Introduction to Serial Review on Heme oxygenase in human disease
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Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling. Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling. Heme is an essential but potentially cytotoxic metallocofactor and signaling molecule (1Chiabrando D. Mercurio S. Tolosano E. Heme and erythropoieis: More than a structural role.Haematologica. 2014; 99: 973-983Google Scholar, 2Chiabrando D. Vinchi F. Fiorito V. Mercurio S. Tolosano E. 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From synthesis to utilization: The ins and outs of mitochondrial heme.Cells. 2020; 9: 579Google Scholar, 15Severance S. Hamza I. Trafficking of heme and porphyrins in metazoa.Chem. Rev. 2009; 109: 4596-4616Google Scholar). Although cell surface heme importers (16Duffy S.P. Shing J. Saraon P. Berger L.C. Eiden M.V. Wilde A. Tailor C.S. The Fowler syndrome-associated protein FLVCR2 is an importer of heme.Mol. Cell. Biol. 2010; 30: 5318-5324Google Scholar) and exporters (17Quigley J.G. Yang Z. Worthington M.T. Phillips J.D. Sabo K.M. Sabath D.E. Berg C.L. Sassa S. Wood B.L. Abkowitz J.L. Identification of a human heme exporter that is essential for erythropoiesis.Cell. 2004; 118: 757-766Google Scholar, 18Quigley J.G. Gazda H. Yang Z. Ball S. Sieff C.A. Abkowitz J.L. Investigation of a putative role for FLVCR, a cytoplasmic heme exporter, in Diamond-Blackfan anemia.Blood Cells Mol. Dis. 2005; 35: 189-192Google Scholar) have been identified, their molecular mechanisms remain poorly characterized and outside of developing red blood cells in the case of heme exporters, the physiological context in which they function is unclear and controversial (19Ponka P. Sheftel A.D. English A.M. Scott Bohle D. Garcia-Santos D. Do mammalian cells really need to export and import heme?.Trends Biochem. Sci. 2017; 42: 395-406Google Scholar). The bioavailability of heme, which is comparatively less well understood, is governed by a poorly characterized network of heme buffering factors, intracellular transporters, and chaperones that ensure heme is made available for heme-dependent processes located throughout the cell. When the cells are confronted with excess heme, heme synthesis is downregulated (11Swenson S.A. Moore C.M. Marcero J.R. Medlock A.E. Reddi A.R. Khalimonchuk O. From synthesis to utilization: The ins and outs of mitochondrial heme.Cells. 2020; 9: 579Google Scholar, 20Yamamoto M. Hayashi N. Kikuchi G. Evidence for the transcriptional inhibition by heme of the synthesis of delta-aminolevulinate synthase in rat liver.Biochem. Biophys. Res. Commun. 1982; 105: 985-990Google Scholar, 21Yamamoto M. Hayashi N. Kikuchi G. Translational inhibition by heme of the synthesis of hepatic delta-aminolevulinate synthase in a cell-free system.Biochem. Biophys. Res. Commun. 1983; 115: 225-231Google Scholar), and heme can be detoxified by storage into lysosome-related organelles (22Chen A.J. Yuan X. Li J. Dong P. Hamza I. Cheng J.-X. Label-free imaging of heme dynamics in living organisms by transient absorption microscopy.Anal. Chem. 2018; 90: 3395-3401Google Scholar, 23Pek R.H. Yuan X. Rietzschel N. Zhang J. Jackson L. Nishibori E. Ribeiro A. Simmons W. Jagadeesh J. Sugimoto H. Alam M.Z. Garrett L. Haldar M. Ralle M. Phillips J.D. et al.Hemozoin produced by mammals confers heme tolerance.Elife. 2019; 8e49503Google Scholar), export (24Keel S.B. Doty R.T. Yang Z. Quigley J.G. Chen J. Knoblaugh S. Kingsley P.D. De Domenico I. Vaughn M.B. Kaplan J. Palis J. Abkowitz J.L. A heme export protein is required for red blood cell differentiation and iron homeostasis.Science. 2008; 319: 825-828Google Scholar, 25Doty R.T. Phelps S.R. Shadle C. Sanchez-Bonilla M. Keel S.B. Abkowitz J.L. Coordinate expression of heme and globin is essential for effective erythropoiesis.J. Clin. Invest. 