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Mahin D. Maines - One of the best experts on this subject based on the ideXlab platform.

  • Cysteine-independent activation/inhibition of Heme Oxygenase-2.
    Medical gas research, 2016
    Co-Authors: Dragic Vukomanovic, Mahin D. Maines, Mona N Rahman, Walter A Szarek, Terence R.s. Ozolinš, Zongchao Jia, Kanji Nakatsu
    Abstract:

    Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The Heme Oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of Heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human Heme Oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for Heme through cys265 and cys282.

  • the coordinated increased expression of biliverdin reductase and Heme Oxygenase 2 promotes cardiomyocyte survival a reductase based peptide counters β adrenergic receptor ligand mediated cardiac dysfunction
    The FASEB Journal, 2011
    Co-Authors: Bo Ding, Peter E. M. Gibbs, Paul S Brookes, Mahin D. Maines
    Abstract:

    HO-2 oxidizes Heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca2-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of β-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ≥96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligand's stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC281 and KYCCSRK296, respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.—Ding, B., Gibbs, P. E. M., Brookes, P. S., Maines, M. D. The coordinated increased expression of biliverdin reductase and Heme Oxygenase-2 promotes cardiomyocyte survival; a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction.

  • Heme Oxygenase-2 interaction with metalloporphyrins: function of Heme regulatory motifs.
    Antioxidants & redox signaling, 2001
    Co-Authors: Tian J. Huang, William K. Mccoubrey, Mahin D. Maines
    Abstract:

    Heme Oxygenase-2 (HO-2) degrades Heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of Heme regulatory motif (...

  • Interaction of Heme Oxygenase‐2 with nitric oxide donors
    European journal of biochemistry, 1999
    Co-Authors: Yan Ding, William K. Mccoubrey, Mahin D. Maines
    Abstract:

    Heme Oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of Heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds Heme at Heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281  Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm  413–419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of Heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased (≈ two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound Heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular ‘sink’ for the Heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.

  • Regulation of Heme Oxygenase-2 by glucocorticoids in neonatal rat brain: characterization of a functional glucocorticoid response element.
    Biochimica et Biophysica Acta, 1997
    Co-Authors: V S Raju, William K. Mccoubrey, Mahin D. Maines
    Abstract:

    Abstract Heme Oxygenase-2 (HO-2) is constitutively expressed in mammalian tissues; together with HO-1 (HSP32) it catalyzes the cleavage of Heme to produce biliverdin IXα, CO and Fe. Detection of a consensus sequence of the glucocorticoid response element (GRE) in the promoter region of the HO-2 gene prompted the present study which has investigated the role of glucocorticoids (Gcs) in the regulation of HO-2 protein and transcript development in the newborn rat brain and has examined the promoter activity of the GRE in HeLa cells. Using in situ hybridization histochemistry, we noted a pronounced increase in signal for HO-2 mRNA in the brain of 14-day-old rats postnatally treated with corticosterone (5 μg/g, 4×, starting 24–36 h after birth). And, using immunohistochemistry, a striking increase in neuronal HO-2 immunostaining in treated brains was detected. The HO-2 GRE was tested for responsiveness to dexamethasone (DX) using both a promoterless CAT expression vector, and a heterologous promoter containing luciferase expression vector in HeLa cells. The HO-2 promoter containing the GRE and transcription start site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Similarly, constructs containing the GRE conferred responsiveness to DX in an orientation-independent manner and increased relative luciferase activity. Further, specific binding of glucocorticoid receptor protein to the GRE was observed; binding could be competed out only by excess cold GRE and not by mutated HO-2 GRE, or AP1. HO-2 mRNAs (∼1.3 and ∼1.9 kb) increased in HeLa cells treated with DX (5 μM), the level reached a maximum at 24 h. DX did not effect HO-1 mRNA level. The increase in the HO-2 transcript was accompanied by an increase in HO-2 protein, as assessed by Western blot analysis, and an increase in HO activity, as measured by bilirubin formation. Also, an increase in intensity of immunostaining was noted in DX-treated HeLa cells. We conclude that the GRE present in the HO-2 gene promoter region is functional, and propose the direct involvement of the adrenal glucocorticoids in modulation of HO-2 gene expression. In the context of biological functions of Heme degradation products, we suggest that this regulation may be of significance, particularly to the neurons.

