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1
Liu, Liu, Arti B. Dumbrepatil, Angela S. Fleischhacker, E. Neil G. Marsh, and Stephen W. Ragsdale. "Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site." Journal of Biological Chemistry 295, no. 50 (October 13, 2020): 17227–40. http://dx.doi.org/10.1074/jbc.ra120.014919.
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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.
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Nakamura, Nozomi, Yoichi Naoe, Akihiro Doi, Yoshitsugu Shiro, and Hiroshi Sugimoto. "Conformational change of periplasmic heme-binding protein in ABC transporter." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1496. http://dx.doi.org/10.1107/s2053273314085039.
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Iron is one of the essential elements for all living organisms. Pathogenic bacteria acquire heme from the host proteins as an iron source. Gram-negative opportunistic pathogen, Burkholderia cenocepacia have ATP-binding cassette (ABC) transporter BhuUV-T complex to permeate heme through inner membrane. BhuT, periplasmic binding protein (PBP), bind and deliver heme(s) to inner membrane transporter BhuUV complex. BhuUV is 2:2 complex of the transmembrane permease subunit and cytoplasmic ATP-binding subunit which couple ATP hydrolysis to solute translocation. The molecular level mechanism of heme recognition and dissociation by PBP and heme transport by transporter complex are not fully understood. Here we describe the crystal structures of the heme-free and two types of heme-bound state of BhuT. These crystals were obtained in different crystallization conditions. Crystals diffracted to high resolution at SPring-8. BhuT is composed of two globular domains linked by a long a-helix. The transport ligand heme is bound between the two domains. A detailed structural comparison of the conformation of the domain and residues involved in the heme binding will be presented.
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Nath, Karl A., Joseph P. Grande, John D. Belcher, Vesna D. Garovic, Anthony J. Croatt, Matthew L. Hillestad, Michael A. Barry, Meryl C. Nath, Raymond F. Regan, and Gregory M. Vercellotti. "Antithrombotic effects of heme-degrading and heme-binding proteins." American Journal of Physiology-Heart and Circulatory Physiology 318, no. 3 (March 1, 2020): H671—H681. http://dx.doi.org/10.1152/ajpheart.00280.2019.
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In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2−/− mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin−/− mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs. NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.
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El-Mashtoly, Samir F., and Teizo Kitagawa. "Structural chemistry involved in information detection and transmission by gas sensor heme proteins: Resonance Raman investigation." Pure and Applied Chemistry 80, no. 12 (January 1, 2008): 2667–78. http://dx.doi.org/10.1351/pac200880122667.
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A variety of heme-containing gas sensor proteins have been discovered by gene analysis from bacteria to mammals. In general, these proteins are composed of an N-terminal heme-containing sensor domain and a C-terminal catalytic domain. Binding of O2, CO, or NO to the heme causes a change in the structure of heme, which alters the protein conformation in the vicinity of the heme, and the conformational change is propagated to the catalytic domain, leading to regulation of the protein activity. This mini-review summarizes the recent resonance Raman studies obtained with both visible and UV excitation sources for two O2 sensor proteins, EcDOS and HemAT-Bs. These investigations have shown the role of heme propionate hydrogen-bonding interactions in communicating the heme structural changes, which occur upon ligand binding, from heme to the protein moiety. Furthermore, it is deduced that the contact interactions between the heme 2-vinyl group and the surrounding residues are also important for signal transmission from heme to protein in EcDOS.
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Tiedemann, Michael T., Naomi Muryoi, David E. Heinrichs, and Martin J. Stillman. "Characterization of IsdH (NEAT domain 3) and IsdB (NEAT domain 2) in Staphylococcus aureus by magnetic circular dichroism spectroscopy and electrospray ionization mass spectrometry." Journal of Porphyrins and Phthalocyanines 13, no. 10 (October 2009): 1006–16. http://dx.doi.org/10.1142/s1088424609001352.
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Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the first two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdB-N2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits significantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fifth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme.
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Freeman, Samuel L., Hanna Kwon, Nicola Portolano, Gary Parkin, Umakhanth Venkatraman Girija, Jaswir Basran, Alistair J. Fielding, et al. "Heme binding to human CLOCK affects interactions with the E-box." Proceedings of the National Academy of Sciences 116, no. 40 (September 16, 2019): 19911–16. http://dx.doi.org/10.1073/pnas.1905216116.
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The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep–wake cycle via 2 basic helix–loop–helix PER-ARNT-SIM (bHLH-PAS) domain proteins—CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.
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Fleischhacker, Angela S., Amanda L. Gunawan, Brent A. Kochert, Liu Liu, Thomas E. Wales, Maelyn C. Borowy, John R. Engen, and Stephen W. Ragsdale. "The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation." Journal of Biological Chemistry 295, no. 16 (March 9, 2020): 5177–91. http://dx.doi.org/10.1074/jbc.ra120.012803.
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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.
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Lechuga, Guilherme C., Franklin Souza-Silva, Carolina Q. Sacramento, Monique R. O. Trugilho, Richard H. Valente, Paloma Napoleão-Pêgo, Suelen S. G. Dias, et al. "SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites." International Journal of Molecular Sciences 22, no. 16 (August 21, 2021): 9035. http://dx.doi.org/10.3390/ijms22169035.
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(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC–MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC–MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus’s adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.
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Jeong, Jinsook, Tracey A. Rouault, and Rodney L. Levine. "Identification of a Heme-sensing Domain in Iron Regulatory Protein 2." Journal of Biological Chemistry 279, no. 44 (August 16, 2004): 45450–54. http://dx.doi.org/10.1074/jbc.m407562200.
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Iron regulatory protein 2 coordinates the cellular regulation of iron metabolism by binding to iron-responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. The mechanisms that prevent degradation during iron deficiency or promote degradation during iron sufficiency are not delineated. Iron regulatory protein 2 contains a domain not present in the closely related iron regulatory protein 1, and we found that this domain binds heme with high affinity. A cysteine within the domain is axially liganded to the heme, as occurs in cytochrome P450. The protein-bound heme reacts with molecular oxygen to mediate the oxidation of cysteine, including β-elimination of the sulfur to yield alanine. This covalent modification may thus mark the protein molecule for degradation by the proteasome system, providing another mechanism by which heme can regulate the level of iron regulatory protein 2.
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Yang, Jianhua, Kevin D. Kim, Andrew Lucas, Karen E. Drahos, Carlo S. Santos, Sean P. Mury, Daniel G. S. Capelluto, and Carla V. Finkielstein. "A Novel Heme-Regulatory Motif Mediates Heme-Dependent Degradation of the Circadian Factor Period 2." Molecular and Cellular Biology 28, no. 15 (May 27, 2008): 4697–711. http://dx.doi.org/10.1128/mcb.00236-08.