2015; 125: 4681-4691Google Scholar), or degradation (26Sassa S. Why heme needs to be degraded to iron, biliverdin IXalpha, and carbon monoxide?.Antioxid. Redox Signal. 2004; 6: 819-824Google Scholar, 27Ayer A. Zarjou A. Agarwal A. Stocker R. Heme oxygenases in cardiovascular health and disease.Physiol. Rev. 2016; 96: 1449-1508Google Scholar, 28Desmard M. Boczkowski J. Poderoso J. Motterlini R. Mitochondrial and cellular heme-dependent proteins as targets for the bioactive function of the heme oxygenase/carbon monoxide system.Antioxid. Redox Signal. 2007; 9: 2139-2155Google Scholar, 29Gozzelino R. Jeney V. Soares M.P. Mechanisms of cell protection by heme oxygenase-1.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 323-354Google Scholar, 30Hill-Kapturczak N. Jarmi T. Agarwal A. Growth factors and heme oxygenase-1: Perspectives in physiology and pathophysiology.Antioxid. Redox Signal. 2007; 9: 2197-2207Google Scholar, 31Munoz-Sanchez J. Chanez-Cardenas M.E. A review on hemeoxygenase-2: Focus on cellular protection and oxygen response.Oxid. Med. Cell. Longev. 2014; 2014: 604981Google Scholar). Arguably, the best understood mechanism for heme detoxification is through the heme catabolism pathway. The first and rate-limiting step of heme degradation is catalyzed by the heme oxygenases (HO) (32Stec D.E. Ishikawa K. Sacerdoti D. Abraham N.G. The emerging role of heme oxygenase and its metabolites in the regulation of cardiovascular function.Int. J. Hypertens. 2012; 2012: 593530Google Scholar, 33Constantin M. Choi A.J. Cloonan S.M. Ryter S.W. Therapeutic potential of heme oxygenase-1/carbon monoxide in lung disease.Int. J. Hypertens. 2012; 2012: 859235Google Scholar). Mammals encode two HO isoforms, inducible HO-1 and constitutive HO-2 (34Maines M.D. The heme oxygenase system: A regulator of second messenger gases.Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Google Scholar, 35Maines M.D. The heme oxygenase system: Update 2005.Antioxid. Redox Signal. 2005; 7: 1761-1766Google Scholar, 36Maines M.D. Trakshel G.M. Kutty R.K. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible.J. Biol. Chem. 1986; 261: 411-419Google Scholar, 37Trakshel G.M. Kutty R.K. Maines M.D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.J. Biol. Chem. 1986; 261: 11131-11137Google Scholar). HO-1 and HO-2 are structurally similar, both in primary sequence and tertiary structure, operate using the same chemical mechanism, and exhibit similar catalytic properties, including Michaelis constants (KM) and maximal velocities (Vmax) (37Trakshel G.M. Kutty R.K. Maines M.D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.J. Biol. Chem. 1986; 261: 11131-11137Google Scholar, 38Bianchetti C.M. Yi L. Ragsdale S.W. Phillips Jr., G.N. Comparison of apo- and heme-bound crystal structures of a truncated human heme oxygenase-2.J. Biol. Chem. 2007; 282: 37624-37631Google Scholar, 39Kochert B.A. Fleischhacker A.S. Wales T.E. Becker D.F. Engen J.R. Ragsdale S.W. Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.J. Biol. Chem. 2019; 294: 8259-8272Google Scholar). HOs, which are primarily anchored into the endoplasmic reticulum (ER) membrane and whose active sites face the cytoplasm, bind oxidized ferric heme in its resting state using a histidine axial ligand. Upon reduction, using electrons from the NADPH-cytochrome P450 reductase (CPR) system and dioxygen binding (O2), HOs catalyze the oxidative degradation of heme to form biliverdin, ferrous iron (Fe2+), and carbon monoxide (CO) (40Trakshel G.M. Kutty R.K. Maines M.D. Cadmium-mediated inhibition of testicular heme oxygenase activity: The role of NADPH-cytochrome c (P-450) reductase.Arch. Biochem. Biophys. 1986; 251: 175-187Google Scholar, 41Matsui T. Iwasaki M. Sugiyama R. Unno M. Ikeda-Saito M. Dioxygen activation for the self-degradation of heme: Reaction mechanism and regulation of heme oxygenase.Inorg. 