Sylvain Doré - One of the best experts on this subject based on the ideXlab platform.

  • Deletion of the hemopexin or Heme Oxygenase-2 gene aggravates brain injury following stroma-free hemoglobin-induced intracerebral hemorrhage.
    Journal of neuroinflammation, 2016
    Co-Authors: Jason Day, Harrison Phillips, Bryan Slootsky, Emanuela Tolosano, Sylvain Doré
    Abstract:

    Background Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic Heme that causes local brain injury. Hemopexin (Hpx) has the highest binding affinity to Heme and participates in its transport, while Heme Oxygenase 2 (HO2) is the rate-limiting enzyme for the degradation of Heme. Microglia are the resident macrophages in the brain; however, the significance and role of HO2 and Hpx on microglial clearance of the toxic Heme (iron-protoporphyrin IX) after ICH still remain understudied. Accordingly, we postulated that global deletion of constitutive HO2 or Hpx would lead to worsening of ICH outcomes.

  • Abstract W MP95: Deletion of the Hemopexin or Heme Oxygenase-2 Gene Aggravates Brain Injury Following Stroma-free-hemoglobin Induced Intracerebral Hemorrhage
    Stroke, 2015
    Co-Authors: Jason Day, Emanuela Tolosano, Sylvain Doré
    Abstract:

    Introduction: The breakdown of Heme-hemoglobin released after intracerebral hemorrhage (ICH) contributes to brain injury. Hemopexin (HPX) has highest binding affinity to Heme as its transporter and Heme Oxygenase 2 (HO2) is Heme rate-limiting enzyme for its degradation. However, the significance and role of HPX and HO2 on hemoglobin clearance in brain remains understudied. Methods: Intracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induced ICH. The HPX or HO2 knockout (KO) mice were injected with 10μL SFHb into the striatum. Behavioral/functional tests were performed along with anatomical changes. Iron deposition and neuronal degeneration was shown by Perl’s or Fluoro Jade B staining, respectively. Immunohistochemistry with anti-Iba1 was used to detect microglial activation around the injured site. Result: This study reveals that microglia contribute to hemoglobin clearance after SFHb injection. Deletion of HPX or HO2 aggravated the SFHb-induced brain injury. Both of HPX or ...

  • Erratum to: Heme Oxygenase-2 Is Neuroprotective in Cerebral Ischemia
    Molecular Medicine, 2013
    Co-Authors: Sylvain Doré, Kenji Sampei, Shozo Goto, Nabil J. Alkayed, Daniel Guastella, Seth Blackshaw, Michela Gallagher, Richard J. Traystman, Patricia D. Hurn, Raymond C. Koehler
    Abstract:

    Sylvain Dore, Kenji Sampei, Shozo Goto, Nabil J Alkayed, Daniel Guastella, Seth Blackshaw, Michela Gallagher, Richard J Traystman, Patricia D Hurn, Raymond C Koehler, and Solomon H Snyder. (1999) Heme Oxygenase-2 Is Neuroprotective in Cerebral Ischemia. Mol. Med. 1999 Oct;5(10):656–663.

  • Heme Oxygenase 2 is neuroprotective against intracerebral hemorrhage.
    Neurobiology of disease, 2006
    Co-Authors: Jian Wang, Hean Zhuang, Sylvain Doré
    Abstract:

    Abstract Recent studies suggest a neuroprotective function for Heme Oxygenase 2 (HO2) in acute brain injury and ischemia. HO2, the main enzyme to degrade the pro-oxidant Heme, was tested for its neuroprotective ability in postnatal neuronal cell cultures and in a model of collagenase-induced intracerebral hemorrhage. Genetic deletion of HO2 rendered cultured neurons 32% (P

  • Catalytically inactive Heme Oxygenase-2 mutant is cytoprotective
    Free radical biology & medicine, 2005
    Co-Authors: Yun-sook Kim, Sylvain Doré
    Abstract:

    Heme Oxygenase (HO) catalyzes the rate-limiting step in Heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45A-overexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant Heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself.