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ABSTRACT Although efforts have been made to identify circadian-controlled genes regulating cell cycle progression and cell death, little is known about the metabolic signals modulating circadian regulation of gene expression. We identify heme, an iron-containing prosthetic group, as a regulatory ligand controlling human Period-2 (hPer2) stability. Furthermore, we define a novel heme-regulatory motif within the C terminus of hPer2 (SC841PA) as necessary for heme binding and protein destabilization. Spectroscopy reveals that whereas the PAS domain binds to both the ferric and ferrous forms of heme, SC841PA binds exclusively to ferric heme, thus acting as a redox sensor. Consequently, binding prevents hPer2 from interacting with its stabilizing counterpart cryptochrome. In vivo, hPer2 downregulation is suppressed by inhibitors of heme synthesis or proteasome activity, while SA841PA is sufficient to stabilize hPer2 in transfected cells. Moreover, heme binding to the SC841PA motif directly impacts circadian gene expression, resulting in altered period length. Overall, the data support a model where heme-mediated oxidation triggers hPer2 degradation, thus controlling heterodimerization and ultimately gene transcription.
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Franceschi, Lucia De, Mariarita Bertoldi, Maria Domenica Cappellini, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Francesco Turrini, Alessandra Colancecco, Clara Camaschella, and Achille Iolascon. "OXIDATIVE STRESS MODULATES HEME LEVELS and INDUCES PEROXIREDOXIN-2 IN β THALASSEMIC ERYTHROPOIESIS as NOVEL CYTOPROTECTIVE RESPONSE." Blood 116, no. 21 (November 19, 2010): 4266. http://dx.doi.org/10.1182/blood.v116.21.4266.4266.
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Abstract Abstract 4266 Beta thalassemia (β-thal) syndromes are worldwide distributed congenital red cell disorders. Increased levels of reactive-oxygen-species (ROS) have been reported to contribute to anemia in β-thal but the mechanism(s) involved in cell protection against ROS damage has only partially investigated. Here, we studied in vitro normal and β-thal erythropoiesis in erythroid cell cultures from CD34+ cells isolated from peripheral blood from adult normal volunteers and from homozygous (bcod39) b-thalassemia patients. We showed increased ROS production in β-thal erythropoiesis and we evaluated the effects of ROS on normal and β-thal erythropoiesis. We carried out a proteomic comparative study, validated by coupling Quantitative-Real time PCR and immunoblot analysis of the differently expressed proteins. We found down-regulation in expression of enzymes involved in heme catabolism such as biliverdin reductase (BVR) and heme-oxygenase-1 (HO-1) and up-regulation of two new cytoprotective cysteine-based-systems: peroxiredoxin-2 (Prx2) and heat-shock-protein-27 (HSP27), while catalase was similarly expressed in both cell models, suggesting a specific pattern of Px2 and HSP27 in β-thal erythroid precursors. We then measured heme levels and during b-thal-erythropoiesis and found that the synthesis of heme was biphasic displaying an increase of heme levels in early phase followed by a decrease in late phase in comparison to controls. Since heme synthesis depends on the erythroid δ-aminolevulinate-synthase isoform (ALAS-2), we evaluated ALAS-2 expression that resulted similar in normal and β-thal erythroid cells. We then showed that ALAS-2 activity was inhibited by both ROS and hemin, suggesting a possible role of heme and ROS levels in regulation of heme biosynthesis in β-thal cells. Since it has been reported that oxidative stress can up-regulate Prx2 expression and that genetically modified cells over-expressing Prx2 are generally more protected from severe oxidative stress (Phalen TJ et al 2006; Rabilloud T et al 2002; Kang SW et al 1998; Zhang P et al 1997), we have hypothesized a cytoprotective role of Prx2 in b-thal-erythropoiesis. We determined that the anti-oxidant Prx2 specifically binds hemin with high and affinity, most likely involving Prx2 cysteine residues. In order to look for the structural determinants to the binding, we noted that both ALAS-2 and Prx2 possess one and two cys-pro motifs, respectively. This motif is generally considered a heme sensor for many proteins able to bind heme and we propose that it could be responsible for heme binding in both enzymes. These data suggest a wider role of Prx2 as both anti-oxidant and heme-binding protein in protective stress-response-systems in β-thal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.
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Choi, Clara Y. H., Jose F. Cerda, Hsiu-An Chu, Gerald T. Babcock, and Michael A. Marletta. "Spectroscopic Characterization of the Heme-Binding Sites inPlasmodium falciparumHistidine-Rich Protein 2†." Biochemistry 38, no. 51 (December 1999): 16916–24. http://dx.doi.org/10.1021/bi991665k.
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Airola, Michael V., Jing Du, John H. Dawson, and Brian R. Crane. "Heme Binding to the Mammalian Circadian Clock Protein Period 2 Is Nonspecific." Biochemistry 49, no. 20 (May 25, 2010): 4327–38. http://dx.doi.org/10.1021/bi901945w.
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Uchida, Takeshi, Julie M. Stevens, Oliver Daltrop, Edgar M. Harvat, Lin Hong, Stuart J. Ferguson, and Teizo Kitagawa. "The Interaction of Covalently Bound Heme with the CytochromecMaturation Protein CcmE." Journal of Biological Chemistry 279, no. 50 (October 1, 2004): 51981–88. http://dx.doi.org/10.1074/jbc.m408963200.
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The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochromecbiogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation ofc-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE fromEscherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr134. In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.
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Zenke-Kawasaki, Yukari, Yoshihiro Dohi, Yasutake Katoh, Tsuyoshi Ikura, Masae Ikura, Toshimasa Asahara, Fuminori Tokunaga, Kazuhiro Iwai, and Kazuhiko Igarashi. "Heme Induces Ubiquitination and Degradation of the Transcription Factor Bach1." Molecular and Cellular Biology 27, no. 19 (August 6, 2007): 6962–71. http://dx.doi.org/10.1128/mcb.02415-06.
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ABSTRACT The transcription repressor Bach1 is a sensor and effector of heme that regulates the expression of heme oxygenase 1 and globin genes. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. We found that hemin further induced the degradation of endogenous Bach1 in NIH 3T3 cells, murine embryonic fibroblasts, and murine erythroleukemia cells. In contrast, succinylacetone, an inhibitor of heme synthesis, caused accumulation of Bach1 in murine embryonic fibroblasts, indicating that physiological levels of heme regulated the Bach1 turnover. Polyubiquitination and rapid degradation of overexpressed Bach1 were induced by hemin treatment. HOIL-1, an ubiquitin-protein ligase which recognizes heme-bound, oxidized iron regulatory protein 2, was found to bind with Bach1 when both were overexpressed in NIH 3T3 cells. HOIL-1 stimulated the polyubiquitination of Bach1 in a purified in vitro ubiquitination system depending on the intact heme binding motifs of Bach1. Expression of dominant-negative HOIL-1 in murine erythroleukemia cells resulted in higher stability of endogenous Bach1, raising the possibility that the heme-regulated degradation involved HOIL-1 in murine erythroleukemia cells. These results suggest that heme within a cell regulates the polyubiquitination and degradation of Bach1.