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The heme synthesis and degradation pathways: Role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties.Free Radic. Biol. Med. 2000; 28: 289-309Google Scholar, 55Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Bilirubin is an antioxidant of possible physiological importance.Science. 1987; 235: 1043-1046Google Scholar). Although the structures and mechanisms of HO-1 and HO-2 are largely the same (37Trakshel G.M. Kutty R.K. Maines M.D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.J. Biol. Chem. 1986; 261: 11131-11137Google Scholar, 38Bianchetti C.M. Yi L. Ragsdale S.W. Phillips Jr., G.N. Comparison of apo- and heme-bound crystal structures of a truncated human heme oxygenase-2.J. Biol. Chem. 2007; 282: 37624-37631Google Scholar, 39Kochert B.A. Fleischhacker A.S. Wales T.E. Becker D.F. Engen J.R. Ragsdale S.W. Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.J. Biol. Chem. 2019; 294: 8259-8272Google Scholar, 41Matsui T. Iwasaki M. Sugiyama R. Unno M. Ikeda-Saito M. Dioxygen activation for the self-degradation of heme: Reaction mechanism and regulation of heme oxygenase.Inorg. Chem. 2010; 49: 3602-3609Google Scholar, 43Kumar D. de Visser S.P. Shaik S. Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.J. Am. Chem. Soc. 2005; 127: 8204-8213Google Scholar, 56Davydov R. Fleischhacker A.S. Bagai I. Hoffman B.M. Ragsdale S.W. Comparison of the mechanisms of heme hydroxylation by heme oxygenases-1 and -2: Kinetic and cryoreduction studies.Biochemistry. 2016; 55: 62-68Google Scholar), the regulation and expression of these two enzymes is very different (27Ayer A. Zarjou A. Agarwal A. Stocker R. Heme oxygenases in cardiovascular health and disease.Physiol. Rev. 2016; 96: 1449-1508Google Scholar, 31Munoz-Sanchez J. Chanez-Cardenas M.E. A review on hemeoxygenase-2: Focus on cellular protection and oxygen response.Oxid. Med. Cell. Longev. 2014; 2014: 604981Google Scholar, 35Maines M.D. The heme oxygenase system: Update 2005.Antioxid. Redox Signal. 2005; 7: 1761-1766Google Scholar). Heme oxygenase-1, which is comparatively far better understood, is induced by excess heme, as well as several nonheme stressors like oxidative stress, infection, and exposure to various xenobiotics (57Prawan A. Kundu J.K. Surh Y.J. Molecular basis of heme oxygenase-1 induction: Implications for chemoprevention and chemoprotection.Antioxid. Redox Signal. 2005; 7: 1688-1703Google Scholar, 58Funes S.C. Rios M. Fernandez-Fierro A. Covian C. Bueno S.M. Riedel C.A. Mackern-Oberti J.P. Kalergis A.M. 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HO-2, on the other hand, is constitutively expressed across all tissues and cell types, being most abundant in the brain and testis (34Maines M.D. The heme oxygenase system: A regulator of second messenger gases.Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Google Scholar, 35Maines M.D. The heme oxygenase system: Update 2005.Antioxid. Redox Signal. 2005; 7: 1761-1766Google Scholar). The current rationale for dual mammalian HO isoforms is that HO-2 provides a baseline level of protection from heme in the absence of cellular stressors that would otherwise induce HO-1. However, the biochemical necessity for expressing constitutive HO-2 is largely dependent on whether sufficient heme is available as a substrate under conditions in which HO-1 is not induced. Total cellular heme in yeast and various nonerythroid human cell lines is on the order of 1 to 20 μM (62Hanna D.A. Hu R. Kim H. Martinez-Guzman O. Torres M.P. Reddi A.R. Heme bioavailability and signaling in response to stress in yeast cells.J. Biol. Chem. 2018; 293: 12378-12393Google Scholar, 63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 64Liu L. Dumbrepatil A.B. Fleischhacker A.S. Marsh E.N.G. Ragsdale S.W. Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site.J. Biol. Chem. 2020; 295: 17227-17240Google Scholar, 65Hopp M.T. Schmalohr B.F. Kuhl T. Detzel M.S. Wissbrock A. Imhof D. Heme determination and quantification methods and their suitability for practical applications and everyday use.Anal. Chem. 2020; 92: 9429-9440Google Scholar, 66Martinez-Guzman O. Willoughby M.M. Saini A. Dietz J.V. Bohovych I. Medlock A.E. Khalimonchuk O. Reddi A.R. Mitochondrial-nuclear heme trafficking in budding yeast is regulated by GTPases that control mitochondrial dynamics and ER contact sites.J. Cell Sci. 2020; 133jcs237917Google Scholar, 67Mestre-Fos S. Ito C. Moore C.M. Reddi A.R. Williams L.D. Human ribosomal G-quadruplexes regulate heme bioavailability.J. Biol. Chem. 2020; 295: 14855-14865Google Scholar). All heme in the cell partitions between exchange inert high affinity hemoproteins, such as cytochromes and other heme enzymes, and certain exchange labile heme (LH) complexes that buffer free heme down to nanomolar concentrations (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 12Reddi A.R. Hamza I. Heme mobilization in animals: A metallolipid's journey.Acc. Chem. Res. 2016; 49: 1104-1110Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 14Chambers I.G. Willoughby M.M. Hamza I. Reddi A.R. One ring to bring them all and in the darkness bind them: The trafficking of heme without deliverers.Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868: 118881Google Scholar, 63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 68Song Y. Yang M. Wegner S.V. Zhao J. Zhu R. Wu Y. He C. Chen P.R. A genetically encoded FRET sensor for intracellular heme.ACS Chem. Biol. 2015; 10: 1610-1615Google Scholar, 69Leung G.C.-H. Fung S.S.-P. Gallio A.E. Blore R. Alibhai D. Raven E.L. Hudson A.J. Unravelling the mechanisms controlling heme supply and demand.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2104008118Google Scholar). The factors that buffer heme are poorly understood, but likely consist of a network of heme-binding proteins, nucleic acids, and lipid membranes (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 12Reddi A.R. Hamza I. Heme mobilization in animals: A metallolipid's journey.Acc. Chem. Res. 2016; 49: 1104-1110Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 14Chambers I.G. Willoughby M.M. Hamza I. Reddi A.R. One ring to bring them all and in the darkness bind them: The trafficking of heme without deliverers.Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868: 118881Google Scholar, 63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 66Martinez-Guzman O. Willoughby M.M. Saini A. Dietz J.V. Bohovych I. Medlock A.E. Khalimonchuk O. Reddi A.R. Mitochondrial-nuclear heme trafficking in budding yeast is regulated by GTPases that control mitochondrial dynamics and ER contact sites.J. Cell Sci. 2020; 133jcs237917Google Scholar, 67Mestre-Fos S. Ito C. Moore C.M. Reddi A.R. Williams L.D. Human ribosomal G-quadruplexes regulate heme bioavailability.J. Biol. Chem. 2020; 295: 14855-14865Google Scholar, 70Sweeny E.A. Singh A.B. Chakravarti R. Martinez-Guzman O. Saini A. Haque M.M. Garee G. Dans P.D. Hannibal L. Reddi A.R. Stuehr D.J. Glyceraldehyde-3-phosphate dehydrogenase is a chaperone that allocates labile heme in cells.J. Biol. Chem. 2018; 293: 14557-14568Google Scholar, 71Gray L.T. Puig Lombardi E. Verga D. Nicolas A. Teulade-Fichou M.P. Londono-Vallejo A. Maizels N. G-quadruplexes sequester free heme in living cells.Cell Chem. Biol. 2019; 26: 1681-1691.e5Google Scholar). Labile heme may act as a reservoir for bioavailable heme that can readily exchange with and populate heme-binding sites in heme dependent or regulated enzymes and proteins. The nature of LH, including its speciation, oxidation state, concentration, and distribution are not well understood but may be relevant for the mobilization and trafficking of heme. It is currently not known what the source of heme is for HOs, that is, whether it is buffered-free heme or a dedicated chaperone system that traffics and channels heme to HO in a manner that bypasses the LH pool. The recent development of fluorescence and activity-based heme sensors has offered unprecedented insights into LH and their diverse roles in physiology (63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 68Song Y. Yang M. Wegner S.V. Zhao J. Zhu R. Wu Y. He C. Chen P.R. A genetically encoded FRET sensor for intracellular heme.ACS Chem. Biol. 2015; 10: 1610-1615Google Scholar, 69Leung G.C.-H. Fung S.S.-P. Gallio A.E. Blore R. Alibhai D. Raven E.L. Hudson A.J. Unravelling the mechanisms controlling heme supply and demand.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2104008118Google Scholar, 72Abshire J.R. Rowlands C.J. Ganesan S.M. So P.T. Niles J.C. Quantification of labile heme in live malaria parasites using a genetically encoded biosensor.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E2068-E2076Google Scholar, 73Yuan X. Rietzschel N. Kwon H. Walter Nuno A.B. Hanna D.A. Phillips J.D. Raven E.L. Reddi A.R. Hamza I. Regulation of intracellular heme trafficking revealed by subcellular reporters.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E5144-E5152Google Scholar). Strictly speaking, these probes report on the availability of heme to the sensor, not necessarily free heme coordinated by water (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 74Bal W. Kurowska E. Maret W. The final frontier of pH and the undiscovered country beyond.PLoS One. 2012; 7e45832Google Scholar). In other words, the heme occupancy of the sensor is dictated by the extent to which LH can exchange with the probe. However, many investigators convert the fractional heme loading of a probe to a buffered-free heme concentration, which can be done if the heme-sensor dissociation constant is known. Although problematic in that the sensor may not be probing "free heme", it nonetheless provides a measure of labile or accessible heme because the calculated concentration of free heme is related to sensor heme occupancy. In intact living yeast and various nonerythroid human cell lines, the estimates of buffered-free heme based on genetically encoded heme sensors are on the order of ∼5 to 20 nM (63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 68Song Y. Yang M. Wegner S.V. Zhao
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Biliverdin
Biliverdin reductase
Hemeprotein
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The effects of heme on the induction of mRNA and protein synthesis for heme oxygenase‐1 have been studied in primary cultures of chick embryo liver cells. Heme increased the amount of mRNA and the rate of heme oxygenase‐1‐gene transcription in a dose‐dependent fashion, with a maximal 20‐fold increase occurring at 20 μM heme. The largest increase in the rate of transcription, measured by nuclear run‐on assays, occurred at 5 h, 2 h earlier than the maximum increase in the amount of mRNA, measured by densitometry of Northern blots. 7–15 h after heme addition, the half‐life of heme‐oxygenase‐1 mRNA was 3.5 h in the presence or absence of actinomycin D. In contrast, addition of cycloheximide markedly increased the stability of the message (half‐life = 18 h), suggesting that a short‐lived protein plays a key role in modulating heme oxygenase‐1 mRNA levels. The half‐life of heme‐induced heme‐oxygenase‐1 protein, measured by [ 35 S]methionine labelling and immunoprecipitation, was 15 h. This long half‐life of the protein can largely account for the additional finding that, following addition of heme, the amount of enzyme protein in the cells increased for 10 h, after which it remained essentially constant for 15 h. A striking finding was that, after an initial burst of heme‐stimulated gene transcription, the cells became refractory to further heme‐mediated induction. This acquired resistance could not be attributed to the following: a longer duration of culture time; cellular toxicity caused by heme; a lack of heme in the medium or the cells; secretion of heme‐binding proteins into the medium, preventing further heme uptake; the induction of cellular heme catabolism sufficient to deplete cellular heme. Instead, the results suggest a down‐regulation of the intracellular machinery required for heme‐dependent induction of heme oxygenase‐1.
Transcription
Hemeprotein
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Biliverdin
Biliverdin reductase
Bile Pigments
Hemeprotein
HMOX1
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Heme oxygenase is an essential enzyme in heme catabolism that cleaves heme to form biliverdin, releasing carbon monoxide and iron. In mammals, biliverdin is subsequently converted to bilirubin by biliverdin reductase. There are two isozymes of heme oxygenase: heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2), each of which is encoded by a separate gene. Both isozymes share a significant similarity in amino acid sequence and catalyze heme breakdown under similar conditions. However, both enzymes are regulated in distinct manners: specifically, HO-1 is inducible by various environmental factors including its own substrate heme, while HO-2 is not inducible at all. Moreover, there has been remarkable progress concerning the physiological roles of the heme catabolites carbon monoxide (CO) and bilirubin, which had previously been considered mere toxic waste products. However, CO was suggested to function as a potential signaling gas, and bilirubin was shown to be an effective radical scavenger under physiological conditions. Thus, the inducibility of HO-1 may represent an important biological response. In this review, the general properties of heme oxygenase are briefly described with emphasis on the current findings regarding the regulation of heme oxygenase gene expression.
Biliverdin
Biliverdin reductase
Catabolism
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Biliverdin
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Imidazole‐based compounds previously synthesized in our laboratory were selected and reconsidered as inhibitors of heme oxygenase‐1 obtained from the microsomal fractions of rat spleens. Most of tested compounds were good inhibitors with IC 50 values in the low micromolar range. Compounds were also assayed on membrane‐free full‐length recombinant human heme oxygenase‐1; all tested compounds were unable to interact with human heme oxygenase‐1 at 100 μ m concentrations with the exception of compounds 11 and 13 that inhibited the enzyme of 54% and 20%, respectively. The binding of the most active compound 11 with heme or heme‐conjugated human heme oxygenase‐1 was also examined by spectral analyses. When heme was not conjugated to human heme oxygenase‐1, compound 11 caused changes in the heme spectrum only at concentration 50‐fold (100 μ m ) higher than that required to inhibit rat heme oxygenase‐1; when heme was conjugated to human heme oxygenase‐1, compound 11 was able to form a heme‐compound 11 complex also at low micromolar concentrations. To obtain information on the binding mode of the tested compounds with enzyme, docking studies and pharmacophore analysis were performed. Template docking results were in agreement with experimental inhibition data and with a structure‐based pharmacophoric model. These data may be exploitable to design new OH‐1 inhibitors.
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Hemeprotein
Imidazole
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Hemeprotein
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Biliverdin
Biliverdin reductase
Hemeprotein
Cytochrome P450 reductase
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