H W Deng - One of the best experts on this subject based on the ideXlab platform.

William K. Mccoubrey - One of the best experts on this subject based on the ideXlab platform.

  • Heme Oxygenase-2 interaction with metalloporphyrins: function of Heme regulatory motifs.
    Antioxidants & redox signaling, 2001
    Co-Authors: Tian J. Huang, William K. Mccoubrey, Mahin D. Maines
    Abstract:

    Heme Oxygenase-2 (HO-2) degrades Heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of Heme regulatory motif (...

  • Interaction of Heme Oxygenase‐2 with nitric oxide donors
    European journal of biochemistry, 1999
    Co-Authors: Yan Ding, William K. Mccoubrey, Mahin D. Maines
    Abstract:

    Heme Oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of Heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds Heme at Heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281  Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm  413–419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of Heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased (≈ two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound Heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular ‘sink’ for the Heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.

  • Regulation of Heme Oxygenase-2 by glucocorticoids in neonatal rat brain: characterization of a functional glucocorticoid response element.
    Biochimica et Biophysica Acta, 1997
    Co-Authors: V S Raju, William K. Mccoubrey, Mahin D. Maines
    Abstract:

    Abstract Heme Oxygenase-2 (HO-2) is constitutively expressed in mammalian tissues; together with HO-1 (HSP32) it catalyzes the cleavage of Heme to produce biliverdin IXα, CO and Fe. Detection of a consensus sequence of the glucocorticoid response element (GRE) in the promoter region of the HO-2 gene prompted the present study which has investigated the role of glucocorticoids (Gcs) in the regulation of HO-2 protein and transcript development in the newborn rat brain and has examined the promoter activity of the GRE in HeLa cells. Using in situ hybridization histochemistry, we noted a pronounced increase in signal for HO-2 mRNA in the brain of 14-day-old rats postnatally treated with corticosterone (5 μg/g, 4×, starting 24–36 h after birth). And, using immunohistochemistry, a striking increase in neuronal HO-2 immunostaining in treated brains was detected. The HO-2 GRE was tested for responsiveness to dexamethasone (DX) using both a promoterless CAT expression vector, and a heterologous promoter containing luciferase expression vector in HeLa cells. The HO-2 promoter containing the GRE and transcription start site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Similarly, constructs containing the GRE conferred responsiveness to DX in an orientation-independent manner and increased relative luciferase activity. Further, specific binding of glucocorticoid receptor protein to the GRE was observed; binding could be competed out only by excess cold GRE and not by mutated HO-2 GRE, or AP1. HO-2 mRNAs (∼1.3 and ∼1.9 kb) increased in HeLa cells treated with DX (5 μM), the level reached a maximum at 24 h. DX did not effect HO-1 mRNA level. The increase in the HO-2 transcript was accompanied by an increase in HO-2 protein, as assessed by Western blot analysis, and an increase in HO activity, as measured by bilirubin formation. Also, an increase in intensity of immunostaining was noted in DX-treated HeLa cells. We conclude that the GRE present in the HO-2 gene promoter region is functional, and propose the direct involvement of the adrenal glucocorticoids in modulation of HO-2 gene expression. In the context of biological functions of Heme degradation products, we suggest that this regulation may be of significance, particularly to the neurons.

  • Multiple transcripts encoding Heme Oxygenase-2 in rat testis: developmental and cell-specific regulation of transcripts and protein.
    Biology of reproduction, 1995
    Co-Authors: William K. Mccoubrey, Benay C. Eke, Mahin D. Maines
    Abstract:

    We report for the first time that Heme Oxygenase-2 (HO-2) expression is regulated by developmental and cell type-specific factors in the testis, and we describe the presence of three unique sizes of HO-2 transcripts in the testis. HO-2, together with HO-1 (HSP32), catalyzes oxidative cleavage of the Heme molecule to biliverdin, carbon monoxide, and iron; HO-2 is the major isozyme of the testis. Northern blot analysis was used to demonstrate the presence of five transcripts for HO-2 in rat testis mRNA; they range from approximately 1.3 to approximately 2.1 kg in length with a predominant 1.45-kb message; three of the transcripts, approximately 1.45 kb, approximately 1.7 kb, and approximately 2.1 kg, are unique to testis. The two other transcripts of approximately 1.3 and approximately 1.9 kb are common to every tissue examined, including the testis. Analysis of three distinct cDNAs isolated from rat libraries in phage lambda indicates that all are identical from -37, relative to translation initiation through the coding region to the first of two poly(A) signals previously identified in the HO-2 gene (McCoubrey and Maines, 1994). Upstream of -37, the 5' untranslated sequences of the isolates differ in both length and sequence. Comparison with the genomic sequence suggests that the multiple transcripts arise by splicing of alternative first exons as well as use of alternate poly(A) signals. Northern hybridization with probes specific for the unique portion of each cDNA are consistent with this interpretation. Further, unlike HO-1, HO-2 messages are developmentally regulated; only approximately 1.3- and approximately 1.9-kb transcripts were detected, at minute levels, in the testis RNA of 7-day-old rats. A pronounced increase in total message level was observed by Day 28 postpartum, although the level had not reached the marked amplification seen in the adult testis. Further, the transcript patterns differed when Day 28 and adult testis were compared to Day 7 testis. The very predominant approximately 1.45-kb band and the approximately 1.7- and 2.1-kb bands were absent from Day 7 testis. Heme Oxygenase activity and HO-2 protein levels, as assessed by Western blot, reflect the increases at the RNA level. Interestingly, although abundant HO-2 mRNA can be detected by in situ hybridization in spermatogonia, spermatocytes, and spermatids, HO-2 protein was detected, by immunocytochemistry, only in spermatids. These observations demonstrate tissue and cell specificity of HO-2 gene expression and suggest that in the testis, HO-2 expression is regulated at the transcriptional and translational levels.

  • Human Heme Oxygenase-2 : characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal
    Archives of biochemistry and biophysics, 1992
    Co-Authors: William K. Mccoubrey, James F. Ewing, Mahin D. Maines
    Abstract:

    We show by Northern blot analysis that human HO-2 is encoded by two transcripts (1.3 and 1.7 kb) and is a single-copy gene as judged by Southern blot analysis. We further provide evidence based on Northern blot and sequence analysis of a cDNA representing the larger transcript that the transcripts differ in the 3' untranslated region. A 274-base-pair DNA fragment from the rat Heme Oxygenase-2 gene (I. Cruse and M.D. Maines, 1988, J. Biol. Chem. 263, 3348-3353) was used to isolate a human HO-2 cDNA from a fetal kidney library in lambda gt11. The clone, designated hK-1, was sequenced and the cDNA insert was determined to be 1625 base pairs in length, encoding a protein of 313 amino acids. Two consensus polyadenylation signals separated by 440 nucleotides were identified in the 3' untranslated region. The size of the cDNA insert closely approximated the larger of two mRNAs. The nucleotide sequence was 88% identical to the rat HO-2 gene within the predicted coding region and the putative translation product was also estimated to be 88% identical to the rat gene product (M. O. Rotenberg and D. Maines, 1990, J. Biol. Chem. 265, 7501). The predicted size, 36 kDa, corresponded well with HO-2 detected in human testis microsomes by Western blot analysis. Further, the fusion protein expressed in Escherichia coli displayed significant Heme Oxygenase activity, which was inhibited by Zn- and Sn-protoporphyrins, known inhibitors of eukaryotic Heme Oxygenase, but not by sulfhydryl reagents.

Stephen W. Ragsdale - One of the best experts on this subject based on the ideXlab platform.

  • Heme Oxygenase-2 is post-translationally regulated by Heme occupancy in the catalytic site
    The Journal of biological chemistry, 2020
    Co-Authors: Liu Liu, Angela S. Fleischhacker, Arti B. Dumbrepatil, E. Neil G. Marsh, Stephen W. Ragsdale
    Abstract:

    Heme Oxygenase-2 (HO2) and -1 (HO1) catalyze Heme degradation to biliverdin, CO, and iron, forming an essential link in the Heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining Heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by Heme. We find that, under Heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that Heme plays a direct role in HO2 regulation. HO2 has three Heme binding sites: one at its catalytic site and the others at its two Heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of Heme binding to the HRMs. Rather, under Heme deficiency, loss of Heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind Heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining Heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in Heme deficiency response.

  • Absence of Heme at the catalytic site of Heme Oxygenase-2 triggers its lysosomal degradation
    2020
    Co-Authors: Liu Liu, Angela S. Fleischhacker, Arti B. Dumbrepatil, E. Neil G. Marsh, Stephen W. Ragsdale
    Abstract:

    Heme Oxygenase-2 (HO2) and -1 (HO1) catalyze Heme degradation to biliverdin, CO, and iron,forming an essential link in the Heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining Heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 levels by Heme is elucidated. Under Heme deficient conditions, HO2 is destabilized andtargeted for degradation. In HO2, three Heme binding sites are potential targets of Heme-dependent regulation: one at its catalytic site; the others at its two Heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of Heme binding to the HRMs. Rather, under Heme deficiency, loss of Heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind Heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular Heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2s role in maintaining Heme homeostasis, paving the way for future investigation into HO2s pathophysiological role in Heme deficiency response.

  • The Heme regulatory motifs of Heme Oxygenase-2 contribute to the transfer of Heme to the catalytic site for degradation
    The Journal of biological chemistry, 2020
    Co-Authors: Angela S. Fleischhacker, Liu Liu, Amanda L. Gunawan, Brent A. Kochert, Thomas E. Wales, Maelyn C. Borowy, John R. Engen, Stephen W. Ragsdale
    Abstract:

    Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human Heme Oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-Heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where Heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of Heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-Heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle-when Fe3+-Heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of Heme between binding sites. Indeed, we observed that HRM-bound Fe3+-Heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of Heme at the core. Moreover, we observed transfer of Heme from the core to the HRMs and equilibration of Heme between the core and HRMs. We therefore propose an Fe3+-Heme transfer model in which HRM-bound Heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain Heme homeostasis.

  • Redox Regulation of Heme Oxygenase-2 and the Transcription Factor, Rev-Erb, Through Heme Regulatory Motifs
    Antioxidants & redox signaling, 2017
    Co-Authors: Angela S. Fleischhacker, Eric L. Carter, Stephen W. Ragsdale
    Abstract:

    Abstract Significance: Heme binds to and serves as a cofactor for a myriad of proteins that are involved in diverse biological processes. Hemoproteins also exhibit varying modes of Heme binding, suggesting that the protein environment contributes to the functional versatility of this prosthetic group. The subject of this review is a subset of hemoproteins that contain at least one Heme regulatory motif (HRM), which is a short sequence containing a Cys-Pro core that, in many cases, binds Heme with the Cys acting as an axial ligand. Recent Advances: As more details about HRM-containing proteins are uncovered, some underlying commonalities are emerging, including a role in regulating protein stability. Further, the cysteines of some HRMs have been shown to form disulfide bonds. Because the cysteines must be in the reduced, dithiol form to act as a Heme axial ligand, Heme binds at these sites in a redox-regulated manner, as demonstrated for Heme Oxygenase-2 (HO2) and Rev-erbβ. Critical Issues: HRM-containing ...

  • The C-Terminal Heme Regulatory Motifs of Heme Oxygenase-2 Are Redox-Regulated Heme Binding Sites
    Biochemistry, 2015
    Co-Authors: Angela S. Fleischhacker, Ajay Sharma, Michelle Choi, Andrea Morris Spencer, Ireena Bagai, Brian M. Hoffman, Stephen W. Ragsdale
    Abstract:

    Heme Oxygenase-2 (HO2), an enzyme that catalyzes the conversion of Heme to biliverdin, contains three Heme regulatory motifs (HRMs) centered at Cys127, Cys265, and Cys282. Previous studies using the soluble form of human HO2 spanning residues 1–288 (HO2sol) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, Heme binds to the catalytic site of HO2sol via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe3+-Heme binding upon reduction of the disulfide bond. In an effort to understand how the HRMs are involved in binding of Heme to disulfide-reduced HO2sol, in the work described here, we further investigated the properties of Fe3+-Heme bound to HO2. Specifically, we investigated binding of Fe3+-Heme to a truncated form of soluble HO2 (residues 213–288; HO2tail) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2tail in the disulfide-reduced state binds Fe3+-Heme and acc...