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Hopp, Marie-Thérèse, Daniel Domingo-Fernández, Yojana Gadiya, Milena S. Detzel, Regina Graf, Benjamin F. Schmalohr, Alpha T. Kodamullil, Diana Imhof, and Martin Hofmann-Apitius. "Linking COVID-19 and Heme-Driven Pathophysiologies: A Combined Computational–Experimental Approach." Biomolecules 11, no. 5 (April 27, 2021): 644. http://dx.doi.org/10.3390/biom11050644.
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The SARS-CoV-2 outbreak was declared a worldwide pandemic in 2020. Infection triggers the respiratory tract disease COVID-19, which is accompanied by serious changes in clinical biomarkers such as hemoglobin and interleukins. The same parameters are altered during hemolysis, which is characterized by an increase in labile heme. We present two computational–experimental approaches aimed at analyzing a potential link between heme-related and COVID-19 pathophysiologies. Herein, we performed a detailed analysis of the common pathways induced by heme and SARS-CoV-2 by superimposition of knowledge graphs covering heme biology and COVID-19 pathophysiology. Focus was laid on inflammatory pathways and distinct biomarkers as the linking elements. In a second approach, four COVID-19-related proteins, the host cell proteins ACE2 and TMPRSS2 as well as the viral proteins 7a and S protein were computationally analyzed as potential heme-binding proteins with an experimental validation. The results contribute to the understanding of the progression of COVID-19 infections in patients with different clinical backgrounds and may allow for a more individual diagnosis and therapy in the future.
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Chen, Jane-Jane. "Regulation of protein synthesis by the heme-regulated eIF2α kinase: relevance to anemias." Blood 109, no. 7 (November 16, 2006): 2693–99. http://dx.doi.org/10.1182/blood-2006-08-041830.
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Abstract During erythroid differentiation and maturation, it is critical that the 3 components of hemoglobin, α-globin, β-globin, and heme, are made in proper stoichiometry to form stable hemoglobin. Heme-regulated translation mediated by the heme-regulated inhibitor kinase (HRI) provides one major mechanism that ensures balanced synthesis of globins and heme. HRI phosphorylates the α-subunit of eukaryotic translational initiation factor 2 (eIF2α) in heme deficiency, thereby inhibiting protein synthesis globally. In this manner, HRI serves as a feedback inhibitor of globin synthesis by sensing the intracellular concentration of heme through its heme-binding domains. HRI is essential not only for the translational regulation of globins, but also for the survival of erythroid precursors in iron deficiency. Recently, the protective function of HRI has also been demonstrated in murine models of erythropoietic protoporphyria and β-thalassemia. In these 3 anemias, HRI is essential in determining red blood cell size, number, and hemoglobin content per cell. Translational regulation by HRI is critical to reduce excess synthesis of globin proteins or heme under nonoptimal disease states, and thus reduces the severity of these diseases. The protective role of HRI may be more common among red cell disorders.
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Shibata, Tomokazu, Eisuke Furuichi, Kiyohiro Imai, Akihiro Suzuki, and Yasuhiko Yamamoto. "Effects of heme modification on oxygen affinity and cooperativity of human adult hemoglobin." Journal of Porphyrins and Phthalocyanines 19, no. 01-03 (January 2015): 301–7. http://dx.doi.org/10.1142/s1088424615500200.
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We substituted strongly electron-withdrawing trifluoromethyl ( CF 3) group(s) as heme side chain(s) of human adult hemoglobin (Hb) to achieve large alterations of the heme electronic structure, in order to elucidate the relationship between the oxygen ( O 2) binding properties of Hb and the electronic properties of heme peripheral side chains. The obtained results were compared with those of similar studies performed on myoglobin (Mb), e.g. (Nishimura R, Matsumoto D, Shibata T, Yanagisawa S, Ogura T, Tai H, Matsuo T, Hirota S, Neya S, Suzuki A, and Yamamoto Y. Inorg. Chem. 2014; 53: 9156–9165). These two proteins shared the common feature of a decrease in O 2 affinity upon the CF 3 substitution(s). Using the P50 value, which is the partial pressure of O 2 required for 50% oxygenation of a protein, and the equilibrium constant ( p K a ) of the "acid-alkaline transition" in the met form of a protein as measures of the O 2 affinity and the electron density of heme Fe atom of the protein, respectively, a linear p K a - log (1/P50) relationship was demonstrated for the Hb and Mb systems. The native Hb, however, deviated from the p K a - log (1/P50) relationship, while the native Mb followed it. These results highlighted the significance of the vinyl side chains of the heme cofactor in the functional control of Hb through tertiary and quaternary structural changes upon the oxygenation of the protein.
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Crosby, John S., Peter J. Chefalo, Irene Yeh, Shong Ying, Irving M. London, Philippe Leboulch, and Jane-Jane Chen. "Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2α kinase." Blood 96, no. 9 (November 1, 2000): 3241–48. http://dx.doi.org/10.1182/blood.v96.9.3241.
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Abstract Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2α kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2α. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.
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Crosby, John S., Peter J. Chefalo, Irene Yeh, Shong Ying, Irving M. London, Philippe Leboulch, and Jane-Jane Chen. "Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2α kinase." Blood 96, no. 9 (November 1, 2000): 3241–48. http://dx.doi.org/10.1182/blood.v96.9.3241.h8003241_3241_3248.
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Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2α kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2α. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.
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Varfaj, Fatbardha, Jed N. Lampe, and Paul R. Ortiz de Montellano. "Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy." Journal of Biological Chemistry 287, no. 42 (August 24, 2012): 35181–91. http://dx.doi.org/10.1074/jbc.m112.378042.
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Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-13C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys282 and Cys265 are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys265 and Cys282 varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys265 appears to be in a relatively hydrophilic, oxidizable environment compared with Cys127 and Cys282. Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.
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Choi, Clara Y. H., Eric L. Schneider, Jin M. Kim, Ilya Y. Gluzman, Daniel E. Goldberg, Jonathan A. Ellman, and Michael A. Marletta. "Interference with Heme Binding to Histidine-Rich Protein-2 as an Antimalarial Strategy." Chemistry & Biology 9, no. 8 (August 2002): 881–89. http://dx.doi.org/10.1016/s1074-5521(02)00183-7.
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Kranz, Robert G., Cynthia Richard-Fogal, John-Stephen Taylor, and Elaine R. Frawley. "Cytochrome c Biogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control." Microbiology and Molecular Biology Reviews 73, no. 3 (September 2009): 510–28. http://dx.doi.org/10.1128/mmbr.00001-09.
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SUMMARY Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.
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Alvarado, Gerardo, Attila Tóth, Éva Csősz, Gergő Kalló, Katalin Dankó, Zoltán Csernátony, Ann Smith, et al. "Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres." International Journal of Molecular Sciences 21, no. 21 (October 31, 2020): 8172. http://dx.doi.org/10.3390/ijms21218172.
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Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20–300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.
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Detzel, Milena Sophie, Benjamin Franz Schmalohr, Francèl Steinbock, Marie-Thérèse Hopp, Anuradha Ramoji, Ajay Abisheck Paul George, Ute Neugebauer, and Diana Imhof. "Revisiting the interaction of heme with hemopexin." Biological Chemistry 402, no. 6 (February 19, 2021): 675–91. http://dx.doi.org/10.1515/hsz-2020-0347.
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Abstract In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a K D value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.
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Lemli, Beáta, Zuzana Lomozová, Tamás Huber, András Lukács, and Miklós Poór. "Effects of Heme Site (FA1) Ligands Bilirubin, Biliverdin, Hemin, and Methyl Orange on the Albumin Binding of Site I Marker Warfarin: Complex Allosteric Interactions." International Journal of Molecular Sciences 23, no. 22 (November 13, 2022): 14007. http://dx.doi.org/10.3390/ijms232214007.
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Human serum albumin (HSA) is the most abundant plasma protein in circulation. The three most important drug-binding sites on HSA are Sudlow’s Site I (subdomain IIA), Sudlow’s Site II (subdomain IIIA), and Heme site (subdomain IB). Heme site and Site I are allosterically coupled; therefore, their ligands may be able to allosterically modulate the binding affinity of each other. In this study, the effects of four Heme site ligands (bilirubin, biliverdin, hemin, and methyl orange) on the interaction of the Site I ligand warfarin with HSA were tested, employing fluorescence spectroscopic, ultrafiltration, and ultracentrifugation studies. Our major results/conclusions are the following. (1) Quenching studies indicated no relevant interaction, while the other fluorescent model used suggested that each Heme site ligand strongly decreases the albumin binding of warfarin. (2) Ultrafiltration and ultracentrifugation studies demonstrated the complex modulation of warfarin–HSA interaction by the different Heme site markers; for example, bilirubin strongly decreased while methyl orange considerably increased the bound fraction of warfarin. (3) Fluorescence spectroscopic studies showed misleading results in these diligand–albumin interactions. (4) Different Heme site ligands can increase or decrease the albumin binding of warfarin and the outcome can even be concentration dependent (e.g., biliverdin and hemin).
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Matsuura, Kenji, Mieko Otani, Masaoki Takano, Keiichi Kadoyama, and Shogo Matsuyama. "Proteomic Analysis of Hippocampus and Cortex in Streptozotocin-Induced Diabetic Model Mice Showing Dementia." Journal of Diabetes Research 2018 (2018): 1–11. http://dx.doi.org/10.1155/2018/8953015.
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Aim. Diabetes with its associated hyperglycemia induces various type of peripheral damage and also impairs the central nervous system (CNS). This study is aimed at clarifying the precise mechanism of diabetes-induced dementia as an impairment of CNS. Methods. The proteomic analysis of the hippocampus and cortex in streptozotocin- (STZ-) treated mouse diabetic model showing dementia was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (n=3/group). Results. Significant changes in the expression of 32 proteins and 7 phosphoproteins were observed in the hippocampus and cortex. These identified proteins and phosphoproteins could be functionally classified as cytoskeletal protein, oxidoreductase, protein deubiquitination, energy metabolism, GTPase activation, heme binding, hydrolase, iron storage, neurotransmitter release, protease inhibitor, transcription, glycolysis, antiapoptosis, calcium ion binding, heme metabolic process, protein degradation, vesicular transport, and unknown in the hippocampus or cortex. Additionally, Western blotting validated the changes in translationally controlled tumor protein, ATP-specific succinyl-CoA synthetase beta subunit, and gamma-enolase isoform 1. Conclusions. These findings showed that STZ-induced diabetes changed the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that alterations in expression levels of these proteins play an important role in diabetes-induced dementia.
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De Simone, Giovanna, Paolo Ascenzi, Alessandra di Masi, and Fabio Polticelli. "Nitrophorins and nitrobindins: structure and function." Biomolecular Concepts 8, no. 2 (May 24, 2017): 105–18. http://dx.doi.org/10.1515/bmc-2017-0013.
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AbstractClassical all α-helical globins are present in all living organisms and are ordered in three lineages: (i) flavohemoglobins and single domain globins, (ii) protoglobins and globin coupled sensors and (iii) truncated hemoglobins, displaying the 3/3 or the 2/2 all α-helical fold. However, over the last two decades, all β-barrel and mixed α-helical-β-barrel heme-proteins displaying heme-based functional properties (e.g. ligand binding, transport and sensing) closely similar to those of all α-helical globins have been reported. Monomeric nitrophorins (NPs) and α1-microglobulin (α1-m), belonging to the lipocalin superfamily and nitrobindins (Nbs) represent prototypical heme-proteins displaying the all β-barrel and mixed α-helical-β-barrel folds. NPs are confined to the Reduviidae and Cimicidae families of Heteroptera, whereas α1-m and Nbs constitute heme-protein families spanning bacteria to Homo sapiens. The structural organization and the reactivity of the stable ferric solvent-exposed heme-Fe atom suggest that NPs and Nbs are devoted to NO transport, storage and sensing, whereas Hs-α1-m participates in heme metabolism. Here, the structural and functional properties of NPs and Nbs are reviewed in parallel with those of sperm whale myoglobin, which is generally taken as the prototype of monomeric globins.
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Nath, Karl A. "Heme, Iron, and the Kidney." Blood 116, no. 21 (November 19, 2010): SCI—26—SCI—26. http://dx.doi.org/10.1182/blood.v116.21.sci-26.sci-26.
