicative stages. Overexpression of a non-functional mutant caused a reduction in heme A content. Moreover, our results clearly showed that this hindrance in the heme A synthesis provoked a reduction on C c O activity and, in consequence, an impairment on T. cruzi survival, proliferation and infectivity. This evidence supports that T. cruzi depends on the respiratory chain activity along its life cycle, being C c O an essential terminal oxidase. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  18. Influence of heme environment structure on dioxygen affinity for the dual function Amphitrite ornata hemoglobin/dehaloperoxidase. Insights into the evolutional structure-function adaptations

    Sun, Shengfang; Sono, Masanori; Wang, Chunxue; Du, Jing; Lebioda, Lukasz; Dawson, John H. [SC

    2014-05-15

    Sea worm, Amphitrite ornata, has evolved its globin (an O2 carrier) also to serves as a dehaloperoxidase (DHP) to detoxify haloaromatic pollutants generated by competing species. A previous mutagenesis study by our groups on both DHP and sperm whale myoglobin (SW Mb) revealed some structural factors that influence the dehaloperoxidase activities (significantly lower for Mb) of both proteins. Using an isocyanide/O2 partition constant measurement method in this study, we have examined the effects of these structural factors on the O2 equilibrium constants (KO2) of DHP, SW Mb, and their mutants. A clear trend of decreasing O2 affinity and increasing catalytic activity along with the increase in the distal His Nεheme iron distance is observed. An H93K/T95H Mb double mutant mimicking the DHP proximal His positioning exhibited markedly enhanced O2 affinity, confirming the essential effect of proximal His rotation on the globin function of DHP. For DHP, the L100F, T56G and M86E variants showed the effects of distal volume, distal His flexibility and proximal electronic push, respectively, on the O2 affinity. This study provides insights into how DHP has evolved its heme environment to gain significantly enhanced peroxidase capability without compromising its primary function as an O2 carrier.

  19. Iron uptake mechanisms in the fish pathogen Tenacibaculum maritimum.

    Avendaño-Herrera, Ruben; Toranzo, Alicia E; Romalde, Jesús L; Lemos, Manuel L; Magariños, Beatriz

    2005-11-01

    We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. Proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.

  20. Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

    Carlo Travaglini-Allocatelli

    2013-01-01

    Full Text Available Cytochromes c (Cyt c are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i heme translocation and delivery, (ii apoCyt thioreductive pathway, and (iii apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria.