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

PubMed

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.

The Janus Face of the Heme Oxygenase/Biliverdin Reductase System in Alzheimer Disease: It’s Time for Reconciliation

PubMed Central

Barone, Eugenio; Di Domenico, Fabio; Mancuso, Cesare; Butterfield, D. Allan

2013-01-01

Alzheimer disease (AD) is the most common form of dementia among the elderly and is characterized by progressive loss of memory and cognition. These clinical features are due in part to the increase of reactive oxygen and nitrogen species that mediate neurotoxic effects. The up-regulation of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system is one of the earlier events in the adaptive response to stress. HO-1/BVR-A reduces the intracellular levels of pro-oxidant heme and generates equimolar amounts of the free radical scavengers biliverdin-IX alpha (BV)/bilirubin-IX alpha (BR) as well as the pleiotropic gaseous neuromodulator carbon monoxide (CO) and ferrous iron. Two main and opposite hypotheses for a role of the HO-1/BVR-A system in AD propose that this system mediates neurotoxic and neuroprotective effects, respectively. This apparent controversy was mainly due to the fact that for over about 20 years HO-1 was the only player on which all the analyses were focused, excluding the other important and essential component of the entire system, BVR. Following studies from the Butterfield laboratory that reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative post-translational modifications in the brain of subjects with AD and amnestic mild cognitive impairment (MCI) subjects, a debate was opened on the real pathophysiological and clinical significance of BVR-A. In this paper we provide a review of the main discoveries about the HO/BVR system in AD and MCI, and propose a mechanism that reconciles these two hypotheses noted above of neurotoxic and the neuroprotective aspects of this important stress responsive system. PMID:24095978

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

EPA Science Inventory

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...

Heme oxygenase activity increases after exercise in healthy volunteers

EPA Science Inventory

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)....

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

PubMed Central

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Jenkins, Jermaine L.; Heimiller, Chelsea; Maines, Mahin D.

2016-01-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1–3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S473 independent of hBVR’s kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S473 independent of hBVR’s kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S230 in hBVR 225RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR’s PDK1 binding 161RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.—Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. PMID:27166089

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

PubMed

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

2016-08-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. © FASEB.

Role of vitamin C transporters and biliverdin reductase in the dual pro-oxidant and anti-oxidant effect of biliary compounds on the placental-fetal unit in cholestasis during pregnancy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez, Maria J.; Castano, Beatriz; Jimenez, Silvia

Maternal cholestasis causes oxidative damage to the placental-fetal unit that may challenge the outcome of pregnancy. This has been associated with the accumulation of biliary compounds able to induce oxidative stress. However, other cholephilic compounds such as ursodeoxycholic acid (UDCA) and bilirubin have direct anti-oxidant properties. In the present study we investigated whether these compounds exert a protective effect on cholestasis-induced oxidative stress in placenta as compared to maternal and fetal livers, and whether this is due in part to the activation of anti-oxidant mechanisms involving vitamin C uptake and biliverdin/bilirubin recycling. In human placenta (JAr) and liver (HepG2) cells,more » deoxycholic acid (DCA) similar rates of free radical generation. In JAr (not HepG2), the mitochondrial membrane potential and cell viability were impaired by low DCA concentrations; this was partly prevented by bilirubin and UDCA. In HepG2, taurocholic acid (TCA) and UDCA up-regulated biliverdin-IX{alpha} reductase (BVR{alpha}) and the vitamin C transporter SVCT2 (not SVCT1), whereas bilirubin up-regulated both SVCT1 and SVCT2. In JAr, TCA and UDCA up-regulated BVR{alpha}, SVCT1 and SVCT2, whereas bilirubin up-regulated only SVCT2. A differential response to these compounds of nuclear receptor expression (SXR, CAR, FXR and SHP) was found in both cell types. When cholestasis was induced in pregnant rats, BVR{alpha}, SVCT1 and SVCT2 expression in maternal and fetal livers was stimulated, and this was further enhanced by UDCA treatment. In placenta, only BVR{alpha} was up-regulated. In conclusion, bilirubin accumulation and UDCA administration may directly and indirectly protect the placental-fetal unit from maternal cholestasis-induced oxidative stress.« less

Isoporphyrin intermediate in heme oxygenase catalysis. Oxidation of alpha-meso-phenylheme.

PubMed

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.

Isoporphyrin Intermediate in Heme Oxygenase Catalysis

PubMed Central

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

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

PubMed

Cremers, Niels A J; Lundvig, Ditte M S; van Dalen, Stephanie C M; Schelbergen, Rik F; van Lent, Peter L E M; Szarek, Walter A; Regan, Raymond F; Carels, Carine E; Wagener, Frank A D T G

2014-10-08

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Kinetics of oxidation of bilirubin and its protein complex by hydrogen peroxide in aqueous solutions

NASA Astrophysics Data System (ADS)

Solomonov, A. V.; Rumyantsev, E. V.; Antina, E. V.

2010-12-01

A comparative study of oxidation reactions of bilirubin and its complex with albumin was carried out in aqueous solutions under the action of hydrogen peroxide and molecular oxygen at different pH values. Free radical oxidation of the pigment in both free and bound forms at pH 7.4 was shown not to lead to the formation of biliverdin, but to be associated with the decomposition of the tetrapyrrole chromophore into monopyrrolic products. The effective and true rate constants of the reactions under study were determined. It was assumed that one possible mechanism of the oxidation reaction is associated with the interaction of peroxyl radicals and protons of the NH groups of bilirubin molecules at the limiting stage with the formation of a highly reactive radical intermediate. The binding of bilirubin with albumin was found to result in a considerable reduction in the rate of the oxidation reaction associated with the kinetic manifestation of the protein protection effect. It was found that the autoxidation of bilirubin by molecular oxygen with the formation of biliverdin at the intermediate stage can be observed with an increase in the pH of solutions.

Clinical potential of phycocyanobilin for induction of T regulatory cells in the management of inflammatory disorders.

PubMed

McCarty, Mark F

2011-12-01

Exposure of human mononuclear cells to phycocyanin in vitro is reported to promote generation of Treg cells. Induction of heme oxygenase-1 (HO-1) in lymphocytes has a similar effect, and it is not likely to be accidental that a key product of HO-1 activity, biliverdin, is homologous to the structure of phycocyanin's chromophore phycocyanobilin (PhyCB). Moreover, Treg induction is observed in mice injected with bilirubin, biliverdin's chief metabolite. These considerations suggest that bilirubin, generated within lymphocytes by HO-1 activation, may play a physiological role in the promotion of Treg immunomodulation. This effect of bilirubin is likely to be independent of NADPH oxidase inhibition, since the NAPDH oxidase activity of macrophages is necessary for Treg induction, possibly because it contributes to HO-1 induction in lymphocytes. In light of numerous reports that oral phycocyanin is beneficial in various rodent models of autoimmune disorders, it is reasonable to suspect that PhyCB-enriched spirulina extracts may have clinical potential for boosting Treg activity in human autoimmune or allergic syndromes, mimicking the physiological role of HO-1 induction in this regard. Copyright © 2011 Elsevier Ltd. All rights reserved.

HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1

PubMed Central

Rodriguez, Andres I.; Gangopadhyay, Archana; Kelley, Eric E.; Pagano, Patrick J.; Zuckerbraun, Brian S.; Bauer, Philip M.

2009-01-01

Objective Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration. Results and Methods The effects of increased HO-1 expression as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches including detection of intracellular superoxide, NADPH oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased PDGF-stimulated, redox-sensitive signaling pathways. Conclusion Herein we demonstrate that increased HO-1 expression and CO decreases PDGF-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury. PMID:19875720

Near infrared fluorescent biliproteins generated from bacteriophytochrome AphB of Nostoc sp. PCC 7120.

PubMed

Yuan, Che; Li, Hui-Zhen; Tang, Kun; Gärtner, Wolfgang; Scheer, Hugo; Zhou, Ming; Zhao, Kai-Hong

2016-04-01

The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1-321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

PubMed

Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

PubMed Central

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Continuous de novo biosynthesis of haem and its rapid turnover to bilirubin are necessary for cytoprotection against cell damage

PubMed Central

Takeda, Taka-aki; Mu, Anfeng; Tai, Tran Tien; Kitajima, Sakihito; Taketani, Shigeru

2015-01-01

It is well known that haem serves as the prosthetic group of various haemoproteins that function in oxygen transport, respiratory chain, and drug metabolism. However, much less is known about the functions of the catabolites of haem in mammalian cells. Haem is enzymatically degraded to iron, carbon monoxide (CO), and biliverdin, which is then converted to bilirubin. Owing to difficulties in measuring bilirubin, however, the generation and transport of this end product remain unclear despite its clinical importance. Here, we used UnaG, the recently identified bilirubin-binding fluorescent protein, to analyse bilirubin production in a variety of human cell lines. We detected a significant amount of bilirubin with many non-blood cell types, which was sensitive to inhibitors of haem metabolism. These results suggest that there is a basal level of haem synthesis and its conversion into bilirubin. Remarkably, substantial changes were observed in the bilirubin generation when cells were exposed to stress insults. Since the stress-induced cell damage was exacerbated by the pharmacological blockade of haem metabolism but was ameliorated by the addition of biliverdin and bilirubin, it is likely that the de novo synthesis of haem and subsequent conversion to bilirubin play indispensable cytoprotective roles against cell damage. PMID:25990790

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

PubMed

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.

Inducible bilirubin oxidase: A novel function for the mouse cytochrome P450 2A5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abu-Bakar, A'edah, E-mail: a.abubakar@uq.edu.au; Arthur, Dionne Maioha; Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide

2011-11-15

We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic 'BR oxidase'. A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR tomore » biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible 'BR oxidase' where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced. -- Highlights: Black-Right-Pointing-Pointer CYP2A5 metabolizes bilirubin to biliverdin and dipyrroles. Black-Right-Pointing-Pointer Bilirubin increased the hepatic CYP2A5 protein and activity levels. Black-Right-Pointing-Pointer Bilirubin does not change the hepatic CYP2A5 mRNA levels. Black-Right-Pointing-Pointer Co-treatment with a protein synthesis inhibitor prolongs CYP2A5 half-life. Black-Right-Pointing-Pointer CYP2A5 is potentially an inducible bilirubin oxidase.« less

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding.

PubMed

Goncharova, Iryna; Jašprová, Jana; Vítek, Libor; Urbanová, Marie

2015-12-01

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA). Copyright © 2015 Elsevier Inc. All rights reserved.

Electrochemical Sensor for Bilirubin Detection Using Screen Printed Electrodes Functionalized with Carbon Nanotubes and Graphene.

PubMed

Thangamuthu, Madasamy; Gabriel, Willimann Eric; Santschi, Christian; Martin, Olivier J F

2018-03-07

Practice oriented point-of-care diagnostics require easy-to-handle, miniaturized, and low-cost analytical tools. In a novel approach, screen printed carbon electrodes (SPEs), which were functionalized with nanomaterials, are employed for selective measurements of bilirubin, which is an important biomarker for jaundice. Multi-walled carbon nanotubes (MWCNT) and graphene separately deposited on SPEs provide the core of an electrochemical sensor for bilirubin. The electrocatalytic activity towards bilirubin oxidation (bilirubin to biliverdin) was observed at +0.25 V. In addition, a further peak corresponding to the electrochemical conversion of biliverdin into purpurin appeared at +0.48 V. When compared to MWCNT, the graphene type shows a 3-fold lower detection limit (0.3 ± 0.022 nM and 0.1 ± 0.018 nM, respectively), moreover, the graphene type exhibits a larger linear range (0.1-600 µM) than MWCNT (0.5-500 µM) with a two-fold better sensitivity, i.e., 30 nA µM -1 cm -2 , and 15 nA µM -1 cm -2 , respectively. The viability is validated through measurements of bilirubin in blood serum samples and the selectivity is ensured by inhibiting common interfering biological substrates using an ionic nafion membrane. The presented approach enables the design and implementation of low cost and miniaturized electrochemical sensors.

Nature’s Palette: Characterization of Shared Pigments in Colorful Avian and Mollusk Shells

PubMed Central

Brennan, Patricia L. R.; Hanley, Daniel; Grim, Tomáš; Hauber, Mark E.; Holford, Mandë

2015-01-01

Pigment-based coloration is a common trait found in a variety of organisms across the tree of life. For example, calcareous avian eggs are natural structures that vary greatly in color, yet just a handful of tetrapyrrole pigment compounds are responsible for generating this myriad of colors. To fully understand the diversity and constraints shaping nature’s palette, it is imperative to characterize the similarities and differences in the types of compounds involved in color production across diverse lineages. Pigment composition was investigated in eggshells of eleven paleognath bird taxa, covering several extinct and extant lineages, and shells of four extant species of mollusks. Birds and mollusks are two distantly related, calcareous shell-building groups, thus characterization of pigments in their calcareous structures would provide insights to whether similar compounds are found in different phyla (Chordata and Mollusca). An ethylenediaminetetraacetic acid (EDTA) extraction protocol was used to analyze the presence and concentration of biliverdin and protoporphyrin, two known and ubiquitous tetrapyrrole avian eggshell pigments, in all avian and molluscan samples. Biliverdin was solely detected in birds, including the colorful eggshells of four tinamou species. In contrast, protoporphyrin was detected in both the eggshells of several avian species and in the shells of all mollusks. These findings support previous hypotheses about the ubiquitous deposition of tetrapyrroles in the eggshells of various bird lineages and provide evidence for its presence also across distantly related animal taxa. PMID:26650398

Heme Oxygenases in Cardiovascular Health and Disease

PubMed Central

Ayer, Anita; Zarjou, Abolfazl; Agarwal, Anupam; Stocker, Roland

2016-01-01

Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies. PMID:27604527

Structural Determinants of Improved Fluorescence in a Family of Bacteriophytochrome-Based Infrared Fluorescent Proteins: Insights from Continuum Electrostatic Calculations and Molecular Dynamics Simulations.

PubMed

Feliks, Mikolaj; Lafaye, Céline; Shu, Xiaokun; Royant, Antoine; Field, Martin

2016-08-09

Using X-ray crystallography, continuum electrostatic calculations, and molecular dynamics simulations, we have studied the structure, protonation behavior, and dynamics of the biliverdin chromophore and its molecular environment in a series of genetically engineered infrared fluorescent proteins (IFPs) based on the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. Our study suggests that the experimentally observed enhancement of fluorescent properties results from the improved rigidity and planarity of the biliverdin chromophore, in particular of the first two pyrrole rings neighboring the covalent linkage to the protein. We propose that the increases in the levels of both motion and bending of the chromophore out of planarity favor the decrease in fluorescence. The chromophore-binding pocket in some of the studied proteins, in particular the weakly fluorescent parent protein, is shown to be readily accessible to water molecules from the solvent. These waters entering the chromophore region form hydrogen bond networks that affect the otherwise planar conformation of the first three rings of the chromophore. On the basis of our simulations, the enhancement of fluorescence in IFPs can be achieved either by reducing the mobility of water molecules in the vicinity of the chromophore or by limiting the interactions of the nearby protein residues with the chromophore. Finally, simulations performed at both low and neutral pH values highlight differences in the dynamics of the chromophore and shed light on the mechanism of fluorescence loss at low pH.

Is it worth expending energy to convert biliverdin into bilirubin?

PubMed

Nam, Joon; Lee, Yonghyun; Yang, Yejin; Jeong, Seongkeun; Kim, Wooseong; Yoo, Jin-Wook; Moon, Jeon-Ok; Lee, Changyong; Chung, Hae Young; Kim, Min-Soo; Jon, Sangyong; Jung, Yunjin

2018-06-10

Bilirubin (BR) is generated by the reduction of biliverdin (BV), a metabolite that results from the catalytic degradation of heme by the isoforms of heme oxygenase (HO). BV is nontoxic and water-soluble but BR is potentially toxic and lipophilic. Therefore, a further metabolic step is required for BR before excretion is possible. The reductive conversion of BV to BR costs energy and is evolutionarily conserved in human physiology. There must be a compelling reason for this apparently nonsensical evolutionary conservation. In addition to the differences between BR and BV-such as water solubility, antioxidant activity, and participation as a receptor ligand-in the present study, we focused on the chemistry of the two metabolites with regard to an electrophilic functional group called a Michael reaction acceptor (MRA). Our data reveal that the BR reacts with thiol compounds forming adducts, whereas no reaction occurs with BV. Furthermore, the binding of biotin-tagged BR to Kelch-like ECH-associated protein 1 (KEAP1)-a biological electrophile sensor-was prevented by pretreatment with BR or a thiol compound, but was not by pretreatment with BV. In cells, BR could bind to KEAP1 to release and activate nuclear factor-erythroid 2 (NF-E2) p45-related factor 2, a cytoprotective transcription factor, leading to the induction of HO-1. These findings may provide a physiological rationale for the energy-consuming conversion of BV to BR. Copyright © 2018. Published by Elsevier Inc.

Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Shasha; Wang, Shuang; Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081

2013-08-01

Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxiamore » in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.« less

The kinetics of oxidation of bilirubin and ascorbic acid in solution

NASA Astrophysics Data System (ADS)

Solomonov, A. V.; Rumyantsev, E. V.; Kochergin, B. A.; Antina, E. V.

2012-07-01

The results of a comparative study of the oxidation of bilirubin, ascorbic acid, and their mixture in aqueous solutions under the action of air oxygen and hydrogen peroxide are presented. The observed and true rate constants for the oxidation reactions were determined. It was shown that the oxidation of tetrapyrrole pigment occurred under these conditions bypassing the stage of biliverdin formation to monopyrrole products. Simultaneous oxidation of bilirubin and ascorbic acid was shown to be accompanied by the inhibition of ascorbic acid oxidation by bilirubin, whereas ascorbic acid itself activated the oxidation of bilirubin.

Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies

PubMed Central

Richie, Christopher T.; Whitaker, Leslie R.; Whitaker, Keith W.; Necarsulmer, Julie; Baldwin, Heather A.; Zhang, Yajun; Fortuno, Lowella; Hinkle, Josh J.; Koivula, Pyry; Henderson, Mark J.; Sun, Wenzhi; Wang, Kai; Smith, Jeffrey C.; Pickel, Jim; Ji, Na; Hope, Bruce T.; Harvey, Brandon K.

2017-01-01

Background The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. New Method Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. Results iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. Conclusions Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo. PMID:28380331

Heme oxygenase-1 system and gastrointestinal tumors

PubMed Central

Zhu, Xiao; Fan, Wen-Guo; Li, Dong-Pei; Lin, Marie CM; Kung, Hsiangfu

2010-01-01

Heme oxygenase-1 (HO-1) system catabolizes heme into three products: carbon monoxide, biliverdin/bilirubin and free iron. It is involved in many physiological and pathophysiological processes. A great deal of data has demonstrated the roles of HO-1 in the formation, growth and metastasis of tumors. The interest in this system by investigators involved in gastrointestinal tumors is fairly recent, and few papers on HO-1 have touched upon this subject. This review focuses on the current understanding of the physiological significance of HO-1 induction and its possible roles in the gastrointestinal tumors studied to date. The implications for possible therapeutic manipulation of HO-1 in gastrointestinal tumors are also discussed. PMID:20518085

Carbon monoxide: from the origin of life to molecular medicine.

PubMed

Bach, Fritz H

2006-08-01

Carbon monoxide, long considered only as a toxic gas, has recently been shown to mediate potent anti-inflammatory and other salutary effects in rodents when it is used at low doses. Carbon monoxide is one of the products of the degradation of heme by heme oxygenase 1. Until recently, these beneficial effects of carbon monoxide were shown only when it was given before a stress stimulus. Hagazi and colleagues have recently shown that this substance is effective even when it is given after a disease process has started. The effects of low doses of carbon monoxide are complemented by the production of biliverdin and probably also by ferritin, which are additional products of heme degradation.

Comparative transcriptome analysis provides clues to molecular mechanisms underlying blue-green eggshell color in the Jinding duck (Anas platyrhynchos).

PubMed

Wang, Zhepeng; Meng, Guohua; Bai, Yun; Liu, Ruifang; Du, Yu; Su, Lihong

2017-09-12

In birds, blue-green eggshell color (BGEC) is caused by biliverdin, a bile pigment derived from the degradation of heme and secreted in the eggshell by the shell gland. Functionally, BGEC might promote the paternal investment of males in the nest and eggs. However, little is known about its formation mechanisms. Jinding ducks (Anas platyrhynchos) are an ideal breed for research into the mechanisms, in which major birds lay BGEC eggs with minor individuals laying white eggs. Using this breed, this study aimed to provide insight into the mechanisms via comparative transcriptome analysis. Blue-shelled ducks (BSD) and white-shelled ducks (WSD) were selected from two populations, forming 4 groups (3 ducks/group): BSD1 and WSD1 from population 1 and BSD2 and WSD2 from population 2. Twelve libraries from shell glands were sequenced using the Illumina RNA-seq platform, generating an average of 41 million clean reads per library, of which 55.9% were mapped to the duck reference genome and assembled into 31,542 transcripts. Expression levels of 11,698 genes were successfully compared between all pairs of 4 groups. Of these, 464 candidate genes were differentially expressed between cross-phenotype groups, but not for between same-phenotype groups. Gene Ontology (GO) annotation showed that 390 candidate genes were annotated with 2234 GO terms. No candidate genes were directly involved in biosynthesis or transport of biliverdin. However, the integral components of membrane, metal ion transport, cholesterol biosynthesis, signal transduction, skeletal system development, and chemotaxis were significantly (P < 0.05) overrepresented by candidate genes. This study identified 464 candidate genes associated with duck BGEC, providing valuable information for a better understanding of the mechanisms underlying this trait. Given the involvement of membrane cholesterol contents, ions and ATP levels in modulating the transport activity of bile pigment transporters, the data suggest a potential association between duck BGEC and the transport activity of the related transporters.

Dinosaur origin of egg color: oviraptors laid blue-green eggs

PubMed Central

Yang, Tzu-Ruei; Sander, Philipp N.; Schneider, Marion; Engeser, Marianne; Kath-Schorr, Stephanie; Müller, Christa E.; Sander, P. Martin

2017-01-01

Protoporphyrin (PP) and biliverdin (BV) give rise to the enormous diversity in avian egg coloration. Egg color serves several ecological purposes, including post-mating signaling and camouflage. Egg camouflage represents a major character of open-nesting birds which accomplish protection of their unhatched offspring against visually oriented predators by cryptic egg coloration. Cryptic coloration evolved to match the predominant shades of color found in the nesting environment. Such a selection pressure for the evolution of colored or cryptic eggs should be present in all open nesting birds and relatives. Many birds are open-nesting, but protect their eggs by continuous brooding, and thus exhibit no or minimal eggshell pigmentation. Their closest extant relatives, crocodiles, protect their eggs by burial and have unpigmented eggs. This phylogenetic pattern led to the assumption that colored eggs evolved within crown birds. The mosaic evolution of supposedly avian traits in non-avian theropod dinosaurs, however, such as the supposed evolution of partially open nesting behavior in oviraptorids, argues against this long-established theory. Using a double-checking liquid chromatography ESI-Q-TOF mass spectrometry routine, we traced the origin of colored eggs to their non-avian dinosaur ancestors by providing the first record of the avian eggshell pigments protoporphyrin and biliverdin in the eggshells of Late Cretaceous oviraptorid dinosaurs. The eggshell parataxon Macroolithus yaotunensis can be assigned to the oviraptor Heyuannia huangi based on exceptionally preserved, late developmental stage embryo remains. The analyzed eggshells are from three Late Cretaceous fluvial deposits ranging from eastern to southernmost China. Reevaluation of these taphonomic settings, and a consideration of patterns in the porosity of completely preserved eggs support an at least partially open nesting behavior for oviraptorosaurs. Such a nest arrangement corresponds with our reconstruction of blue-green eggs for oviraptors. According to the sexual signaling hypothesis, the reconstructed blue-green eggs support the origin of previously hypothesized avian paternal care in oviraptorid dinosaurs. Preserved dinosaur egg color not only pushes the current limits of the vertebrate molecular and associated soft tissue fossil record, but also provides a perspective on the potential application of this unexplored paleontological resource. PMID:28875070

Dinosaur origin of egg color: oviraptors laid blue-green eggs.

PubMed

Wiemann, Jasmina; Yang, Tzu-Ruei; Sander, Philipp N; Schneider, Marion; Engeser, Marianne; Kath-Schorr, Stephanie; Müller, Christa E; Sander, P Martin

2017-01-01

Protoporphyrin (PP) and biliverdin (BV) give rise to the enormous diversity in avian egg coloration. Egg color serves several ecological purposes, including post-mating signaling and camouflage. Egg camouflage represents a major character of open-nesting birds which accomplish protection of their unhatched offspring against visually oriented predators by cryptic egg coloration. Cryptic coloration evolved to match the predominant shades of color found in the nesting environment. Such a selection pressure for the evolution of colored or cryptic eggs should be present in all open nesting birds and relatives. Many birds are open-nesting, but protect their eggs by continuous brooding, and thus exhibit no or minimal eggshell pigmentation. Their closest extant relatives, crocodiles, protect their eggs by burial and have unpigmented eggs. This phylogenetic pattern led to the assumption that colored eggs evolved within crown birds. The mosaic evolution of supposedly avian traits in non-avian theropod dinosaurs, however, such as the supposed evolution of partially open nesting behavior in oviraptorids, argues against this long-established theory. Using a double-checking liquid chromatography ESI-Q-TOF mass spectrometry routine, we traced the origin of colored eggs to their non-avian dinosaur ancestors by providing the first record of the avian eggshell pigments protoporphyrin and biliverdin in the eggshells of Late Cretaceous oviraptorid dinosaurs. The eggshell parataxon Macroolithus yaotunensis can be assigned to the oviraptor Heyuannia huangi based on exceptionally preserved, late developmental stage embryo remains. The analyzed eggshells are from three Late Cretaceous fluvial deposits ranging from eastern to southernmost China. Reevaluation of these taphonomic settings, and a consideration of patterns in the porosity of completely preserved eggs support an at least partially open nesting behavior for oviraptorosaurs. Such a nest arrangement corresponds with our reconstruction of blue-green eggs for oviraptors. According to the sexual signaling hypothesis, the reconstructed blue-green eggs support the origin of previously hypothesized avian paternal care in oviraptorid dinosaurs. Preserved dinosaur egg color not only pushes the current limits of the vertebrate molecular and associated soft tissue fossil record, but also provides a perspective on the potential application of this unexplored paleontological resource.

YERSINIA PSEUDOTUBERCULOSIS, SEROGROUP O:1A, INFECTION IN TWO AMAZON PARROTS (AMAZONA AESTIVA AND AMAZONA ORATRIX) WITH HEPATIC HEMOSIDEROSIS.

PubMed

Galosi, Livio; Farneti, Silvana; Rossi, Giacomo; Cork, Susan Catherine; Ferraro, Stefano; Magi, Gian Enrico; Petrini, Stefano; Valiani, Andrea; Cuteri, Vincenzo; Attili, Anna-Rita

2015-09-01

Necropsies were conducted on a female blue-fronted Amazon (Amazona aestiva) and a female yellow-headed Amazon (Amazona oratrix) that died after depression, ruffled feathers, diarrhea, and biliverdin in the urine. Gross and microscopic examinations revealed multifocal necrosis in the liver, spleen, lungs, kidneys, intestines, and heart caused by acute bacteremia. Yersinia pseudotuberculosis, serogroup O:1a, was isolated by culturing from the visceral lesions in the liver, intestines, and spleen. Virulence gene analysis showed the presence of the inv gene and the complete pathogenicity island: IS100, psn, yptE, irp1, irp2 ybtP-ybtQ, ybtX-ybtS, and int asnT-Int. Histopathologic findings and chemical analysis also demonstrated hepatic hemosiderosis. As has been demonstrated in other species, hemosiderosis may predispose Amazona spp. to systemic infection with Y. pseudotuberculosis after enteric disease.

Heme oxygenase: the key to renal function regulation

PubMed Central

Cao, Jian; Sacerdoti, David; Li, Xiaoying; Drummond, George

2009-01-01

Heme oxygenase (HO) plays a critical role in attenuating the production of reactive oxygen species through its ability to degrade heme in an enzymatic process that leads to the production of equimolar amounts of carbon monoxide and biliverdin/bilirubin and the release of free iron. The present review examines the beneficial role of HO-1 (inducible form of HO) that is achieved by increased expression of this enzyme in renal tissue. The influence of the HO system on renal physiology, obesity, vascular dysfunction, and blood pressure regulation is reviewed, and the clinical potential of increased levels of HO-1 protein, HO activity, and HO-derived end products of heme degradation is discussed relative to renal disease. The use of pharmacological and genetic approaches to investigate the role of the HO system in the kidney is key to the development of therapeutic approaches to prevent the adverse effects that accrue due to an impairment in renal function. PMID:19570878

Assessment of bile fluorescence patterns in a tropical fish, Nile tilapia (Oreochromis niloticus) exposed to naphthalene, phenanthrene, pyrene and chrysene using fixed wavelength fluorescence and synchronous fluorescence spectrometry.

PubMed

Pathiratne, A; Hemachandra, C K; Pathiratne, K A S

2010-05-01

Bile fluorescence patterns in Nile tilapia, a potential fish for biomonitoring tropical water pollution were assessed following exposure to selected polycyclic aromatic hydrocarbons (PAHs): naphthalene, phenanthrene, pyrene and chrysene. Non-normalized fixed wavelength fluorescence signals in the fish exposed to these PAHs reflected dose and/or time response relationships of their metabolism. Normalizing signals to biliverdin introduced deviations to these response patterns. The optimal wavelength pairs (excitation/emission) for synchronous fluorescence scanning measurements of bile metabolites of naphthalene, phenanthrene, pyrene and chrysene were identified as 284/326, 252/357, 340/382 and 273/382 respectively. This study supports the use of bile fluorescence in Nile tilapia by fixed wavelength fluorescence and synchronous fluorescence spectrometry with non-normalized data as a simple method for screening bioavailability of these PAHs.

An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging.

PubMed

Yu, Dan; Gustafson, William Clay; Han, Chun; Lafaye, Céline; Noirclerc-Savoye, Marjolaine; Ge, Woo-Ping; Thayer, Desiree A; Huang, Hai; Kornberg, Thomas B; Royant, Antoine; Jan, Lily Yeh; Jan, Yuh Nung; Weiss, William A; Shu, Xiaokun

2014-05-15

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.

Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay.

PubMed

Rücker, Hannelore; Amslinger, Sabine

2015-01-01

The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of lipopolysaccharide and the specific HO-1 inhibitor tin protoporphyrin IX. Taken together, we developed a convenient and highly sensitive ELISA-based HO-1 enzyme activity assay, allowing the identification and characterization of molecules potentially useful for the treatment of inflammatory and autoimmune diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography.

PubMed

Unno, Masaki; Ishikawa-Suto, Kumiko; Kusaka, Katsuhiro; Tamada, Taro; Hagiwara, Yoshinori; Sugishima, Masakazu; Wada, Kei; Yamada, Taro; Tomoyori, Katsuaki; Hosoya, Takaaki; Tanaka, Ichiro; Niimura, Nobuo; Kuroki, Ryota; Inaka, Koji; Ishihara, Makiko; Fukuyama, Keiichi

2015-04-29

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O(+). These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.

Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome.

PubMed

Piatkevich, Kiryl D; Subach, Fedor V; Verkhusha, Vladislav V

2013-01-01

The ability to modulate the fluorescence of optical probes can be used to enhance signal-to-noise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here, we report two bacteriophytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

PubMed

Smirnov, Valeriy V; Roth, Justine P

2014-10-01

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

PubMed Central

Rodriguez, Erik A.; Tran, Geraldine N.; Gross, Larry A.; Crisp, Jessica L.; Shu, Xiaokun; Lin, John Y.; Tsien, Roger Y.

2016-01-01

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP. PMID:27479328

Phototropism and gravitropism in transgenic lines of Arabidopsis altered in the phytochrome pathway.

PubMed

Hopkins, Jane A; Kiss, John Z

2012-07-01

Phytochromes are a family of photoreceptor molecules, absorbing primarily in red and far-red, that are important in many aspects of plant development. These studies investigated the role of phytochromes in phototropism and gravitropism of seedlings of Arabidopsis thaliana. We used two transgenic lines, one which lacked phytochromes specifically in the roots (M0062/UASBVR) and the other lacked phytochromes in the shoots (CAB3::pBVR). These transgenic plants are deficient in the phytochrome chromophore in specific tissues due the expression of biliverdin IXa reductase (BVR), which binds to precursors of the chromophore. Experiments were performed in both light and dark conditions to determine whether roots directly perceive light signals or if the signal is perceived in the shoot and then transmitted to the root during tropistic curvature. Kinetics of tropisms and growth were assayed by standard methods or with a computer-based feedback system. We found that the perception of red light occurs directly in the root during phototropism in this organ and that signaling also may occur from root to shoot in gravitropism. Copyright © Physiologia Plantarum 2012.

STATINS MORE THAN CHOLESTEROL LOWERING AGENTS IN ALZHEIMER DISEASE: THEIR PLEIOTROPIC FUNCTIONS AS POTENTIAL THERAPEUTIC TARGETS

PubMed Central

Barone, Eugenio; Domenico, Fabio Di; Butterfield, D. Allan

2013-01-01

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment, inability to perform activities of daily living and mood changes. Statins, long known to be beneficial in conditions where dyslipidemia occurs by lowering serum cholesterol levels, also have been proposed for use in neurodegenerative conditions, including AD. However, it is not clear that the purported effectiveness of statins in neurodegenerative disorders is directly related to cholesterol-lowering effects of these agents; rather, the pleiotropic functions of statins likely play critical roles. The aim of this review is to provide an overview on the new discoveries about the effects of statin therapy on the oxidative ad nitrosative stress levels as well as on the modulation of the heme oxygenase/biliverdin reductase (HO/BVR) system in the brain. We propose a novel mechanism of action for atorvastatin which, through the activation of HO/BVR-A system, may contribute to the neuroprotective effects thus suggesting a potential therapeutic role in AD and potentially accounting for the observation of decreased AD incidence with persons on statin. PMID:24231510

Study on bioactive compounds of in vitro cultured Calculus Suis and natural Calculus Bovis.

PubMed

Wan, Tien-Chun; Cheng, Fu-Yuan; Liu, Yu-Tse; Lin, Liang-Chuan; Sakata, Ryoichi

2009-12-01

The purpose of the study was to investigate bioactive compounds of in vitro cultured Calculus Suis and natural Calculus Bovis obtained as valuable by-products from animals used for meat production. The results showed that the components of natural Calculus Bovis were rich in bilirubin and biliverdin and had higher content of essential amino acids. The major amino acids of in vitro cultured Calculus Suis were identified as glycine, alanine, glutamic acid and aspartic acid, and those for natural Calculus Bovis were found to be glutamic acid, aspartic acid, proline, and arginine. The methionine and cysteine contents of precursors for glutathione in natural Calculus Bovis were significantly higher than those of in vitro cultured Calculus Suis. The mineral contents of zinc, iron and manganese of natural Calculus Bovis were significantly higher than those of in vitro cultured Calculus Suis. The major bile acids in both products were cholic acid and dehydrocholic acid, respectively. The chenodeoxycholic and ursodeoxycholic acid content of in vitro cultured Calculus Suis was significantly higher than that of natural Calculus Bovis.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

PubMed

Rodriguez, Erik A; Tran, Geraldine N; Gross, Larry A; Crisp, Jessica L; Shu, Xiaokun; Lin, John Y; Tsien, Roger Y

2016-09-01

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

The cuticle modulates ultraviolet reflectance of avian eggshells

PubMed Central

Fecheyr-Lippens, Daphne C.; Igic, Branislav; D'Alba, Liliana; Hanley, Daniel; Verdes, Aida; Holford, Mande; Waterhouse, Geoffrey I. N.; Grim, Tomas; Hauber, Mark E.; Shawkey, Matthew D.

2015-01-01

ABSTRACT Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour. PMID:25964661

Heme oxygenase-1 in tumor biology and therapy.

PubMed

Was, Halina; Dulak, Jozef; Jozkowicz, Alicja

2010-12-01

Heme oxygenase-1 (HO-1) degrades heme to carbon monoxide (CO), biliverdin, and ferrous iron. As HO-1 expression is highly increased by stressful conditions, the major role of the enzyme is the protection against oxidative injury. Additionally, it regulates cell proliferation, modulates inflammatory response and facilitates angiogenesis. Beneficial activities of HO-1 have been recognized in many pathological states e.g. atherosclerosis, diabetes, ischemia/reperfusion injury or organ transplantation. Interestingly HO-1 expression is very often boosted in tumor tissues and could be further elevated in response to radio-, chemo-, or photodynamic therapy. A growing body of evidence suggests that HO-1 may play a role in tumor induction and can potently improve the growth and spread of tumors. This review discusses the implications of HO-1 properties for tumor proliferation and cell death, differentiation, angiogenesis and metastasis, and tumor-related inflammation. Finally, it suggests that pharmacological agents that regulate HO activity or HO-1 gene silencing may become powerful tools for preventing the onset or progression of various cancers and sensitize them to anticancer therapies.

Optical classification of bruises

NASA Astrophysics Data System (ADS)

Randeberg, Lise L.; Winnem, Andreas M.; Blindheim, Sandra; Haugen, Olav A.; Svaasand, Lars O.

2004-07-01

Determining the age of injuries is an important aspect of forensic medicine. Currently, visual inspection and colorimetric measurements are the most common techniques used to assess the age of bruises on a victim's body. Bruises are caused by trauma to the skin and vasculature, and the color will depend on the age, depth, and anatomic site of the hemorrhage. Breakdown products of hemoglobin e.g. biliverdin and bilirubin possess various colors, which can be determined spectrometrically. This study presents reflection spectra collected from bruises in otherwise healthy subjects. A total of 73 spectra of 25 bruises were measured on 13 individuals in the 400-850 nm wavelength region. All injuries were caused by sports activities such as judo and soccer. The bruises were classified according to visual appearance, bilirubin content, oxygenation, and age of the injury. Only bruises with known age and cause were included in the study. Spectral changes of each hematoma were recorded over several days. Preliminary results show large variation in the spectra, caused by differences in age and depth of the bruises. This data may provide a basis for developing an algorithm to determine the age of injuries in e.g. child abuse cases.

Purification and Biochemical Properties of Phytochromobilin Synthase from Etiolated Oat Seedlings1

PubMed Central

McDowell, Michael T.; Lagarias, J. Clark

2001-01-01

Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (PΦB) for photoactivity. In planta, biliverdin IXα (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme PΦB synthase to yield 3Z-PΦB. Here, we describe the >50,000-fold purification of PΦB synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, PΦB synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s−1, which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat PΦB synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of PΦB synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A Km for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 μm. PΦB synthase has a high affinity for its bilin substrate, with a sub-micromolar Km for BV. PMID:11500553

Peptides in low molecular weight fraction of serum associated with hepatocellular carcinoma.

PubMed

An, Yanming; Bekesova, Slavka; Edwards, Nathan; Goldman, Radoslav

2010-01-01

The incidence of hepatocellular carcinoma (HCC) in the United States is increasing and the increase is projected to continue for several decades. The overall survival of HCC patients is poor and treatments are not effective in part because most of the diagnoses come at a late stage. The development of new markers for detection of HCC would significantly improve patient prognosis. This paper describes identification of candidate markers previously reported in our serologic study of an Egyptian population by quantitative comparison of matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectra. To identify these marker candidates, we performed LC-MS/MS sequencing that identified nine native peptides associated with HCC, including two reported previously. Four truncations of N terminus of complement C3f and a fibrinopeptide increased in control sera; two complement C4alpha peptides, a zyxin peptide, and a coagulation factor XIII peptide increased in cancer patient sera. We have also identified increased biliverdin diglucuronide in the sera of cancer patients. These peptides could potentially serve as markers of HCC following additional validation studies; however, association of similar peptides with other diseases and cancers dictates a very cautious approach.

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

PubMed

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.

Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism.

PubMed

Loboda, Agnieszka; Damulewicz, Milena; Pyza, Elzbieta; Jozkowicz, Alicja; Dulak, Jozef

2016-09-01

The multifunctional regulator nuclear factor erythroid 2-related factor (Nrf2) is considered not only as a cytoprotective factor regulating the expression of genes coding for anti-oxidant, anti-inflammatory and detoxifying proteins, but it is also a powerful modulator of species longevity. The vertebrate Nrf2 belongs to Cap 'n' Collar (Cnc) bZIP family of transcription factors and shares a high homology with SKN-1 from Caenorhabditis elegans or CncC found in Drosophila melanogaster. The major characteristics of Nrf2 are to some extent mimicked by Nrf2-dependent genes and their proteins including heme oxygenase-1 (HO-1), which besides removing toxic heme, produces biliverdin, iron ions and carbon monoxide. HO-1 and their products exert beneficial effects through the protection against oxidative injury, regulation of apoptosis, modulation of inflammation as well as contribution to angiogenesis. On the other hand, the disturbances in the proper HO-1 level are associated with the pathogenesis of some age-dependent disorders, including neurodegeneration, cancer or macular degeneration. This review summarizes our knowledge about Nrf2 and HO-1 across different phyla suggesting their conservative role as stress-protective and anti-aging factors.

Heme oxygenase-1 regulates mitochondrial quality control in the heart

PubMed Central

Hull, Travis D.; Boddu, Ravindra; Guo, Lingling; Tisher, Cornelia C.; Traylor, Amie M.; Patel, Bindiya; Joseph, Reny; Prabhu, Sumanth D.; Suliman, Hagir B.; Piantadosi, Claude A.; George, James F.

2016-01-01

The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control. PMID:27110594

Proteomic analysis of acetaminophen-induced hepatotoxicity and identification of heme oxygenase 1 as a potential plasma biomarker of liver injury.

PubMed

Gao, Yuan; Cao, Zhijun; Yang, Xi; Abdelmegeed, Mohamed A; Sun, Jinchun; Chen, Si; Beger, Richard D; Davis, Kelly; Salminen, William F; Song, Byoung-Joon; Mendrick, Donna L; Yu, Li-Rong

2017-01-01

Overdose of acetaminophen (APAP) is a major cause of acute liver failure. This study was aimed to identify pathways related to hepatotoxicity and potential biomarkers of liver injury. Rats were treated with low (100 mg/kg) and high (1250 mg/kg) doses of APAP, and liver tissues at 6 and 24 h post-treatment were analyzed using a proteomic approach of 16O/18O labeling and 2D-LC-MS/MS. Molecular pathways evolved progressively from scattered and less significant perturbations to more focused and significant alterations in a dose- and time-dependent manner upon APAP treatment. Imbalanced expression of hemeoxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA) was associated with hepatotoxicity. Protein abundance changes of a total of 31 proteins were uniquely correlated to liver damage, among which a dramatic increase of HMOX1 levels in plasma was observed. Liver injury-associated significant elevation of plasma HMOX1 was further validated in mice treated with APAP. This study unveiled molecular changes associated with APAP-induced liver toxicity at the pathway levels and identified HMOX1 as a potential plasma biomarker of liver injury. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Proteomic analysis of acetaminophen-induced hepatotoxicity and identification of heme oxygenase 1 as a potential plasma biomarker of liver injury

PubMed Central

Gao, Yuan; Cao, Zhijun; Yang, Xi; Abdelmegeed, Mohamed A.; Sun, Jinchun; Chen, Si; Beger, Richard D.; Davis, Kelly; Salminen, William F.; Song, Byoung-Joon; Mendrick, Donna L.; Yu, Li-Rong

2017-01-01

Purpose Overdose of acetaminophen (APAP) is a major cause of acute liver failure. This study was aimed to identify pathways related to hepatotoxicity and potential biomarkers of liver injury. Experimental design Rats were treated with low (100 mg/kg) and high (1250 mg/kg) doses of APAP, and liver tissues at 6 and 24 h post-treatment were analyzed using a proteomic approach of 16O/18O labeling and 2D-LC-MS/MS. Results Molecular pathways evolved progressively from scattered and less significant perturbations to more focused and significant alterations in a dose- and time-dependent manner upon APAP treatment. Imbalanced expression of hemeoxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA) was associated with hepatotoxicity. Protein abundance changes of a total of 31 proteins were uniquely correlated to liver damage, among which a dramatic increase of HMOX1 levels in plasma was observed. Liver injury-associated significant elevation of plasma HMOX1 was further validated in mice treated with APAP. Conclusions and clinical relevance This study unveiled molecular changes associated with APAP-induced liver toxicity at the pathway levels and identified HMOX1 as a potential plasma biomarker of liver injury. PMID:27634590

Heme oxygenase and carbon monoxide protect from muscle dystrophy.

PubMed

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.

1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure

PubMed Central

Ma, Li-Hua; Liu, Yangzhong; Zhang, Xuhong; Yoshida, Tadashi; La Mar, Gerd N.

2009-01-01

Heme oxygenase carries out stereospecific catabolism of protohemin to yield iron, CO and biliverdin. Instability of the physiological oxy complex has necessitated the use of model ligands, of which cyanide and azide are amenable to solution NMR characterization. Since cyanide and azide are contrasting models for bound oxygen, it is of interest to characterize differences in their molecular and/or electronic structures. We report on detailed 2D NMR comparison of the azide and cyanide substrate complexes of heme oxygenase from Neisseria meningitidis, which reveals significant and widespread differences in chemical shifts between the two complexes. To differentiate molecular from electronic structural changes between the two complexes, the anisotropy and orientation of the paramagnetic susceptibility tensor were determined for the azide complex for comparison with those for the cyanide complex. Comparison of the predicted and observed dipolar shifts reveals that shift differences are strongly dominated by differences in electronic structure and do not provide any evidence for detectable differences in molecular structure or hydrogen bonding except in the immediate vicinity of the distal ligand. The readily cleaved C-terminus interacts with the active site and saturation-transfer allows difficult heme assignments in the high-spin aquo complex. PMID:18976815

Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

PubMed Central

Jison, Maria L.; Munson, Peter J.; Barb, Jennifer J.; Suffredini, Anthony F.; Talwar, Shefali; Logun, Carolea; Raghavachari, Nalini; Beigel, John H.; Shelhamer, James H.; Danner, Robert L.; Gladwin, Mark T.

2016-01-01

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury. PMID:15031206

Serial Metabolome Changes in a Prospective Cohort of Subjects with Influenza Viral Infection and Comparison with Dengue Fever.

PubMed

Cui, Liang; Fang, Jinling; Ooi, Eng Eong; Lee, Yie Hou

2017-07-07

Influenza virus infection (IVI) and dengue virus infection (DVI) are major public health threats. Between IVI and DVI, clinical symptoms can be overlapping yet infection-specific, but host metabolome changes are not well-described. Untargeted metabolomics and targeted oxylipinomic analyses were performed on sera serially collected at three phases of infection from a prospective cohort study of adult subjects with either H3N2 influenza infection or dengue fever. Untargeted metabolomics identified 26 differential metabolites, and major perturbed pathways included purine metabolism, fatty acid biosynthesis and β-oxidation, tryptophan metabolism, phospholipid catabolism, and steroid hormone pathway. Alterations in eight oxylipins were associated with the early symptomatic phase of H3N2 flu infection, were mostly arachidonic acid-derived, and were enriched in the lipoxygenase pathway. There was significant overlap in metabolome profiles in both infections. However, differences specific to IVI and DVI were observed. DVI specifically attenuated metabolites including serotonin, bile acids and biliverdin. Additionally, metabolome changes were more persistent in IVI in which metabolites such as hypoxanthine, inosine, and xanthine of the purine metabolism pathway remained significantly elevated at 21-27 days after fever onset. This study revealed the dynamic metabolome changes in IVI subjects and provided biochemical insights on host physiological similarities and differences between IVI and DVI.

Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance.

PubMed

Ryter, Stefan W; Otterbein, Leo E; Morse, Danielle; Choi, Augustine M K

2002-01-01

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO exists as constitutive (HO-2, HO-3) and inducible isoforms (HO-1), the latter which responds to regulation by multiple stress-stimuli. HO-1 confers protection in vitro and in vivo against oxidative cellular stress. Although the redox active compounds that are generated from HO activity (i.e. iron, biliverdin-IXalpha, and bilirubin-IXa) potentially modulate oxidative stress resistance, increasing evidence points to cytoprotective roles for CO. Though not reactive, CO regulates vascular processes such as vessel tone, smooth muscle proliferation, and platelet aggregation, and possibly functions as a neurotransmitter. The latter effects of CO depend on the activation of guanylate cyclase activity by direct binding to the heme moiety of the enzyme, stimulating the production of cyclic 3':5'-guanosine monophosphate. CO potentially interacts with other intracellular hemoprotein targets, though little is known about the functional significance of such interactions. Recent progress indicates that CO exerts novel anti-inflammatory and anti-apoptotic effects dependent on the modulation of the p38 mitogen activated protein kinase (MAPK)-signaling pathway. By virtue of these effects, CO confers protection in oxidative lung injury models, and likely plays a role in HO-1 mediated tissue protection.

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Restless Legs Syndrome

PubMed Central

García-Martín, Elena; Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A.G.

2015-01-01

Abstract Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Heme-oxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls. The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs. The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population. PMID:26313808

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Restless Legs Syndrome.

PubMed

García-Martín, Elena; Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A G

2015-08-01

Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Heme-oxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS.We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls.The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs.The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population.

Heme Oxygenase in the Regulation of Vascular Biology: From Molecular Mechanisms to Therapeutic Opportunities

PubMed Central

Kim, Young-Myeong; Pae, Hyun-Ock; Park, Jeong Euy; Lee, Yong Chul; Woo, Je Moon; Kim, Nam-Ho; Choi, Yoon Kyung; Lee, Bok-Soo; Kim, So Ri

2011-01-01

Abstract Heme oxygenases (HOs) are the rate-limiting enzymes in the catabolism of heme into biliverdin, free iron, and carbon monoxide. Two genetically distinct isoforms of HO have been characterized: an inducible form, HO-1, and a constitutively expressed form, HO-2. HO-1 is a kind of stress protein, and thus regarded as a sensitive and reliable indicator of cellular oxidative stress. The HO system acts as potent antioxidants, protects endothelial cells from apoptosis, is involved in regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in angiogenesis and vasculogenesis. Endothelial integrity and activity are thought to occupy the central position in the pathogenesis of cardiovascular diseases. Cardiovascular disease risk conditions converge in the contribution to oxidative stress. The oxidative stress leads to endothelial and vascular smooth muscle cell dysfunction with increases in vessel tone, cell growth, and gene expression that create a pro-thrombotic/pro-inflammatory environment. Subsequent formation, progression, and obstruction of atherosclerotic plaque may result in myocardial infarction, stroke, and cardiovascular death. This background provides the rationale for exploring the potential therapeutic role for HO system in the amelioration of vascular inflammation and prevention of adverse cardiovascular outcomes. Antioxid. Redox Signal. 14, 137–167. PMID:20624029

Heme oxygenase 1 defects lead to reduced chlorophyll in Brassica napus.

PubMed

Zhu, Lixia; Yang, Zonghui; Zeng, Xinhua; Gao, Jie; Liu, Jie; Yi, Bin; Ma, Chaozhi; Shen, Jinxiong; Tu, Jinxing; Fu, Tingdong; Wen, Jing

2017-04-01

We previously described a Brassica napus chlorophyll-deficient mutant (ygl) with yellow-green seedling leaves and mapped the related gene, BnaC.YGL, to a 0.35 cM region. However, the molecular mechanisms involved in this chlorophyll defect are still unknown. In this study, the BnaC07.HO1 gene (equivalent to BnaC.YGL) was isolated by the candidate gene approach, and its function was confirmed by genetic complementation. Comparative sequencing analysis suggested that BnaC07.HO1 was lost in the mutant, while a long noncoding-RNA was inserted into the promoter of the homologous gene BnaA07.HO1. This insert was widely present in B. napus cultivars and down-regulated BnaA07.HO1 expression. BnaC07.HO1 was highly expressed in the seedling leaves and encoded heme oxygenase 1, which was localized in the chloroplast. Biochemical analysis showed that BnaC07.HO1 can catalyze heme conversion to form biliverdin IXα. RNA-seq analysis revealed that the loss of BnaC07.HO1 impaired tetrapyrrole metabolism, especially chlorophyll biosynthesis. According, the levels of chlorophyll intermediates were reduced in the ygl mutant. In addition, gene expression in multiple pathways was affected in ygl. These findings provide molecular evidences for the basis of the yellow-green leaf phenotype and further insights into the crucial role of HO1 in B. napus.

Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.

PubMed

Hontani, Yusaku; Shcherbakova, Daria M; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav V; Kennis, John T M

2016-11-18

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

RNAi knockdown of rice SE5 gene is sensitive to the herbicide methyl viologen by the down-regulation of antioxidant defense.

PubMed

Xu, Sheng; Wang, Lijuan; Zhang, Bo; Han, Bin; Xie, Yanjie; Yang, Jie; Zhong, Weigong; Chen, Huiping; Wang, Ren; Wang, Ning; Cui, Weiti; Shen, Wenbiao

2012-09-01

Plant heme oxygenase (HO) catalyzes the oxygenation of heme to biliverdin, carbon monoxide (CO), and free iron (Fe(2+))-and Arabidopsis and rice (Oryza sativa) HOs are involved in light signaling. Here, we identified that the rice PHOTOPERIOD SENSITIVITY 5 (SE5) gene, which encoded a putative HO with high similarity to HO-1 from Arabidopsis (HY1), exhibited HO activity, and localized in the chloroplasts. Rice RNAi mutants silenced for SE5 were generated and displayed early flowering under long-day conditions, consistent with phenotypes of the null mutation in SE5 gene reported previously (se5 and s73). The herbicide methyl viologen (MV), which produces reactive oxygen species (ROS), was applied to determine whether SE5 regulates oxidative stress response. Compared with wild-type, SE5 RNAi transgenic plants aggravated seedling growth inhibition, chlorophyll loss and ROS overproduction, and decreased the transcripts of some representative antioxidative genes. By contrast, administration of exogenous CO partially rescued corresponding MV hypersensitivity in the SE5 RNAi plants. Alleviation of seed germination inhibition, chlorophyll loss and ROS overproduction, as well as the induction of antioxidant defense were further observed when SE5 or HY1 was overexpressed in transgenic Arabidopsis plants, indicating that SE5 may be useful for molecular breeding designed to improve plant tolerance to oxidative stress.

The non-canonical functions of the heme oxygenases

PubMed Central

Tibullo, Daniele; Forte, Stefano; Zappalà, Agata; Volti, Giovanni Li

2016-01-01

Heme oxygenase (HO) isoforms catalyze the conversion of heme to carbon monoxide (CO) and biliverdin with a concurrent release of iron, which can drive the synthesis of ferritin for iron sequestration. Most of the studies so far were directed at evaluating the protective effect of these enzymes because of their ability to generate antioxidant and antiapoptotic molecules such as CO and bilirubin. Recent evidences are suggesting that HO may possess other important physiological functions, which are not related to its enzymatic activity and for which we would like to introduce for the first time the term “non canonical functions”. Recent evidence suggest that both HO isoforms may form protein-protein interactions (i.e. cytochrome P450, adiponectin, CD91) thus serving as chaperone-like protein. In addition, truncated HO-1 isoform was localized in the nuclear compartment under certain experimental conditions (i.e. excitotoxicity, hypoxia) regulating the activity of important nuclear transcription factors (i.e. Nrf2) and DNA repair. In the present review, we discuss three potential signaling mechanisms that we refer to as the non-canonical functions of the HO isoforms: protein-protein interaction, intracellular compartmentalization, and extracellular secretion. The aim of the present review is to describe each of this mechanism and all the aspects warranting additional studies in order to unravel all the functions of the HO system. PMID:27626166

In vivo regulation of the heme oxygenase-1 gene in humanized transgenic mice

PubMed Central

Kim, Junghyun; Zarjou, Abolfazl; Traylor, Amie M.; Bolisetty, Subhashini; Jaimes, Edgar A.; Hull, Travis D.; George, James F.; Mikhail, Fady M.; Agarwal, Anupam

2012-01-01

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation but whether such control occurs in vivo is not known. To enable such analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome Conformation Capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases. PMID:22495295

A Central Role for Heme Oxygenase-1 in the Control of Intestinal Epithelial Chemokine Expression.

PubMed

Onyiah, Joseph C; Schaefer, Rachel E M; Colgan, Sean P

2018-05-23

In mucosal inflammatory disorders, the protective influence of heme oxygenase-1 (HO-1) and its metabolic byproducts, carbon monoxide (CO) and biliverdin, is a topic of significant interest. Mechanisms under investigation include the regulation of macrophage function and mucosal cytokine expression. While there is an increasing recognition of the importance of epithelial-derived factors in the maintenance of intestinal mucosal homeostasis, the contribution of intestinal epithelial cell (IEC) HO-1 on inflammatory responses has not previously been investigated. We examined the influence of modulating HO-1 expression on the inflammatory response of human IECs. Engineered deficiency of HO-1 in Caco-2 and T84 IECs led to increased proinflammatory chemokine expression in response to pathogenic bacteria and inflammatory cytokine stimulation. Crosstalk with activated leukocytes also led to increased chemokine expression in HO-1-deficient cells in an IL-1β dependent manner. Treatment of Caco-2 cells with a pharmacological inducer of HO-1 led to the inhibition of chemokine expression. Mechanistic studies suggest that HO-1 and HO-1-related transcription factors, but not HO-1 metabolic products, are partly responsible for the influence of HO-1 on chemokine expression. In conclusion, our data identify HO-1 as a central regulator of IEC chemokine expression that may contribute to homeo-stasis in the intestinal mucosa. © 2018 S. Karger AG, Basel.

Insight Into the Radical Mechanism of Phycocyanobilin-Ferredoxin Oxidoreductase (Pcya) Revealed By X-Ray Crystallography And Biochemical Measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu, S.-L.; Rockwell, N.; Lagarias, J.C.

2007-07-13

The X-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) from the cyanobacterium Nostoc sp. PCC7120 has been solved at 2.5 angstrom resolution. A comparative analysis of this structure with those recently reported for substrate-bound and substrate-free forms of PcyA from the cyanobacterium Synechocystis sp. PCC6803 (Hagiwara et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 27-32; Hagiwara et al. (2006) FEBS Lett. 580, 3823-3828) provides a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. On the basis of these structures and the biochemical analysis ofmore » site-directed mutants of Nostoc PcyA, including mutants reported in recent studies (Tu et al. (2006) J. Biol. Chem. 281, 3127-3136) as well as mutants described in this study, a revised mechanism for the PcyA-mediated four-electron reduction of biliverdin IX{alpha} to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates is proposed. The mechanistic insight of these studies, along with homology modeling, have provided new insight into the catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family that are widespread in oxygenic photosynthetic organisms.« less

Electronic transitions and heterogeneity of the bacteriophytochrome Pr absorption band: An angle balanced polarization resolved femtosecond VIS pump–IR probe study

PubMed Central

Linke, Martin; Yang, Yang; Zienicke, Benjamin; Hammam, Mostafa A.S.; von Haimberger, Theodore; Zacarias, Angelica; Inomata, Katsuhiko; Lamparter, Tilman; Heyne, Karsten

2013-01-01

Photoisomerization of biliverdin (BV) chromophore triggers the photoresponse in native Agp1 bacteriophytochrome. We discuss heterogeneity in phytochrome Pr form to account for the shape of the absorption profile. We investigated different regions of the absorption profile by angle balanced polarization resolved femtosecond VIS pump–IR probe spectroscopy. We studied the Pr form of Agp1 with its natural chromophore and with a sterically locked 18Et-BV (locked Agp1). We followed the dynamics and orientations of the carbonyl stretching vibrations of ring D and ring A in their ground and electronically excited states. Photoisomerization of ring D is reflected by strong signals of the ring D carbonyl vibration. In contrast, orientational data on ring A show no rotation of ring A upon photoexcitation. Orientational data allow excluding a ZZZasa geometry and corroborates a nontwisted ZZZssa geometry of the chromophore. We found no proof for heterogeneity but identified a new, to our knowledge, electronic transition in the absorption profile at 644 nm (S0→S2). Excitation of the S0→S2 transition will introduce a more complex photodynamics compared with S0→S1 transition. Our approach provides fundamental information on disentanglement of absorption profiles, identification of chromophore structures, and determination of molecular groups involved in the photoisomerization process of photoreceptors. PMID:24138851

Heme oxygenase-1: a metabolic nike.

PubMed

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.

The hmuQ and hmuD Genes from Bradyrhizobium japonicum Encode Heme-Degrading Enzymes

PubMed Central

Puri, Sumant; O'Brian, Mark R.

2006-01-01

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a Kd value of 0.8 μM and showed spectral properties consistent with a heme oxygenase. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. The findings show that hmuQ and hmuD encode heme oxygenases and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to gram-positive pathogenic bacteria. PMID:16952937

Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence

NASA Astrophysics Data System (ADS)

Rumyantsev, Konstantin A.; Shcherbakova, Daria M.; Zakharova, Natalia I.; Emelyanov, Alexander V.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

2015-12-01

Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

Parental conflict and blue egg coloration in a seabird

NASA Astrophysics Data System (ADS)

Morales, Judith; Torres, Roxana; Velando, Alberto

2010-02-01

When both parents provide offspring care, equal sharing of costly parental duties may enhance reproductive success. This is crucial for longlived species, where increased parental effort in current reproduction profoundly affects future reproduction. Indication of reproductive value or willingness to invest in reproduction may promote matching responses by mates, thus reducing the conflict over care. In birds with biparental care, blue-green eggshell color may function as a signal of reproductive value that affects parental effort, as predicted by the signaling hypothesis of blue-green eggshell coloration. However, this hypothesis has not been explored during incubation, when the potential stimulus of egg color is present, and has been little studied in longlived birds. We experimentally studied if egg color affected incubation patterns in the blue-footed booby, a longlived species with biparental care and blue eggs. We exchanged fresh eggs between nests of the same laying date and recorded parental incubation effort on the following 4 days. Although egg color did not affect male effort, original eggshell color was correlated with pair matching in incubation. Exchanged eggshell color did not affect incubation patterns. This suggests that biliverdin-based egg coloration reflects female quality features that are associated with pair incubation effort or that blue-footed boobies mate assortatively high-quality pairs incubating more colorful clutches. An intriguing possibility is that egg coloration facilitates an equal sharing of incubation, the signal being functional only during a short period close to laying. Results also suggest that indication of reproductive value reduces the conflict over care.

Microarray genomic profile of mitochondrial and oxidant response in Manganese Chloride treated PC12 cells

PubMed Central

Taka, Equar; Mazzio, Elizabeth; Soliman, Karam FA; Reams, R. Renee

2012-01-01

Environmental or occupational exposure to high levels of manganese (Mn) can lead to manganism, a symptomatic neuro-degenerative disorder similar to idiopathic Parkinson’s disease. The underlying mechanism of Mn neurotoxicity remains unclear. In this study, we evaluate the primary toxicological events associated with MnCl2 toxicity in rat PC12 cells using whole genome cDNA microarray, RT-PCR, western blot and functional studies. The results show that a sub-lethal dose range (38–300 µM MnCl2) initiated slight metabolic stress evidenced by heightened glycolytic rate and induction of enolase / aldolase - gene expression. The largest shift observed in the transcriptome was MnCl2 induction of heme-oxygenase 1 (HO-1) [7.7 fold, p <0.001], which was further corroborated by RT-PCR and western blot studies. Concentrations in excess of 300 µM corresponded to dose dependent loss of cell viability which was associated with enhanced production of H2O2 concomitant to elevation of of gene expression for diverse antioxidant enzymes; biliverdin reductase, arsenite inducible RNA associated protein, dithiolethione-inducible gene-1 (DIG-1) and .thioredoxin reductase 1. Moreover, Mn initiated significant reduction of gene expression of mitochondrial glutaryl-coenzyme A dehydrogenase (GCDH) -, an enzyme involved with glutaric acidemia, oxidative stress, lipid peroxidation and striatal degeneration observed in association with severe dystonic dyskinetic movement disorder. Future research will be required to elucidate a defined role for HO-1 and GCDH in Mn toxicity. PMID:22281203

Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

PubMed

Assunção-Miranda, I; Cruz-Oliveira, C; Neris, R L S; Figueiredo, C M; Pereira, L P S; Rodrigues, D; Araujo, D F F; Da Poian, A T; Bozza, M T

2016-03-01

To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV. © 2016 The Society for Applied Microbiology.

Plasma metabolomic study in Chinese patients with wet age-related macular degeneration.

PubMed

Luo, Dan; Deng, Tingting; Yuan, Wei; Deng, Hui; Jin, Ming

2017-09-06

Age-related macular degeneration (AMD) is a leading disease associated with blindness. It has a high incidence and complex pathogenesis. We aimed to study the metabolomic characteristics in Chinese patients with wet AMD by analyzing the morning plasma of 20 healthy controls and 20 wet AMD patients for metabolic differences. We used ultra-high-pressure liquid chromatography and quadrupole-time-of-flight mass spectrometry for this analysis. The relationship of these differences with AMD pathophysiology was also assessed. Remaining data were normalized using Pareto scaling, and then valid data were handled using multivariate data analysis with MetaboAnalysis software, including unsupervised principal component analysis and supervised partial least squares-discriminate analysis. The purpose of the present work was to identify significant metabolites for the analyses. Hierarchical clustering was conducted to identify metabolites that differed between the two groups. Significant metabolites were then identified using the established database, and features were mapped on the Kyoto Encyclopedia of Genes and Genomes. A total of 5443 ion peaks were detected, all of them attributable to the same 10 metabolites. These included some amino acids, isomaltose, hydrocortisone, and biliverdin. The heights of these peaks differed significantly between the two groups. The biosynthesis of amino acids pathways also differed profoundly between patients with wet AMD and controls. These findings suggested that metabolic profiles and and pathways differed between wet AMD and controls and may provide promising new targets for AMD-directed therapeutics and diagnostics.

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

PubMed

Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

1995-04-04

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Heme Oxygenase-1: A Metabolic Nike

PubMed Central

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

2014-01-01

Abstract Significance: 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. Recent Advances: 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. Critical Issues: 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. Future Directions: 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. Antioxid. Redox Signal. 20, 1709–1722. PMID:24180257

The acute impact of polyphenols from Hibiscus sabdariffa in metabolic homeostasis: an approach combining metabolomics and gene-expression analyses.

PubMed

Beltrán-Debón, Raúl; Rodríguez-Gallego, Esther; Fernández-Arroyo, Salvador; Senan-Campos, Oriol; Massucci, Francesco A; Hernández-Aguilera, Anna; Sales-Pardo, Marta; Guimerà, Roger; Camps, Jordi; Menendez, Javier A; Joven, Jorge

2015-09-01

We explored the acute multifunctional effects of polyphenols from Hibiscus sabdariffa in humans to assess possible consequences on the host's health. The expected dynamic response was studied using a combination of transcriptomics and metabolomics to integrate specific functional pathways through network-based methods and to generate hypotheses established by acute metabolic effects and/or modifications in the expression of relevant genes. Data were obtained from healthy male volunteers after 3 hours of ingestion of an aqueous Hibiscus sabdariffa extract. The data were compared with data obtained prior to the ingestion, and the overall findings suggest that these particular polyphenols had a simultaneous role in mitochondrial function, energy homeostasis and protection of the cardiovascular system. These findings suggest beneficial actions in inflammation, endothelial dysfunction, and oxidation, which are interrelated mechanisms. Among other effects, the activation of the heme oxygenase-biliverdin reductase axis, the systemic inhibition of the renin-angiotensin system, the inhibition of the angiotensin-converting enzyme, and several actions mirroring those of the peroxisome proliferator-activated receptor agonists further support this notion. We also found concordant findings in the serum of the participants, which include a decrease in cortisol levels and a significant increase in the active vasodilator metabolite of bradykinin (des-Arg(9)-bradykinin). Therefore, our data support the view that polyphenols from Hibiscus sabdariffa play a regulatory role in metabolic health and in the maintenance of blood pressure, thus implying a multi-faceted impact in metabolic and cardiovascular diseases.

Does egg colouration reflect male condition in birds?

NASA Astrophysics Data System (ADS)

Martínez-Padilla, Jesús; Dixon, Heather; Vergara, Pablo; Pérez-Rodríguez, Lorenzo; Fargallo, Juan A.

2010-05-01

How colouration provides information about individuals in birds has been a central issue in recent decades. Although much information has been derived, little is known about the adaptive significance of egg colouration in birds. A recent idea suggests that biliverdin- and porphyrin-pigmented eggs may act as a post-mating sexual signal for males to assess female quality. In birds, it is common for males to influence prelaying female condition by courtship feeding. Using Eurasian kestrels, a species that lays protoporphyrin-pigmented eggs, we descriptively assessed the influence of male feeding on egg pigmentation by considering female phenotype, condition, breeding parameters and male body condition. We found that older females and females with greyer tails (an index of individual quality) produce highly pigmented eggs. However, male body condition was the only variable that explained egg colouration when considered together with the female-related variables. Therefore, females that mated with males in better condition laid highly pigmented eggs. With the same species, we also explored the cost of producing protoporphyrin-pigmented eggs using a food-supply experiment before the laying period. Food supplementation did not increase egg pigmentation, but hatching success and egg mass were positively related to egg colouration only in food supplied pairs. We suggest that egg colouration might be costly to produce and probably suggests egg quality. However, this cost cannot be explained by female quality, but by male condition instead. In general, our results do not support the theory that egg colouration is a post-mating sexual signal in species where males determine female condition at the time of laying.

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

PubMed

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.

Cerebroprotective functions of HO-2.

PubMed

Parfenova, Helena; Leffler, Charles W

2008-01-01

The constitutive isoform of heme oxygenase, HO-2, is highly expressed in the brain and in cerebral vessels. HO-2 functions in the brain have been evaluated using pharmacological inhibitors of the enzyme and HO-2 gene deletion in in vivo animal models and in cultured cells (neurons, astrocytes, cerebral vascular endothelial cells). Rapid activation of HO-2 via post-translational modifications without upregulation of HO-2 expression or HO-1 induction coincides with the increase in cerebral blood flow aimed at maintaining brain homeostasis and neuronal survival during seizures, hypoxia, and hypotension. Pharmacological inhibition or gene deletion of brain HO-2 exacerbates oxidative stress induced by seizures, glutamate, and inflammatory cytokines, and causes cerebral vascular injury. Carbon monoxide (CO) and bilirubin, the end products of HO-catalyzed heme degradation, have distinct cytoprotective functions. CO, by binding to a heme prosthetic group, regulates the key components of cell signaling, including BK(Ca) channels, guanylyl cyclase, NADPH oxidase, and the mitochondria respiratory chain. Cerebral vasodilator effects of CO are mediated via activation of BK(Ca) channels and guanylyl cyclase. CO, by inhibiting the major components of endogenous oxidant-generating machinery, NADPH oxidase and the cytochrome C oxidase of the mitochondrial respiratory chain, blocks formation of reactive oxygen species. Bilirubin, via redox cycling with biliverdin, is a potent oxidant scavenger that removes preformed oxidants. Overall, HO-2 has dual housekeeping cerebroprotective functions by maintaining autoregulation of cerebral blood flow aimed at improving neuronal survival in a changing environment, and by providing an effective defense mechanism that blocks oxidant formation and prevents cell death caused by oxidative stress.

An Integrated Clinico-transcriptomic Approach Identifies a Central Role of the Heme Degradation Pathway for Septic Complications after Trauma.

PubMed

Rittirsch, Daniel; Schoenborn, Veit; Lindig, Sandro; Wanner, Elisabeth; Sprengel, Kai; Günkel, Sebastian; Blaess, Markus; Schaarschmidt, Barbara; Sailer, Patricia; Märsmann, Sonja; Simmen, Hans-Peter; Cinelli, Paolo; Bauer, Michael; Claus, Ralf A; Wanner, Guido A

2016-12-01

The present study was aimed to identify mechanisms linked to complicated courses and adverse events after severe trauma by a systems biology approach. In severe trauma, overwhelming systemic inflammation can result in additional damage and the development of complications, including sepsis. In a prospective, longitudinal single-center study, RNA samples from circulating leukocytes from patients with multiple injury (injury severity score ≥17 points; n = 81) were analyzed for dynamic changes in gene expression over a period of 21 days by whole-genome screening (discovery set; n = 10 patients; 90 samples) and quantitative RT-PCR (validation set; n = 71 patients, 517 samples). Multivariate correlational analysis of transcripts and clinical parameters was used to identify mechanisms related to sepsis. Transcriptome profiling of the discovery set revealed the strongest changes between patients with either systemic inflammation or sepsis in gene expression of the heme degradation pathway. Using quantitative RT-PCR analyses (validation set), the key components haptoglobin (HP), cluster of differentiation (CD) 163, heme oxygenase-1 (HMOX1), and biliverdin reductase A (BLVRA) showed robust changes following trauma. Upregulation of HP was associated with the severity of systemic inflammation and the development of sepsis. Patients who received allogeneic blood transfusions had a higher incidence of nosocomial infections and sepsis, and the amount of blood transfusion as source of free heme correlated with the expression pattern of HP. These findings indicate that the heme degradation pathway is associated with increased susceptibility to septic complications after trauma, which is indicated by HP expression in particular.

Comparison of the vanadate oxidase method with the diazo method for serum bilirubin determination in dog, monkey, and rat.

PubMed

Ameri, Mehrdad; Schnaars, Henry; Sibley, John; Honor, David

2011-01-01

The most widely used method for bilirubin concentration determination is the diazo method, which measures the color of azobilirubin. The vanadate oxidase method is based on oxidation of bilirubin to biliverdin by vanadate. The objective of this study was to compare total and direct bilirubin concentration ([Bt] and [Bd], respectively) determined by the diazo and vanadate oxidase methods in pooled serum samples from dogs, monkeys, and rats spiked with panels of different concentrations of bilirubin standards. Pooled serum samples from 40 dogs, 40 monkeys, and 60 rats were spiked with either ditaurine conjugates of bilirubin or a standard reference material. The results obtained from both assays were compared using Deming regression analysis. The intra- and interassay precision, expressed as a percentage of the coefficient of variation (%CV), was determined for [Bt] and [Bd], and the mean percentage of recovery was calculated. The vanadate oxidase method displayed an excellent correlation (r  =  0.99-1.00) with the diazo method. Using Deming regression, there were minimal negative or positive constant and proportional biases for [Bt] and [Bd]. The precision studies revealed that the vanadate oxidase method has comparable between-run and within-run CVs to those of the diazo method. The recovery study demonstrated that the diazo method more closely approximates the expected values of [Bt]. In conclusion, the vanadate oxidase method is a simple and rapid method that can be employed as an alternative to the diazo method when interfering substances are present in the serum samples of dog, monkey, and rat.

Molecular cloning, characterization, and expression of an alfalfa (Medicago sativa L.) heme oxygenase-1 gene, MsHO1, which is pro-oxidants-regulated.

PubMed

Fu, Guang-Qing; Xu, Sheng; Xie, Yan-Jie; Han, Bin; Nie, Li; Shen, Wen-Biao; Wang, Ren

2011-07-01

It has been documented that plant heme oxygenase-1 (HO-1; EC 1.14.99.3) is both development- and stress-regulated, thus it plays a vital role in light signalling and stress responses. In this study, an alfalfa (Medica sativa L.) HO-1 gene MsHO1 was isolated and sequenced. It contains four exons and three introns within genomic DNA sequence and encodes a polypeptide with 283 amino acids. MsHO1 had a conserved HO signature sequence and showed high similarity to other HOs in plants, especially HO-1 isoform. The MsHO1:GFP fusion protein was localized in the chloroplast. Further biochemical activity analysis of mature MsHO1, which was expressed in Escherichia coli, showed that the Vmax was 48.78 nmol biliverdin-IXα (BV) h⁻¹ nmol⁻¹ protein with an apparent Km value for hemin of 2.33 μM, and the optimum Tm and pH were 37 °C and 7.2, respectively. Results of semi-quantitative RT-PCR and western blot showed that the expressions of MsHO1 were higher in alfalfa stems and leaves than those in germinating seeds and roots. Importantly, MsHO1 gene expression and protein level were induced significantly by some pro-oxidant compounds, including hemin and nitric oxide (NO) donor sodium nitroprusside (SNP). In conclusion, MsHO1 may play an important role in oxidative responses. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease

PubMed Central

Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

2015-01-01

Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

Orthodontic Forces Induce the Cytoprotective Enzyme Heme Oxygenase-1 in Rats.

PubMed

Suttorp, Christiaan M; Xie, Rui; Lundvig, Ditte M S; Kuijpers-Jagtman, Anne Marie; Uijttenboogaart, Jasper Tom; Van Rheden, René; Maltha, Jaap C; Wagener, Frank A D T G

2016-01-01

Orthodontic forces disturb the microenvironment of the periodontal ligament (PDL), and induce craniofacial bone remodeling which is necessary for tooth movement. Unfortunately, orthodontic tooth movement is often hampered by ischemic injury and cell death within the PDL (hyalinization) and root resorption. Large inter-individual differences in hyalinization and root resorption have been observed, and may be explained by differential protection against hyalinization. Heme oxygenase-1 (HO-1) forms an important protective mechanism by breaking down heme into the strong anti-oxidants biliverdin/bilirubin and the signaling molecule carbon monoxide. These versatile HO-1 products protect against ischemic and inflammatory injury. We postulate that orthodontic forces induce HO-1 expression in the PDL during experimental tooth movement. Twenty-five 6-week-old male Wistar rats were used in this study. The upper three molars at one side were moved mesially using a Nickel-Titanium coil spring, providing a continuous orthodontic force of 10 cN. The contralateral side served as control. After 6, 12, 72, 96, and 120 h groups of rats were killed. On parasagittal sections immunohistochemical staining was performed for analysis of HO-1 expression and quantification of osteoclasts. Orthodontic force induced a significant time-dependent HO-1 expression in mononuclear cells within the PDL at both the apposition- and resorption side. Shortly after placement of the orthodontic appliance HO-1 expression was highly induced in PDL cells but dropped to control levels within 72 h. Some osteoclasts were also HO-1 positive but this induction was shown to be independent of time- and mechanical stress. It is tempting to speculate that differential induction of tissue protecting- and osteoclast activating genes in the PDL determine the level of bone resorption and hyalinization and, subsequently, "fast" and "slow" tooth movers during orthodontic treatment.

Cupping therapy: An analysis of the effects of suction on skin and the possible influence on human health.

PubMed

Lowe, Duane T

2017-11-01

Cupping therapy is a traditional therapy that has been employed worldwide for thousands of years. Despite a lack of quality clinical studies evaluating the efficacy of cupping therapy, its long history and widespread use throughout the world suggests the commonly claimed health benefits should not be completely discounted as without merit. The goal of this paper is to present the research detailing what is known concerning the effects of suction on skin and underlying tissue, and the reaction of the body to that stimulus. Understanding the literature on the physiological effects of this mechanical force may help elaborate an explanation for the advertised local and systemic effects of cupping therapy. Negative pressure causes stretching of the skin and underlying tissue and dilation of the capillaries. This stimulates an increase in tissue blood flow, eventually leading to capillary rupture and ecchymosis. Macrophages phagocytize the erythrocytes in the extravascular space which stimulates the production of Heme Oxygenase-1 (HO-1) to metabolize the heme. Heme catalysis results in the production of carbon monoxide (CO), biliverdin(BV)/bilirubin(BR) and iron. HO-1, BV, BR, and CO has been shown to have antioxidant, anti-inflammatory, antiproliferative, and neuromodulatory effects in animal and human systems. These substances also stimulate a shift of macrophages to the anti-inflammatory M2 phenotype. There is evidence that the effects are both local and systemic. Besides the mechanical effect of cupping increasing the local blood flow and stretching underlying tissue, activation of the HO-1 system could account for many of cupping therapy's claimed local and systemic health benefits. Published by Elsevier Ltd.

Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

2012-03-01

B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

Comparative Salivary Proteome of Hepatitis B- and C-Infected Patients

PubMed Central

Gonçalves, Lorena Da Rós; Campanhon, Isabele Batista; Domingues, Romênia R.; Paes Leme, Adriana F.; Soares da Silva, Márcia Regina

2014-01-01

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection. PMID:25423034

Hypochlorous acid-induced heme oxygenase-1 gene expression promotes human endothelial cell survival

PubMed Central

Wei, Yong; Liu, Xiao-ming; Peyton, Kelly J.; Wang, Hong; Johnson, Fruzsina K.; Johnson, Robert A.

2009-01-01

Hypochlorous acid (HOCl) is a unique oxidant generated by the enzyme myeloperoxidase that contributes to endothelial cell dysfunction and death in atherosclerosis. Since myeloperoxidase localizes with heme oxygenase-1 (HO-1) in and around endothelial cells of atherosclerotic lesions, the present study investigated whether there was an interaction between these two enzymes in vascular endothelium. Treatment of human endothelial cells with the myeloperoxidase product HOCl stimulated a concentration- and time-dependent increase in HO-1 protein that resulted in a significant rise in carbon monoxide (CO) production. The induction of HO-1 protein was preceded by a prominent increase in HO-1 mRNA and total and nuclear factor-erythroid 2-related factor 2 (Nrf2). In addition, HOCl induced a significant rise in HO-1 promoter activity that was blocked by mutating the antioxidant response element (ARE) in the promoter or by overexpressing a dominant-negative mutant of Nrf2. The HOCl-mediated induction of Nrf2 or HO-1 was blocked by the glutathione donor N-acetyl-l-cysteine but was unaffected by ascorbic or uric acid. Finally, treatment of endothelial cells with HOCl stimulated mitochondrial dysfunction, caspase-3 activation, and cell death that was potentiated by the HO inhibitor, tin protoporphyrin-IX, or by the knockdown of HO-1, and reversed by the exogenous administration of biliverdin, bilirubin, or CO. These results demonstrate that HOCl induces HO-1 gene transcription via the activation of the Nrf2/ARE pathway to counteract HOCl-mediated mitochondrial dysfunction and cell death. The ability of HOCl to activate HO-1 gene expression may represent a critical adaptive response to maintain endothelial cell viability at sites of vascular inflammation and atherosclerosis. PMID:19625608

Design of metal cofactors activated by a protein–protein electron transfer system

PubMed Central

Ueno, Takafumi; Yokoi, Norihiko; Unno, Masaki; Matsui, Toshitaka; Tokita, Yuichi; Yamada, Masako; Ikeda-Saito, Masao; Nakajima, Hiroshi; Watanabe, Yoshihito

2006-01-01

Protein-to-protein electron transfer (ET) is a critical process in biological chemistry for which fundamental understanding is expected to provide a wealth of applications in biotechnology. Investigations of protein–protein ET systems in reductive activation of artificial cofactors introduced into proteins remains particularly challenging because of the complexity of interactions between the cofactor and the system contributing to ET. In this work, we construct an artificial protein–protein ET system, using heme oxygenase (HO), which is known to catalyze the conversion of heme to biliverdin. HO uses electrons provided from NADPH/cytochrome P450 reductase (CPR) through protein–protein complex formation during the enzymatic reaction. We report that a FeIII(Schiff-base), in the place of the active-site heme prosthetic group of HO, can be reduced by NADPH/CPR. The crystal structure of the Fe(10-CH2CH2COOH-Schiff-base)·HO composite indicates the presence of a hydrogen bond between the propionic acid carboxyl group and Arg-177 of HO. Furthermore, the ET rate from NADPH/CPR to the composite is 3.5-fold faster than that of Fe(Schiff-base)·HO, although the redox potential of Fe(10-CH2CH2COOH-Schiff-base)·HO (−79 mV vs. NHE) is lower than that of Fe(Schiff-base)·HO (+15 mV vs. NHE), where NHE is normal hydrogen electrode. This work describes a synthetic metal complex activated by means of a protein–protein ET system, which has not previously been reported. Moreover, the result suggests the importance of the hydrogen bond for the ET reaction of HO. Our Fe(Schiff-base)·HO composite model system may provide insights with regard to design of ET biosystems for sensors, catalysts, and electronics devices. PMID:16769893

Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells.

PubMed

Foresti, Roberta; Bains, Sandip K; Pitchumony, Tamil Selvi; de Castro Brás, Lisandra E; Drago, Filippo; Dubois-Randé, Jean-Luc; Bucolo, Claudio; Motterlini, Roberto

2013-10-01

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5-20 μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: a role of combined treatment versus monotherapy

PubMed Central

Ivanov, Vladimir N.; Hei, Tom K.

2015-01-01

Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20–40 μM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5–20 μM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation. PMID:21910007

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

PubMed

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.

Isocyanides inhibit human heme oxygenases at the verdoheme stage.

PubMed

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.

Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

PubMed Central

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

Rapid induction of heme oxygenase 1 mRNA and protein by hyperthermia in rat brain: heme oxygenase 2 is not a heat shock protein.

PubMed Central

Ewing, J F; Maines, M D

1991-01-01

Catalytic activity of heme oxygenase (heme, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.3) isozymes, HO-1 and HO-2, permits production of physiologic isomers of bile pigments. In turn, bile pigments biliverdin and bilirubin are effective antioxidants in biological systems. In the rat brain we have identified only the HO-1 isozyme of heme oxygenase as a heat shock protein and defined hyperthermia as a stimulus that causes an increase in brain HO-1 protein. Exposure of male rats to 42 degrees C for 20 min caused a rapid and marked increase in brain 1.8-kilobase HO-1 mRNA. Specifically, a 33-fold increase in brain HO-1 mRNA was observed within 1 h and sustained for at least 6 h posttreatment. In contrast, the two HO-2 homologous transcripts (1.3 and 1.9 kilobases) did not respond to heat shock; neither the ratio nor the level of the two messages differed from that of the control when measured either at 1, 6, or 24 h after hyperthermia. The induction of a 1.8-kilobase HO-1 mRNA resulted in a pronounced increase in HO-1 protein 6 h after hyperthermia, as detected by both Western immunoblot and RIA. Immunocytochemistry of rat brain showed discrete localization of HO-1-like protein only in neurons of select brain regions. Six hours after heat shock, an intense increase in HO-1-like protein was observed in both Purkinje cells of the cerebellum and epithelial cells lining the cerebral aqueduct of the brain. We suggest that the increase in HO-1 protein, hence increased capacity to form bile pigments, represents a neuronal defense mechanism against heat shock stress. Images PMID:2052613

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

PubMed

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.

Heme oxygenase-1 posttranslational modifications in the brain of subjects with Alzheimer disease and mild cognitive impairment.

PubMed

Barone, Eugenio; Di Domenico, Fabio; Sultana, Rukhsana; Coccia, Raffaella; Mancuso, Cesare; Perluigi, Marzia; Butterfield, D Allan

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Oxidative and nitrosative stress plays a principal role in the pathogenesis of AD. The induction of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system in the brain represents one of the earliest mechanisms activated by cells to counteract the noxious effects of increased reactive oxygen species and reactive nitrogen species. Although initially proposed as a neuroprotective system in AD brain, the HO-1/BVR-A pathophysiological features are under debate. We previously reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative posttranslational modifications in the brain of subjects with AD and those with mild cognitive impairment (MCI). Furthermore, other groups proposed the observed increase in HO-1 in AD brain as a possible neurotoxic mechanism. Here we provide new insights about HO-1 in the brain of subjects with AD and MCI, the latter condition being the transitional phase between normal aging and early AD. HO-1 protein levels were significantly increased in the hippocampus of AD subjects, whereas HO-2 protein levels were significantly decreased in both AD and MCI hippocampi. In addition, significant increases in Ser-residue phosphorylation together with increased oxidative posttranslational modifications were found in the hippocampus of AD subjects. Interestingly, despite the lack of oxidative stress-induced AD neuropathology in cerebellum, HO-1 demonstrated increased Ser-residue phosphorylation and oxidative posttranslational modifications in this brain area, suggesting HO-1 as a target of oxidative damage even in the cerebellum. The significance of these findings is profound and opens new avenues into the comprehension of the role of HO-1 in the pathogenesis of AD. Copyright © 2012 Elsevier Inc. All rights reserved.

Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

PubMed Central

Peyton, Kelly J.; Shebib, Ahmad R.; Azam, Mohammad A.; Liu, Xiao-ming; Tulis, David A.; Durante, William

2012-01-01

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G2M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G0/G1 phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease. PMID:22470341

Orthodontic Forces Induce the Cytoprotective Enzyme Heme Oxygenase-1 in Rats

PubMed Central

Suttorp, Christiaan M.; Xie, Rui; Lundvig, Ditte M. S.; Kuijpers-Jagtman, Anne Marie; Uijttenboogaart, Jasper Tom; Van Rheden, René; Maltha, Jaap C.; Wagener, Frank A. D. T. G.

2016-01-01

Orthodontic forces disturb the microenvironment of the periodontal ligament (PDL), and induce craniofacial bone remodeling which is necessary for tooth movement. Unfortunately, orthodontic tooth movement is often hampered by ischemic injury and cell death within the PDL (hyalinization) and root resorption. Large inter-individual differences in hyalinization and root resorption have been observed, and may be explained by differential protection against hyalinization. Heme oxygenase-1 (HO-1) forms an important protective mechanism by breaking down heme into the strong anti-oxidants biliverdin/bilirubin and the signaling molecule carbon monoxide. These versatile HO-1 products protect against ischemic and inflammatory injury. We postulate that orthodontic forces induce HO-1 expression in the PDL during experimental tooth movement. Twenty-five 6-week-old male Wistar rats were used in this study. The upper three molars at one side were moved mesially using a Nickel-Titanium coil spring, providing a continuous orthodontic force of 10 cN. The contralateral side served as control. After 6, 12, 72, 96, and 120 h groups of rats were killed. On parasagittal sections immunohistochemical staining was performed for analysis of HO-1 expression and quantification of osteoclasts. Orthodontic force induced a significant time-dependent HO-1 expression in mononuclear cells within the PDL at both the apposition- and resorption side. Shortly after placement of the orthodontic appliance HO-1 expression was highly induced in PDL cells but dropped to control levels within 72 h. Some osteoclasts were also HO-1 positive but this induction was shown to be independent of time- and mechanical stress. It is tempting to speculate that differential induction of tissue protecting- and osteoclast activating genes in the PDL determine the level of bone resorption and hyalinization and, subsequently, “fast” and “slow” tooth movers during orthodontic treatment. PMID:27486402

Blue and green egg-color intensity is associated with parental effort and mating system in passerines: support for the sexual selection hypothesis.

PubMed

Soler, Juan J; Moreno, Juan; Avilés, Jesús M; Møller, Anders P

2005-03-01

Among several adaptive explanations proposed to account for variation in avian egg color, that related to sexual selection is of particular interest because of its possible generality. Briefly, it proposes that because biliverdin (the pigment responsible for blue-green eggshell coloration) is an antioxidant, deposition in the eggshell by laying females may signal the capacity of females to control free radicals, despite the handicap of removing this antioxidant from their body. If males adjust parental effort in response to the intensity of the blue coloration of eggs, thereby investing more in the offspring of high-quality mates, blue eggs may represent a postmating sexually selected signal in females. Here, by image and spectrophotometric analyses of the eggs of European passerines, we tested two different predictions of the hypothesis. First, variables related to intraspecific variation in parental effort (i.e., the duration of the nestling period controlled for body mass) should be positively related to the intensity of blue-green color of the eggshell across species. Second, there should be a positive relationship between intensity of blue-green color of eggs and degree of polygyny. These predictions were supported: intensity of blue-green coloration (i.e., chroma) was significantly related to the duration of the nestling period and to degree of polygyny after controlling for possible confounding variables (i.e., body mass, incubation period, and nest type) and similarity due to common descent. Nest type (hole or nonhole) also explained a significant proportion of variation in egg chroma, perhaps reflecting different selection pressures (i.e., light conditions, risk of parasitism) affecting species with the two types of nests.

Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.

PubMed

Campbell, James H; Heikkila, John J

2018-04-23

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl 2 . In the present study, CdCl 2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl 2 -induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl 2 -induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl 2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures. Copyright © 2018 Elsevier Inc. All rights reserved.

NMR structure and dynamics of the engineered fluorescein-binding lipocalin FluA reveal rigidification of beta-barrel and variable loops upon enthalpy-driven ligand binding.

PubMed

Mills, Jeffrey L; Liu, Gaohua; Skerra, Arne; Szyperski, Thomas

2009-08-11

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel beta-strands forming a beta-barrel with an alpha-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP.biliverdin IX(gamma) and FluA.fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the beta-barrel as well as adjoining beta-strand segments. An "induced fit" became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse (15)N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA.fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called "anticalins".

Inflammatory Microenvironment Persists After Bone Healing in Intra-articular Ankle Fractures.

PubMed

Adams, Samuel B; Leimer, Elizabeth M; Setton, Lori A; Bell, Richard D; Easley, Mark E; Huebner, Janet L; Stabler, Thomas V; Kraus, Virginia B; Olson, Steven A; Nettles, Dana L

2017-05-01

Post-traumatic osteoarthritis (PTOA) is responsible for the majority of cases of ankle arthritis. While acute and end-stage intra-articular inflammation has previously been described, the state of the joint between fracture healing and end-stage PTOA remains undefined. This study characterized synovial fluid (SF) composition of ankles after bone healing of an intra-articular fracture to identify factors that may contribute to the development of PTOA. Of an original 21 patients whose SF was characterized acutely following intra-articular ankle fractures, 7 returned for planned hardware (syndesmotic screw) removal after bone healing (approximately 6 months) and consented to a second bilateral SF collection. SF concentrations of 15 cytokines and matrix metalloproteinases (MMPs) and 2 markers each of cartilage catabolism (CTXII and glycosaminoglycan) and hemarthrosis (biliverdin and bilirubin) were compared for previously fractured and contralateral, uninjured ankles from the same patient. Analysis was also performed to determine the effect of the number of fracture lines and involvement of soft tissue on SF composition. Interleukin (IL)-6, IL-8, MMP-1, MMP-2, and MMP-3 were significantly elevated in the SF from healed ankles compared to matched contralateral uninjured ankles at approximately 6 months after fracture. There were no differences in markers of cartilage catabolism or hemarthrosis. Only IL-1α was affected by the number of fracture lines while differences were not detected for other analytes or with respect to the involvment of soft tissue. Sustained intra-articular inflammation, even after complete bone healing, was suggested by elevations of pro-inflammatory cytokines (IL-6 and IL-8). In addition, elevated concentrations of MMPs were also noted and were consistent with a persistent inflammatory environment. This study suggests new evidence of persistent intra-articular inflammation after intra-articular ankle fracture healing and suggests potential mediators for PTOA development. This work may be relevant to the clinical diagnosis and treatment of post-traumatic osteoarthritis.

Compound C Stimulates Heme Oxygenase-1 Gene Expression via the Nrf2-ARE Pathway to Preserve Human Endothelial Cell Survival

PubMed Central

Liu, Xiao-ming; Peyton, Kelly J.; Shebib, Ahmad R.; Wang, Hong; Durante, William

2011-01-01

We recently identified adenosine monophosphate-activated protein kinase (AMPK) as a novel inducer of heme oxygenase-1 (HO-1) and surprisingly found that compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine), a cell-permeable inhibitor of AMPK, could also elevate HO-1 suggesting other AMPK-independent actions for this agent. In this study, we investigated the biochemical mechanism by which compound C stimulates HO-1 expression in human endothelial cells (ECs) and determined the biological significance of the induction of HO-1 by compound C in these cells. Compound C stimulated a concentration- and time-dependent increase in HO-1 expression and an increase in HO-1 promoter activity that was abrogated by mutating the antioxidant responsive elements (AREs) in the HO-1 promoter or by overexpressing a dominant negative mutant of NF-E2-related factor-2 (Nrf2). Compound C also stimulated Nrf2 expression and this was associated with an increase in the production of reactive oxygen species and with a decline in intracellular glutathione levels. Interestingly, the glutathione donor N-acetyl-L-cysteine or the NADPH oxidase inhibitor apocynin blocked the induction of HO-1 by compound C. Finally, compound C stimulated EC death and this was potentiated by silencing HO-1 expression and reversed by the administration of CO, biliverdin, or bilirubin. In conclusion, this study demonstrates that compound C stimulates HO-1 gene expression in human vascular endothelium via the activation of the Nrf2/ARE signaling pathway to counteract compound C-mediated cell death. The ability of compound C to induce HO-1 expression may contribute to the pleiotropic actions of this agent and suggest caution when using compound C to probe for AMPK functions. PMID:21635873

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages

PubMed Central

Sawle, Philip; Foresti, Roberta; Mann, Brian E; Johnson, Tony R; Green, Colin J; Motterlini, Roberto

2005-01-01

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10–100 μM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-α, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation. PMID:15880142

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, Amit V., E-mail: amit@pandeylab.org; Flueck, Christa E.; Mullis, Primus E.

2010-09-24

Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare formmore » of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.« less

Renal Heme Oxygenase-1 Induction with Hemin Augments Renal Hemodynamics, Renal Autoregulation, and Excretory Function

PubMed Central

Botros, Fady T.; Dobrowolski, Leszek; Navar, L. Gabriel

2012-01-01

Heme oxygenases (HO-1; HO-2) catalyze conversion of heme to free iron, carbon monoxide, and biliverdin/bilirubin. To determine the effects of renal HO-1 induction on blood pressure and renal function, normal control rats (n = 7) and hemin-treated rats (n = 6) were studied. Renal clearance studies were performed on anesthetized rats to assess renal function; renal blood flow (RBF) was measured using a transonic flow probe placed around the left renal artery. Hemin treatment significantly induced renal HO-1. Mean arterial pressure and heart rate were not different (115 ± 5 mmHg versus 112 ± 4 mmHg and 331 ± 16 versus 346 ± 10 bpm). However, RBF was significantly higher (9.1 ± 0.8 versus 7.0 ± 0.5 mL/min/g, P < 0.05), and renal vascular resistance was significantly lower (13.0 ± 0.9 versus 16.6 ± 1.4 [mmHg/(mL/min/g)], P < 0.05). Likewise, glomerular filtration rate was significantly elevated (1.4 ± 0.2 versus 1.0 ± 0.1 mL/min/g, P < 0.05), and urine flow and sodium excretion were also higher (18.9 ± 3.9 versus 8.2 ± 1.0 μL/min/g, P < 0.05 and 1.9 ± 0.6 versus 0.2 ± 0.1 μmol/min/g, P < 0.05, resp.). The plateau of the autoregulation relationship was elevated, and renal vascular responses to acute angiotensin II infusion were attenuated in hemin-treated rats reflecting the vasodilatory effect of HO-1 induction. We conclude that renal HO-1 induction augments renal function which may contribute to the antihypertensive effects of HO-1 induction observed in hypertension models. PMID:22518281

Ultraviolet-A1 irradiation therapy for systemic lupus erythematosus.

PubMed

McGrath, H

2017-10-01

Systemic lupus erythematosus (lupus, SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, which bind to antigens and are deposited within tissues to fix complement, resulting in widespread systemic inflammation. The studies presented herein are consistent with hyperpolarized, adenosine triphosphate (ATP)-deficient mitochondria being central to the disease process. These hyperpolarized mitochondria resist the depolarization required for activation-induced apoptosis. The mitochondrial ATP deficits add to this resistance to apoptosis and also reduce the macrophage energy that is needed to clear apoptotic bodies. In both cases, necrosis, the alternative pathway of cell death, results. Intracellular constituents spill into the blood and tissues, eliciting inflammatory responses directed at their removal. What results is "autoimmunity." Ultraviolet (UV)-A1 photons have the capacity to remediate this aberrancy. Exogenous exposure to low-dose, full-body, UV-A1 radiation generates singlet oxygen. Singlet oxygen has two major palliative actions in patients with lupus and the UV-A1 photons themselves have several more. Singlet oxygen depolarizes the hyperpolarized mitochondrion, triggering non-ATP-dependent apoptosis that deters necrosis. Next, singlet oxygen activates the gene encoding heme oxygenase (HO-1), a major governor of systemic homeostasis. HO-1 catalyzes the degradation of the oxidant heme into biliverdin (converted to bilirubin), Fe, and carbon monoxide (CO), the first three of these exerting powerful antioxidant effects, and in conjunction with a fourth, CO, protecting against injury to the coronary arteries, the central nervous system, and the lungs. The UV-A1 photons themselves directly attenuate disease in lupus by reducing B cell activity, preventing the suppression of cell-mediated immunity, slowing an epigenetic progression toward SLE, and ameliorating discoid and subacute cutaneous lupus. Finally, a combination of these mechanisms reduces levels of anticardiolipin antibodies and protects during lupus pregnancy. Capping all of this is that UV-A1 irradiation is an essentially innocuous, highly manageable, and comfortable therapeutic agency.

Ultraviolet-A1 irradiation therapy for systemic lupus erythematosus

PubMed Central

2017-01-01

Systemic lupus erythematosus (lupus, SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, which bind to antigens and are deposited within tissues to fix complement, resulting in widespread systemic inflammation. The studies presented herein are consistent with hyperpolarized, adenosine triphosphate (ATP)-deficient mitochondria being central to the disease process. These hyperpolarized mitochondria resist the depolarization required for activation-induced apoptosis. The mitochondrial ATP deficits add to this resistance to apoptosis and also reduce the macrophage energy that is needed to clear apoptotic bodies. In both cases, necrosis, the alternative pathway of cell death, results. Intracellular constituents spill into the blood and tissues, eliciting inflammatory responses directed at their removal. What results is “autoimmunity.” Ultraviolet (UV)-A1 photons have the capacity to remediate this aberrancy. Exogenous exposure to low-dose, full-body, UV-A1 radiation generates singlet oxygen. Singlet oxygen has two major palliative actions in patients with lupus and the UV-A1 photons themselves have several more. Singlet oxygen depolarizes the hyperpolarized mitochondrion, triggering non-ATP-dependent apoptosis that deters necrosis. Next, singlet oxygen activates the gene encoding heme oxygenase (HO-1), a major governor of systemic homeostasis. HO-1 catalyzes the degradation of the oxidant heme into biliverdin (converted to bilirubin), Fe, and carbon monoxide (CO), the first three of these exerting powerful antioxidant effects, and in conjunction with a fourth, CO, protecting against injury to the coronary arteries, the central nervous system, and the lungs. The UV-A1 photons themselves directly attenuate disease in lupus by reducing B cell activity, preventing the suppression of cell-mediated immunity, slowing an epigenetic progression toward SLE, and ameliorating discoid and subacute cutaneous lupus. Finally, a combination of these mechanisms reduces levels of anticardiolipin antibodies and protects during lupus pregnancy. Capping all of this is that UV-A1 irradiation is an essentially innocuous, highly manageable, and comfortable therapeutic agency. PMID:28480786

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Soon Won; Sohn, Eun Jeong; Kim, Dae Won

Highlights: {yields} Recombinant PEP-1 heme oxygenase-1 expression vector was constructed and overexpressed. {yields} We investigated transduction efficiency of PEP-1-HO-1 protein in Raw 264.7 cells. {yields} PEP-1-HO-1 was efficiently transduced into Raw 264.7 cells in a dose and time dependent manner. {yields} PEP-1-HO-1 exerted anti-inflammatory activity in Raw 264.7 cells and in a mice edema model. {yields} 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{sup 2+}), is up-regulated by several cellular stress and cell injuries, including inflammation,more » 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.« less

Interaction of nitric oxide with human heme oxygenase-1.

PubMed

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.

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

PubMed Central

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

Interaction of the carbon monoxide-releasing molecule Ru(CO)3Cl(glycinate) (CORM-3) with Salmonella enterica serovar Typhimurium: in situ measurements of carbon monoxide binding by integrating cavity dual-beam spectrophotometry.

PubMed

Rana, Namrata; McLean, Samantha; Mann, Brian E; Poole, Robert K

2014-12-01

Carbon monoxide (CO) is a toxic gas that binds to haems, but also plays critical signalling and cytoprotective roles in mammalian systems; despite problems associated with systemic delivery by inhalation of the gas, it may be employed therapeutically. CO delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas; CO-RMs are also attractive candidates as novel antimicrobial agents. Salmonella enterica serovar Typhimurium is an enteropathogen causing gastroenteritis in humans. Recent studies have implicated haem oxygenase-1 (HO-1), the protein that catalyses the degradation of haem into biliverdin, free iron and CO, in the host immune response to Salmonella infection. In several studies, CO administration via CO-RMs elicited many of the protective roles of HO-1 induction and so we investigated the effects of a well-characterized water-soluble CO-RM, Ru(CO)3Cl(glycinate) (CORM-3), on Salmonella. CORM-3 exhibits toxic effects at concentrations significantly lower than those reported to cause toxicity to RAW 264.7 macrophages. We demonstrated here, through oxyhaemoglobin assays, that CORM-3 did not release CO spontaneously in phosphate buffer, buffered minimal medium or very rich medium. CORM-3 was, however, accumulated to high levels intracellularly (as shown by inductively coupled plasma MS) and released CO inside cells. Using growing Salmonella cultures without prior concentration, we showed for the first time that sensitive dual-beam integrating cavity absorption spectrophotometry can detect directly the CO released from CORM-3 binding in real-time to haems of the bacterial electron transport chain. The toxic effects of CO-RMs suggested potential applications as adjuvants to antibiotics in antimicrobial therapy. © 2014 The Authors.

Structure of the Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme and enzymatic inactivation by mutation of the heme coordinating residue His-193

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suits,M.; Jaffer, N.; Jia, Z.

2006-01-01

Heme oxygenases catalyze the oxidation of heme to biliverdin, CO, and free iron. For pathogenic microorganisms, heme uptake and degradation are critical mechanisms for iron acquisition that enable multiplication and survival within hosts they invade. Here we report the first crystal structure of the pathogenic Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme at 1.45 {angstrom} resolution. When compared with other heme oxygenases, ChuS has a unique fold, including structural repeats and a {beta}-sheet core. Not surprisingly, the mode of heme coordination by ChuS is also distinct, whereby heme is largely stabilized by residues from the C-terminal domain,more » assisted by a distant arginine from the N-terminal domain. Upon heme binding, there is no large conformational change beyond the fine tuning of a key histidine (His-193) residue. Most intriguingly, in contrast to other heme oxygenases, the propionic side chains of heme are orientated toward the protein core, exposing the {alpha}-meso carbon position where O{sub 2} is added during heme degradation. This unique orientation may facilitate presentation to an electron donor, explaining the significantly reduced concentration of ascorbic acid needed for the reaction. Based on the ChuS-heme structure, we converted the histidine residue responsible for axial coordination of the heme group to an asparagine residue (H193N), as well as converting a second histidine to an alanine residue (H73A) for comparison purposes. We employed spectral analysis and CO measurement by gas chromatography to analyze catalysis by ChuS, H193N, and H73A, demonstrating that His-193 is the key residue for the heme-degrading activity of ChuS.« less

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

PubMed

Mishina, Kei; Shinkai, Masaharu; Shimokawaji, Tadasuke; Nagashima, Akimichi; Hashimoto, Yusuke; Inoue, Yoriko; Inayama, Yoshiaki; Rubin, Bruce K; Ishigatsubo, Yoshiaki; Kaneko, Takeshi

2015-12-01

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 μM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme

DOE PAGES

Bagai, Ireena; Sarangi, Ritimukta; Fleischhacker, Angela S.; ...

2015-05-05

Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O₂₋ and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs).more » While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2 O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2 R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme.« less

Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bagai, Ireena; Sarangi, Ritimukta; Fleischhacker, Angela S.

Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O₂₋ and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs).more » While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2 O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2 R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme.« less

EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

PubMed Central

Huber, Warren J.; Backes, Wayne L.

2009-01-01

Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

PubMed

Huber, Warren J; Backes, Wayne L

2007-10-30

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

Ferulic Acid Regulates the Nrf2/Heme Oxygenase-1 System and Counteracts Trimethyltin-Induced Neuronal Damage in the Human Neuroblastoma Cell Line SH-SY5Y

PubMed Central

Catino, Stefania; Paciello, Fabiola; Miceli, Fiorella; Rolesi, Rolando; Troiani, Diana; Calabrese, Vittorio; Santangelo, Rosaria; Mancuso, Cesare

2016-01-01

Over the past years, several lines of evidence have pointed out the efficacy of ferulic acid (FA) in counteracting oxidative stress elicited by β-amyloid or free radical initiators, based on the ability of this natural antioxidant to up-regulate the heme oxygenase-1 (HO-1) and biliverdin reductase (BVR) system. However, scarce results can be found in literature regarding the cytoprotective effects of FA in case of damage caused by neurotoxicants. The aim of this work is to investigate the mechanisms through which FA exerts neuroprotection in SH-SY5Y neuroblastoma cells exposed to the neurotoxin trimethyltin (TMT). FA (1–10 μM for 6 h) dose-dependently increased both basal and TMT (10 μM for 24 h)-induced HO-1 expression in SH-SY5Y cells by fostering the nuclear translocation of the transcriptional activator Nrf2. In particular, the co-treatment of FA (10 μM) with TMT was also responsible for the nuclear translocation of HO-1 in an attempt to further increase cell stress response in SH-SY5Y cells. In addition to HO-1, FA (1–10 μM for 6 h) dose-dependently increased the basal expression of BVR. The antioxidant and neuroprotective features of FA, through the increase of HO activity, were supported by the evidence that FA inhibited TMT (10 μM)-induced lipid peroxidation (evaluated by detecting 4-hydroxy-nonenal) and DNA fragmentation in SH-SY5Y cells and that this antioxidant effect was reversed by the HO inhibitor Zinc-protoporphyrin-IX (5 μM). Among the by-products of the HO/BVR system, carbon monoxide (CORM-2, 50 nM) and bilirubin (BR, 50 nM) significantly inhibited TMT-induced superoxide anion formation in SH-SY5Y cells. All together, these results corroborate the neuroprotective effect of FA through the up-regulation of the HO-1/BVR system, via carbon monoxide and BR formation, and provide the first evidence on the role of HO-1/Nrf2 axis in FA-related enhancement of cell stress response in human neurons. PMID:26779023

Solution NMR study of environmental effects on substrate seating in human heme oxygenase: influence of polypeptide truncation, substrate modification and axial ligand.

PubMed

Zhu, Wenfeng; Li, Yiming; Wang, Jinling; Ortiz de Montellano, Paul R; La Mar, Gerd N

2006-01-01

Solution proton NMR has been used here to show that, as either the high-spin ferric, protohemin (PH) substrate complex at neutral pH, or the low-spin ferric, cyanide-inhibited PH substrate complex, the active site electronic and molecular structure of the 233- and 265-residue recombinant constructs of human heme oxygenase-1, hHO, are essentially indistinguishable. It is shown, moreover, that the equilibrium PH orientational isomerism about the alpha,gamma-meso axis is 1:1 in the water-ligated, resting-state complex, but changes to a 4:1 equilibrium ratio as the cyanide-inhibited complex, with the minor species in solution corresponding to the only one found in crystals. The introduction of significant PH orientational preference in the cyanide over the aquo complex is rationalized by the crystallographic observation for the same H2O and CN ligated complexes of rat heme oxygenase (rHO), where the steric tilt of the Fe-CN unit resulted in a approximately 1 A transition of PH into the hydrophobic interior, and stronger interaction of the vinyls with the HO matrix [M. Sugishima, H. Sakamoto, M. Noguchi, K. Fukugama, Biochemistry 42 (2003) 9898-9905]. 1H NMR spectra of the cyanide-inhibited PH complex are the most used, and most useful, for determining the distribution of orientational isomerism for PH in complexes of HO. Hence, it is imperative that the time-course of the spectra after sample preparation be considered in order to reach conclusions that relate isomeric seating of the heme with variable isomeric biliverdin products. The natural orientational isomerism of PH leads to spectral congestion that has prompted the use of a synthetic, twofold symmetric substrate, 2,4-dimethyldeuterohemin, DMDH. While the hyperfine shift pattern for non-ligated residues are very similar and are consistent with largely conserved molecular structure with the alternate substrates, the steric tilt of the Fe-CN vector towards the protein interior, as determined by the orientation of the major magnetic axes, is 2 degrees smaller for DMDH than PH, and is rationalized by the substrate translating even further into the hydrophobic interior in the cyanide complex when the bulky vinyl groups are replaced by methyl groups.

Impact of higher-order heme degradation products on hepatic function and hemodynamics.

PubMed

Seidel, Raphael A; Claudel, Thierry; Schleser, Franziska A; Ojha, Navin K; Westerhausen, Matthias; Nietzsche, Sandor; Sponholz, Christoph; Cuperus, Frans; Coldewey, Sina M; Heinemann, Stefan H; Pohnert, Georg; Trauner, Michael; Bauer, Michael

2017-08-01

Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Detection and Localization of Markers of Oxidative Stress by In Situ Methods: Application in the Study of Alzheimer Disease

PubMed Central

Moreira, Paula I.; Sayre, Lawrence M.; Zhu, Xiongwei; Nunomura, Akihiko; Smith, Mark A.; Perry, George

2018-01-01

Oxidative stress is a key factor involved in the development and progression of Alzheimer disease (AD), and it is well documented that free radical oxidative damage, particularly of neuronal lipids, proteins, nucleic acids, and sugars, is extensive in brains of AD patients. The complex chemistry of peroxynitrite has been the subject of intense study and is now evident that there are two principal pathways for protein modification: the first one involves homolytic hydroxyl radical-like chemistry that results in protein-based carbonyls and the second involves electrophilic nitration of vulnerable side chains, in particular the electron-rich aromatic rings of Tyr and Trp. In the presence of buffering bicarbonate, peroxynitrite forms a CO2 adduct, which augments its reactivity. Formation of 3-nitrotyrosine by this route has become the classical protein marker specifically for the presence of peroxynitrite. Protein-based carbonyls can be detected by two methods: (i) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection of the protein-bound hydrazones using an enzyme-linked anti-2,4-dinitrophenyl antibody and (ii) derivatization with biotin-hydrazide and detection of the protein-bound acyl hydrazone with enzyme-linked avidin or streptavidin. Glycation of proteins by reducing sugars (Maillard reaction) results in a profile of time-dependent adduct evolution rendering susceptibility to oxidative elaboration. In addition, oxidative stress can result in oxidized sugar derivatives which can subsequently modify protein through a process known as glycoxidation. Of more general importance, oxidative stress results in lipid peroxidation and the production of a range of electrophilic and mostly bifunctional aldehydes that modify numerous proteins. The more important protein modifications are referred to as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Protein modification can result in both non-cross-link and cross-link AGEs and ALEs, the latter arising from the potential bifunctional reactivity, such as that of the lipid-derived modifiers 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA). Oxidative damage to nucleic acids results in base modification, substitutions, and deletions. Among the most common modifications, 8-hydroxyguanosine (8OHG) is considered a signature of oxidative damage to nucleic acid. Cells are not passive to increased oxygen radical production but rather upregulate protective responses. In neurodegenerative diseases, heme oxygenase-1 (HO-1) induction is coincident with the formation of neurofibrillary tangles. This enzyme thatconverts heme, a prooxidant, to biliverdin/bilirubin (antioxidants) and free iron has been considered an antioxidant enzyme. But seen in the context of arresting apoptosis, HO-1 and tau may play a role in maintaining the neurons free from the apoptotic signal (cytochrome c), since tau has strong iron-binding sites. Given the importance of iron as a catalyst for the generation of reactive oxygen species, changes in proteins associated with iron homeostasis can be used as an index of cellular responses. One such class of proteins is the iron regulatory proteins (IRPs) that respond to cellular iron concentrations by regulating the translation of proteins involved in iron uptake, storage, and utilization. Therefore, IRPs are considered to be the central control components of cellular iron concentration. PMID:20013193

Detection and localization of markers of oxidative stress by in situ methods: application in the study of Alzheimer disease.

PubMed

Moreira, Paula I; Sayre, Lawrence M; Zhu, Xiongwei; Nunomura, Akihiko; Smith, Mark A; Perry, George

2010-01-01

Oxidative stress is a key factor involved in the development and progression of Alzheimer disease (AD), and it is well documented that free radical oxidative damage, particularly of neuronal lipids, proteins, nucleic acids, and sugars, is extensive in brains of AD patients. The complex chemistry of peroxynitrite has been the subject of intense study and is now evident that there are two principal pathways for protein modification: the first one involves homolytic hydroxyl radical-like chemistry that results in protein-based carbonyls and the second involves electrophilic nitration of vulnerable side chains, in particular the electron-rich aromatic rings of Tyr and Trp. In the presence of buffering bicarbonate, peroxynitrite forms a CO(2) adduct, which augments its reactivity. Formation of 3-nitrotyrosine by this route has become the classical protein marker specifically for the presence of peroxynitrite. Protein-based carbonyls can be detected by two methods: (i) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection of the protein-bound hydrazones using an enzyme-linked anti-2,4-dinitrophenyl antibody and (ii) derivatization with biotin-hydrazide and detection of the protein-bound acyl hydrazone with enzyme-linked avidin or streptavidin. Glycation of proteins by reducing sugars (Maillard reaction) results in a profile of time-dependent adduct evolution rendering susceptibility to oxidative elaboration. In addition, oxidative stress can result in oxidized sugar derivatives which can subsequently modify protein through a process known as glycoxidation. Of more general importance, oxidative stress results in lipid peroxidation and the production of a range of electrophilic and mostly bifunctional aldehydes that modify numerous proteins. The more important protein modifications are referred to as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Protein modification can result in both non-cross-link and cross-link AGEs and ALEs, the latter arising from the potential bifunctional reactivity, such as that of the lipid-derived modifiers 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA). Oxidative damage to nucleic acids results in base modification, substitutions, and deletions. Among the most common modifications, 8-hydroxyguanosine (8OHG) is considered a signature of oxidative damage to nucleic acid.Cells are not passive to increased oxygen radical production but rather upregulate protective responses. In neurodegenerative diseases, heme oxygenase-1 (HO-1) induction is coincident with the formation of neurofibrillary tangles. This enzyme that converts heme, a prooxidant, to biliverdin/bilirubin (antioxidants) and free iron has been considered an antioxidant enzyme. But seen in the context of arresting apoptosis, HO-1 and tau may play a role in maintaining the neurons free from the apoptotic signal (cytochrome c), since tau has strong iron-binding sites. Given the importance of iron as a catalyst for the generation of reactive oxygen species, changes in proteins associated with iron homeostasis can be used as an index of cellular responses. One such class of proteins is the iron regulatory proteins (IRPs) that respond to cellular iron concentrations by regulating the translation of proteins involved in iron uptake, storage, and utilization. Therefore, IRPs are considered to be the central control components of cellular iron concentration.

Coordination Chemistry of Linear Oligopyrrolic Fragments Inspired by Heme Metabolites

NASA Astrophysics Data System (ADS)

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 at 635 nm. This reaction also explains the antioxidant properties of the linear tripyrrin-1,14-dione ligand, which acts as a scavenger of O2•-. In Chapter 4, the zinc binding properties of the tripyrrin-1,14-dione ligand are described. The tripyrrolic ligand coordinates as a dianionic ligand with the divalent Zn(II) ion in both organic and aqueous buffered conditions. The complex formed is highly fluorescent with a long wavelength emission band at 648 nm. The X-Ray crystallography analysis indicates the existence of dinuclear complex [Zn(TD1•)(H2O)]2, featuring a distorted square planar geometry around the Zn(II) center. In Chapter 5, the coordination chemistry of the dipyrrin-1,9-dione fragment of propentdyopent ligand is shown with a series of transition metals like (e.g., Co(II), Ni(II), Cu(II) and Zn(II)), which form homoleptic tetrahedral complexes. The spectroscopic and electrochemical characterization confirms that the complexes shows ligand-based redox chemistry and acts as reservoirs for unpaired electrons. Chapter 6 describes the formation of the fluorescent BODIPY complex of propentdyopent ligand. The dipyrrin-1,9-dione scaffold of heme metabolite propendyopent undergoes a one-pot reaction with borontrifluoride etherate in toluene to form a green fluorescent [(pdp)BF2] complex. Spectroscopic studies reveal that the meso-unsubstituted [(pdp)BF2] complex is stable in tetrahydrofuran and has a quantum yield of 0.13. Electrochemical studies confirm that the complex undergoes ligand-based reduction and acts as a host for an unpaired electron.