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Abstract Abstract SCI-26 The kidney is often the target of injury by heme proteins and states of iron overload. Exogenous heme proteins, such as hemoglobin delivered to the kidney in hemolytic states, or myoglobin imposed upon the kidney following rhabdomyolysis, can injure the kidney via pathways that include vasoconstriction, direct cytotoxicity, and tubular cast formation. The former two pathways critically involve the heme prosthetic group: heme can bind vasodilator gases such as nitric oxide and carbon monoxide, while free heme can be cytotoxic because it is lipophilic, lipid membrane-transmissible, lipid membrane-destabilizing, prooxidant, and proinflammatory. Renal injury can also arise from endogenous heme proteins, such as cytochrome P450 enzymes, which can be destabilized by ischemic and nephrotoxic insults; such destabilization leads to the release of heme and incurs heme-dependent injury. The toxicity of heme may also reflect, at least in part, the release of iron and the attendant increase in labile cellular iron, the latter representing a potent catalyst for oxidative stress; increased levels of cellular iron may also originate from intracellular nonheme sources. Heme-iron can not only induce acute kidney injury, but also can provoke chronic kidney disease by virtue of its proinflammatory and profibrotic effects. Mechanisms that protect against heme-dependent and iron-dependent toxicity include heme oxygenase (HO), the rate-limiting enzyme in heme degradation, and increased synthesis of ferritin. Induction of HO-1, the inducible HO isoform, is protective against renal injury because of the following mechanisms: 1) the prevention of acute elevation in cellular heme concentrations otherwise incurred by cell injury; 2) the safe sequestration of iron in iron-binding proteins such as ferritin, or the cellular extrusion of iron by iron-exporting proteins; 3) the generation of antioxidant, anti-inflammatory metabolites such as bile pigments; and 4) the generation of carbon monoxide which is an antiapoptotic, anti-inflammatory, and vasorelaxant gas. Carbon monoxide can also be cytoprotective by binding cytochrome P450 enzymes, and thereby preventing their destabilization and the release of heme that subsequently occurs. In addition to these areas, this presentation discusses the pathobiologic and clinical significance of the siderophore-binding protein, NGAL (Neutrophil gelatinase-associated lipocalin): NGAL protects against renal ischemic injury through mechanisms that require the induction of HO-1; NGAL is increasingly utilized as a biomarker of acute kidney injury. The presentation concludes by discussing the use of iron supplementation in the treatment of anemia of chronic kidney disease and therapeutic strategies that may be designed from understanding endogenous and adaptive mechanisms that protect against the toxicity of heme-iron. Disclosures: No relevant conflicts of interest to declare.
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Szigeti, Andras, Szabolcs Bellyei, Balazs Gasz, Arpad Boronkai, Eniko Hocsak, Orsolya Minik, Zita Bognar, Gabor Varbiro, Balazs Sumegi, and Ferenc Gallyas. "Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL." FEBS Letters 580, no. 27 (November 7, 2006): 6447–54. http://dx.doi.org/10.1016/j.febslet.2006.10.067.
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Machado, Ednildo A., Pedro L. Oliveira, Monica F. Moreira, Wanderley de Souza, and Hatisaburo Masuda. "Uptake ofRhodnius heme-binding protein (RHBP) by the ovary ofRhodnius prolixus." Archives of Insect Biochemistry and Physiology 39, no. 4 (1998): 133–43. http://dx.doi.org/10.1002/(sici)1520-6327(1998)39:4<133::aid-arch1>3.0.co;2-d.
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Zhang, Ping, John D. Belcher, Julia Nguyen, Fuad Abdulla, and Gregory M. Vercellotti. "Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells." Blood 134, Supplement_1 (November 13, 2019): 208. http://dx.doi.org/10.1182/blood-2019-122912.
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Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences -- hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p<0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p<0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p<0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p<0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner. To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p<0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p<0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p<0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p<0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p<0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.
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Hayashi, Takashi, Hideaki Sato, Takashi Matsuo, Takaaki Matsuda, Yutaka Hitomi, and Yoshio Hisaeda. "Enhancement of enzymatic activity for myoglobins by modification of heme-propionate side chains." Journal of Porphyrins and Phthalocyanines 08, no. 03 (March 2004): 255–64. http://dx.doi.org/10.1142/s1088424604000246.
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The modification of myoglobin is an attractive process not only for understanding its molecular mechanism but also for engineering the protein function. The strategy of myoglobin functionalization can be divided into at least two approaches: site-directed mutagenesis and reconstitution with a non-natural prosthetic group. The former method enables us to mainly modulate the physiological function, while the latter has the advantage of introducing a new function on the protein. Particularly, replacement of the native hemin with an artificially created hemin having hydrophobic moieties at the terminal of the heme-propionate side chains serves as an appropriate substrate-binding site near the heme pocket, and consequently enhances the peroxidase and peroxygenase activities for the reconstituted myoglobin. In addition, the incorporation of the synthetic hemin bearing modified heme-propionates into an appropriate apomyoglobin mutant drastically enhances the peroxidase activity. In contrast, to convert myoglobin into a cytochrome P450 enzyme, a flavin moiety as an electron transfer mediator was introduced at the terminal of the heme-propionate side chain. The flavomyoglobin catalyzes the deformylation of 2-phenylpropanal in the presence of NADH under aerobic conditions through the peroxoanion formation from the oxygenated species. In addition, modification of the heme-propionate side chains has an significant influence on regulating the reactivity of the horseradish peroxidase. Furthermore, the heme-propionate side chain can form a metal binding site with a carboxylate residue in the heme pocket. These studies indicate that modification of the heme-propionate side chains can be a new and effective way to engineer functions for the hemoproteins.
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Zhou, Huchen, and John T. Groves. "Host-guest interactions of cyclodextrins and metalloporphyrins: supramolecular building blocks toward artificial heme proteins." Journal of Porphyrins and Phthalocyanines 08, no. 02 (February 2004): 125–40. http://dx.doi.org/10.1142/s108842460400012x.
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Cyclodextrins are versatile building blocks for a variety of macromolecules due to the inclusion complexes that are formed with hydrophobic organic molecules. Cyclodextrin-porphyrin interactions are of particular interest since cyclodextrins can serve as a non-covalent binding pocket while metalloporphyrins could serve as the heme analogs in the construction of heme protein model compounds. Various approaches to the design and assembly of biomimetic porphyrin constructs are compared and contrasted in this minireview with a particular emphasis on self-assembled and porphyrin-cyclodextrin systems. Several recent advances from our laboratories are described in this context. A sensitive fluorescent binding probe, 6A-N-dansyl-permethylated-β-cyclodextrin (Dan-NH-TMCD), was found to form 2:1 complexes with the meso-tetraphenylporphyrins Mn(III)TCPP , Mn(III)TPPS and Mn(III)TF 4 TMAP with high binding constants. A perPEGylated cyclodextrin, heptakis(2,3,6-tri-O-2-(2-(2-methoxyethoxy)ethoxy)ethyl)-β-cyclodextrin (TPCD), has been shown by 1 H NMR spectroscopy to form a 1:1 complex with H 2 TCPP with a binding constant above 108M-1. Such a strong binding constant is the largest found for a 1:1 complex between a monomeric cyclodextrin and a guest. TPCD was also found to bind Mn(III)TCPP with a binding constant of 1.2 × 106 M -1. A novel, self-assembled hemoprotein model, hemodextrin is also described. The molecular design is based on a PEGylated cyclodextrin scaffold that bears both a heme-binding pocket and an axial ligand that binds an iron porphyrin. The binding constant for Fe (III) TPPS (iron(III) meso-tetra(4-sulfonatophenyl)porphyrin) by py-PPCD was determined to be 2 × 106 M -1. The pyridyl nitrogen of py-PPCD was shown to ligate to the iron center by observing signal changes in the Fe(II) -porphyrin 1 H NMR spectrum. This hemodextrin ensemble, a minimalist myoglobin, was shown to bind dioxygen reversibly and to form a stable ferryl species.
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Nakagawa, Toshiyuki, and Kazunori Ohta. "Quercetin Regulates the Integrated Stress Response to Improve Memory." International Journal of Molecular Sciences 20, no. 11 (June 5, 2019): 2761. http://dx.doi.org/10.3390/ijms20112761.
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The initiation of protein synthesis is suppressed under several stress conditions, inducing phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2α), thereby inactivating the GTP-GDP recycling protein eIF2B. By contrast, the mammalian activating transcription factor 4 (ATF4, also known as cAMP response element binding protein 2 (CREB2)) is still translated under stress conditions. Four protein kinases (general control nonderepressible-2 (GCN2) kinase, double-stranded RNA-activated protein kinase (PKR), PKR-endoplasmic reticulum (ER)-related kinase (PERK), and heme-regulated inhibitor kinase (HRI)) phosphorylate eIF2α in the presence of stressors such as amino acid starvation, viral infection, ER stress, and heme deficiency. This signaling reaction is known as the integrated stress response (ISR). Here, we review ISR signaling in the brain in a mouse model of Alzheimer’s disease (AD). We propose that targeting ISR signaling with quercetin has therapeutic potential, because it suppresses amyloid-β (Aβ) production in vitro and prevents cognitive impairments in a mouse model of AD.
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Endo, R., H. Ishikawa, K. Iwai, I. Morishima, and K. Ishimori. "2P063 Spectroscopic Characterization of Heme Binding and Coordination Structure in Iron Regulatory Protein 2(IRP2)." Seibutsu Butsuri 44, supplement (2004): S125. http://dx.doi.org/10.2142/biophys.44.s125_3.
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Lok, Chun-Nam, and Prem Ponka. "Stimulation of Transferrin Receptor Expression by Enhanced Heme Biosynthesis in Murine Erythroleukemia Cells." Blood 104, no. 11 (November 16, 2004): 3200. http://dx.doi.org/10.1182/blood.v104.11.3200.3200.
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Abstract In erythroid cells iron uptake from transferrin (Tf) is utilized largely for heme synthesis. Here we provide evidence that Tf receptor (TfR) expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) resulted in an increase in TfR expression accompanied by enhanced uptake of iron from Tf and incorporation of iron into heme. ALA-mediated enhancement of TfR mRNA expression was completely prevented by succinylacetone, an inhibitor of ALA dehydratase, and N-methylprotoporphyrin, an inhibitor of ferrochelatase, indicating that the effect of ALA required its metabolism to heme. Treatment of cells with ALA was associated with enhanced iron regulatory protein-2 (IRP-2) binding activity, which could be blocked by inhibitors of heme synthesis and supplementation of the culture medium with a permeable iron chelate or Tf. In all cases, IRP-2 activities were correlated exactly with TfR mRNA levels. Thus, in addition to the previously characterized transcriptional up-regulation of TfR expression in differentiating erythroid cells, increased TfR expression mediated by enhanced heme biosynthesis may ensure sufficient iron availability for optimal heme synthesis and prevent possible protoporphyrin accumulation under conditions of inadequate iron supply.
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Ma, Jing, Xianfeng Zhang, Yanbin Feng, Hui Zhang, Xiaojun Wang, Yonghui Zheng, Wentao Qiao, and Xinqi Liu. "Structural and Functional Study of Apoptosis-linked Gene-2·Heme-binding Protein 2 Interactions in HIV-1 Production." Journal of Biological Chemistry 291, no. 52 (October 26, 2016): 26670–85. http://dx.doi.org/10.1074/jbc.m116.752444.
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Zager, Richard A., Ali C. M. Johnson, and Kirsten Frostad. "An evaluation of the antioxidant protein α1-microglobulin as a renal tubular cytoprotectant." American Journal of Physiology-Renal Physiology 311, no. 3 (September 1, 2016): F640—F651. http://dx.doi.org/10.1152/ajprenal.00264.2016.
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α1-Microglobulin (A1M) is a low-molecular-weight heme-binding antioxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought “proof of concept” in this regard. Cultured proximal tubule (HK-2) cells or isolated mouse proximal tubule segments were challenged with a variety of prooxidant insults: 1) hemin, 2) myoglobin; 3) “catalytic” iron, 4) H2O2/Fenton reagents, 5) a Ca2+ ionophore, 6) antimycin A, or 7) hypoxia (with or without rA1M treatment). HK-2 injury was gauged by the percent lactate dehydrogenase release and 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake. In vivo protection was sought in rA1M-treated mice subjected to 1) graded myohemoglobinura (2, 4, 8, or 9 ml/kg glycerol injection), 2) purified myoglobinemia/uria, or 3) endotoxemia. In vivo injury was assessed by blood urea nitrogen, creatinine, and the expression of redox-sensitive genes (heme oxygenase-1, neutrophil gelatinase-associated lipocalin, and monocyte chemoattractant protein-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder) or 10% serum induced equal protection. rA1M failed to mitigate any nonhemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (by Western blot analysis). Surprisingly, rA1M exerted select injury-promoting effects (increased in vitro catalytic iron/antimycin toxicities and increased in vivo monocyte chemoattractant protein-1/neutrophil gelatinase-associated lipocalin mRNA expression after glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g., albumin) binders.
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Muñoz-Sánchez, Jorge, and María Elena Chánez-Cárdenas. "A Review on Hemeoxygenase-2: Focus on Cellular Protection and Oxygen Response." Oxidative Medicine and Cellular Longevity 2014 (2014): 1–16. http://dx.doi.org/10.1155/2014/604981.
Full textAbstract:
Hemeoxygenase (HO) system is responsible for cellular heme degradation to biliverdin, iron, and carbon monoxide. Two isoforms have been reported to date. Homologous HO-1 and HO-2 are microsomal proteins with more than 45% residue identity, share a similar fold and catalyze the same reaction. However, important differences between isoforms also exist. HO-1 isoform has been extensively studied mainly by its ability to respond to cellular stresses such as hemin, nitric oxide donors, oxidative damage, hypoxia, hyperthermia, and heavy metals, between others. On the contrary, due to its apparently constitutive nature, HO-2 has been less studied. Nevertheless, its abundance in tissues such as testis, endothelial cells, and particularly in brain, has pointed the relevance of HO-2 function. HO-2 presents particular characteristics that made it a unique protein in the HO system. Since attractive results on HO-2 have been arisen in later years, we focused this review in the second isoform. We summarize information on gene description, protein structure, and catalytic activity of HO-2 and particular facts such as its cellular impact and activity regulation. Finally, we call attention on the role of HO-2 in oxygen sensing, discussing proposed hypothesis on heme binding motifs and redox/thiol switches that participate in oxygen sensing as well as evidences of HO-2 response to hypoxia.
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Sanchez, Mayka, Bruno Galy, Bjoern Schwanhaeusser, Jonathon Blake, Tomi Bähr-Ivacevic, Vladimir Benes, Matthias Selbach, Martina U. Muckenthaler, and Matthias W. Hentze. "Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins." Blood 118, no. 22 (November 24, 2011): e168-e179. http://dx.doi.org/10.1182/blood-2011-04-343541.
Full textAbstract:
Abstract Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.
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Noh, Seung-Jae, Y. Terry Lee, Colleen Byrnes, Antoinette Rabel, and Jeffery L. Miller. "Trafficking Kinesin Binding Protein Is Essential for Human Erythropoiesis." Blood 118, no. 21 (November 18, 2011): 683. http://dx.doi.org/10.1182/blood.v118.21.683.683.
Full textAbstract:
Abstract Abstract 683 Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that <1% of the CD36+ progenitors cells differentiated into glycophorin A erythroblasts compared with >90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis. Disclosures: No relevant conflicts of interest to declare.
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Trawick, J. D., N. Kraut, F. R. Simon, and R. O. Poyton. "Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements." Molecular and Cellular Biology 12, no. 5 (May 1992): 2302–14. http://dx.doi.org/10.1128/mcb.12.5.2302-2314.1992.
Full textAbstract:
Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.
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Trawick, J. D., N. Kraut, F. R. Simon, and R. O. Poyton. "Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements." Molecular and Cellular Biology 12, no. 5 (May 1992): 2302–14. http://dx.doi.org/10.1128/mcb.12.5.2302.
Full textAbstract:
Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.
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Zhou, Suiping, David Gell, Yi Kong, Jianqing Li, Joel P. Mackay, Mitchell J. Weiss, and Andres J. Gow. "Mechanisms of Alpha Hemoglobin Stabilizing Protein (AHSP) Actions." Blood 104, no. 11 (November 16, 2004): 499. http://dx.doi.org/10.1182/blood.v104.11.499.499.
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Abstract Alpha hemoglobin stabilizing protein (AHSP) is an abundant erythroid factor that specifically binds and stabilizes free α hemoglobin (Kihm et al., Nature 417:758, Gell et al., JBC, 277:40602). In mice, AHSP deficiency causes oxidative stress, mild anemia, and reticulocytosis. Furthermore, loss of AHSP exacerbates α globin precipitation and anemia in a murine model for β thalassemia. These data suggest that AHSP operates as a molecular chaperone to stabilize free α hemoglobin and inhibit its ability to catalyze redox chemistry. We investigated how AHSP associates with α hemoglobin and thereby reduces its oxidative capacity. By NMR and mutational analysis, we determined the structure of AHSP and defined its α globin-interacting surfaces. AHSP consists of three linked α helices. Key α globin-binding residues lie along helices 1 and 2 in a region that resembles the G and H helical fold of β globin. Hence, AHSP presents a surface resembling that of β hemoglobin and thus promotes the formation of a “pseudo α/β interface”. To investigate how formation of this complex affects α hemoglobin-mediated redox chemistry, we examined the ability of AHSP to inhibit peroxide-mediated oxidation of the redox-sensitive dye TMPD. Oxy-α hemoglobin alone catalyzed TMPD oxidation at a rate of 140 μMmin−1; preincubation with AHSP reduced this rate significantly, to 65 μMmin−1. Furthermore, AHSP reduced both heme loss from α hemoglobin and its ability to oxidize hemoglobin A. AHSP binding to oxy- α hemoglobin induced a spectral shift in the UV/visible range, suggesting that stabilization might be associated with structural alterations in the heme moiety. Specifically, the Soret peak was reduced and shifted leftward, while the 541 and 576 nm peaks of the visible region were reduced with concomitant rises at 500 nm and at the region above 580 nm. These alterations suggest that heme-bound iron is converted to a ferric (Fe3+) form that is liganded at all 6 coordinate positions, and therefore, can no longer catalyze redox chemistry. Consistent with this possibility, addition of KCN to the α hemoglobin-AHSP complex produced cyanomethemoglobin-AHSP, but at a slower rate than reaction of KCN with ferric-α hemoglobin alone. In addition, electron paramagnetic resonance and resonance raman spectroscopy showed that the heme iron exists in a low spin state within the α hemoglobin-AHSP complex. The oxidized complex was insensitive to pH alteration, indicating that iron was not bound by hydroxyl ion, which typically occurs at alkaline conditions. Together, our findings illustrate a potential mechanism whereby AHSP renders α hemoglobin chemically inert by inhibiting the reactivity of heme-bound iron. We propose that within the AHSP-α hemoglobin dimer, the oxidized heme is held within a bi-histidyl form, thus blocking its ability to act as a redox catalyst.
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Golonka, Rachel, Beng San Yeoh, and Matam Vijay-Kumar. "The Iron Tug-of-War between Bacterial Siderophores and Innate Immunity." Journal of Innate Immunity 11, no. 3 (2019): 249–62. http://dx.doi.org/10.1159/000494627.
Full textAbstract:
Iron is necessary for the survival of almost all aerobic organisms. In the mammalian host, iron is a required cofactor for the assembly of functional iron-sulfur (Fe-S) cluster proteins, heme-binding proteins and ribonucleotide reductases that regulate various functions, including heme synthesis, oxygen transport and DNA synthesis. However, the bioavailability of iron is low due to its insolubility under aerobic conditions. Moreover, the host coordinates a nutritional immune response to restrict the accessibility of iron against potential pathogens. To counter nutritional immunity, most commensal and pathogenic bacteria synthesize and secrete small iron chelators termed siderophores. Siderophores have potent affinity for iron, which allows them to seize the essential metal from the host iron-binding proteins. To safeguard against iron thievery, the host relies upon the innate immune protein, lipocalin 2 (Lcn2), which could sequester catecholate-type siderophores and thus impede bacterial growth. However, certain bacteria are capable of outmaneuvering the host by either producing “stealth” siderophores or by expressing competitive antagonists that bind Lcn2 in lieu of siderophores. In this review, we summarize the mechanisms underlying the complex iron tug-of-war between host and bacteria with an emphasis on how host innate immunity responds to siderophores.
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Chen, Jane-Jane. "Heme-Regulated eIF2α Kinase in Erythropoiesis and Oxidative Stress." Blood 118, no. 21 (November 18, 2011): SCI—23—SCI—23. http://dx.doi.org/10.1182/blood.v118.21.sci-23.sci-23.
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Abstract SCI-23 Besides serving as a prosthetic group for hemoglobin, heme also regulates translation by inhibiting the kinase activity of heme-regulated translational inhibitor (HRI). HRI is the only known protein kinase that senses the intracellular heme concentration and does so through its two heme-binding domains. HRI is activated in heme deficiency by autophosphorylation, and phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), which impairs the recycling of eIF2 for further translational initiation and results in cessation of protein synthesis. In this manner, HRI serves as a feedback inhibitor and coordinates the translation of globin mRNAs with the intracellular heme concentration to ensure that globin proteins are not made in excess of the heme available for the formation of hemoglobin. Excess of globin chains or heme is cytotoxic. In the absence of HRI, uncontrolled protein synthesis in heme deficiency results in globin aggregation and precipitation in red blood cells and their precursors. HRI is responsible for the physiological adaptation that produces hypochromic, microcytic erythrocytes in iron deficiency. The expression of HRI was increased during late stages of erythropoiesis with higher expression in Ter119high erythroblasts and reticulocytes, correlating with the active synthesis and regulation of globins at these stages of erythroid differentiation. In addition to inhibiting global protein synthesis, the second important function of eIF2α phosphorylation is to reprogram translation and the subsequent transcription of genes required for stress response. In mammalian cells, translation of the transcription factor ATF4 mRNA is upregulated specifically by eIF2α phosphorylation via upstream open reading frames in the 5’UTR. Activation of the HRI-ATF4 stress response pathway in nucleated erythroid precursors is required for adaptation to acute and chronic oxidative stress. Furthermore, this HRI-dependent ATF4 pathway is also operative and necessary for erythroid differentiation, especially under stress conditions. In chronic iron deficiency, HRI is necessary for adaptive gene expression for erythroid differentiation as well as for iron heme and redox homeostasis. Beyond heme deficiency and oxidative stress, HRI is also activated by osmotic shock and heat shock. HRI deficiency in mice exacerbates erythropoietic protoporphyria and renders β-thalassemia intermedia embryonically lethal. Hri−/− mice also develop ineffective erythropoiesis during iron/heme deficiency and are severely compromised upon phenylhydrazine-induced acute hemolytic anemia. Thus, translational regulation by HRI plays a critical role in the manifestation of red cell diseases in mice and may be a significant modifier of such diseases in humans. Disclosures: No relevant conflicts of interest to declare.
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Pajarillo, Edward, Asha Rizor, Deok-Soo Son, Michael Aschner, and Eunsook Lee. "The transcription factor REST up-regulates tyrosine hydroxylase and antiapoptotic genes and protects dopaminergic neurons against manganese toxicity." Journal of Biological Chemistry 295, no. 10 (January 30, 2020): 3040–54. http://dx.doi.org/10.1074/jbc.ra119.011446.
Full textAbstract:
Dopaminergic functions are important for various biological activities, and their impairment leads to neurodegeneration, a hallmark of Parkinson's disease (PD). Chronic manganese (Mn) exposure causes the neurological disorder manganism, presenting symptoms similar to those of PD. Emerging evidence has linked the transcription factor RE1-silencing transcription factor (REST) to PD and also Alzheimer's disease. But REST's role in dopaminergic neurons is unclear. Here, we investigated whether REST protects dopaminergic neurons against Mn-induced toxicity and enhances expression of the dopamine-synthesizing enzyme tyrosine hydroxylase (TH). We report that REST binds to RE1 consensus sites in the TH gene promoter, stimulates TH transcription, and increases TH mRNA and protein levels in dopaminergic cells. REST binding to the TH promoter recruited the epigenetic modifier cAMP-response element-binding protein–binding protein/p300 and thereby up-regulated TH expression. REST relieved Mn-induced repression of TH promoter activity, mRNA, and protein levels and also reduced Mn-induced oxidative stress, inflammation, and apoptosis in dopaminergic neurons. REST reduced Mn-induced proinflammatory cytokines, including tumor necrosis factor α, interleukin 1β (IL-1β), IL-6, and interferon γ. Moreover, REST inhibited the Mn-induced proapoptotic proteins Bcl-2–associated X protein (Bax) and death-associated protein 6 (Daxx) and attenuated an Mn-induced decrease in the antiapoptotic proteins Bcl-2 and Bcl-xL. REST also enhanced the expression of antioxidant proteins, including catalase, NF-E2–related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). Our findings indicate that REST activates TH expression and thereby protects neurons against Mn-induced toxicity and neurological disorders associated with dopaminergic neurodegeneration.
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Mikasa, Taisuke, Masami Kugo, Seigo Nishimura, Sigeru Taketani, Sumio Ishijima, and Ikuko Sagami. "Thermodynamic Characterization of the Ca2+-Dependent Interaction Between SOUL and ALG-2." International Journal of Molecular Sciences 19, no. 12 (November 29, 2018): 3802. http://dx.doi.org/10.3390/ijms19123802.
Full textAbstract:
SOUL, a heme-binding protein-2 (HEBP-2), interacts with apoptosis-linked gene 2 protein (ALG-2) in a Ca2+-dependent manner. To investigate the properties of the interaction of SOUL with ALG-2, we generated several mutants of SOUL and ALG-2 and analyzed the recombinant proteins using pulldown assay and isothermal titration calorimetry. The interaction between SOUL and ALG-2 (delta3-23ALG-2) was an exothermic reaction, with 1:1 stoichiometry and high affinity (Kd = 32.4 nM) in the presence of Ca2+. The heat capacity change (ΔCp) of the reaction showed a large negative value (−390 cal/K·mol), which suggested the burial of a significant nonpolar surface area or disruption of a hydrogen bond network that was induced by the interaction (or both). One-point mutation of SOUL Phe100 or ALG-2 Trp57 resulted in complete loss of heat change, supporting the essential roles of these residues for the interaction. Nevertheless, a truncated mutant of SOUL1-143 that deleted the domain required for the interaction with ALG-2 Trp57 still showed 1:1 binding to ALG-2 with an endothermic reaction. These results provide a better understanding of the target recognition mechanism and conformational change of SOUL in the interaction with ALG-2.
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Jones, Richard C., Joanna Deck, Ricky D. Edmondson, and Mark E. Hart. "Relative Quantitative Comparisons of the Extracellular Protein Profiles of Staphylococcus aureus UAMS-1 and Its sarA, agr, and sarA agr Regulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry." Journal of Bacteriology 190, no. 15 (June 6, 2008): 5265–78. http://dx.doi.org/10.1128/jb.00383-08.
Full textAbstract:
ABSTRACT One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.