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Ann Smith - One of the best experts on this subject based on the ideXlab platform.
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Identification of oxidative modifications of Hemopexin and their predicted physiological relevance.
Journal of Biological Chemistry, 2017Co-Authors: Peter Hahl, Rachel Hunt, Edward S. Bjes, Andrew Skaff, J. Andrew Keightley, Ann SmithAbstract:Abstract Hemopexin protects against heme toxicity in hemolytic diseases and conditions, sepsis and sickle cell disease. This protection is sustained by heme-Hemopexin complexes present in biological fluids that resist oxidative damage during heme-driven inflammation. However, apo-Hemopexin is vulnerable to inactivation by reactive nitrogen and oxygen species that covalently modify amino acids. The consequent nitration of amino acids is considered a selective and specific effect reflecting biological events. Using liquid chromatography-tandem mass spectrometry, we discovered low endogenous levels of tyrosine nitration in the peptide YYCFQGNQFLR of human Hemopexin, which was similarly nitrated in rabbit and rat Hemopexins. Immuno-blotting and selective reaction monitoring were used to quantitate tyrosine nitration in these in vivo samples and when Hemopexin was incubated in vitro with a nitrating system of nitrite/myeloperoxidase/glucose oxidase. Significantly, heme binding by Hemopexin declined as tyrosine nitration proceeded in vitro. Three nitrated tyrosines reside in the heme binding site of heme-Hemopexin and one, Tyr199, in YY199CFQGNQFLR interacts directly with the heme ring D propionate. We also investigated the oxidative modifications of amino acids in Hemopexin after incubation with tert-butyl hydroperoxide and hypochlorous acid in vitro. We identified additional covalent oxidative modifications on four tyrosine residues and one tryptophan residue of Hemopexin. Importantly, three of the four modified tyrosines, some of which having more than one modification, resided closely together in the heme-binding site, supporting a hierarchy of amino acids vulnerable to oxidative damage. Based on these in vivo and in vitro data, we propose that during inflammation nitration and oxidative modification of apo-Hemopexin occurs in niches of the body where there are activated immune and endothelial cells that generate RNS and ROS, with the potential to impair the protective extracellular anti-oxidant function of Hemopexin.
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Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis
Molecular Medicine, 2016Co-Authors: Feng Huang, Tjitske S R Engelen, Sujatha R Thundivalappil, Frank E Riley, Alexander L Watters, Nathan Brinkman, Michael Super, Donald E. Ingber, Ann Smith, H Shaw WarrenAbstract:Infusion of the heme-binding protein Hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma Hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-Hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived Hemopexin and recombinant Fc-Hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between Hemopexins. Recombinant Fc-Hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any Hemopexin preparation on chemotaxis, and purified Hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and Hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that Hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.
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mechanisms of neuroprotection by Hemopexin modeling the control of heme and iron homeostasis in brain neurons in inflammatory states
Journal of Neurochemistry, 2013Co-Authors: Peter Hahl, Taron Davis, Cecilia Washburn, Jack T Rogers, Ann SmithAbstract:Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase-1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme-Hemopexin complexes, and physiologically relevant ROS, for example, H(2)O(2) and HOCl, to provide novel insights into the underlying mechanism whereby Hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme-Hemopexin endocytosis by iron from heme catabolism via the iron-regulatory element of hAPP mRNA. Heme-Hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert-butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H(2)O(2) in vitro. Apo-Hemopexin, which predominates in non-hemolytic states, resists damage by H(2)O(2) and HOCl, except for the highest concentrations likely in vivo. Heme-albumin and albumin are preferential targets for ROS; thus, albumin protects Hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that Hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.
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An alternative view of the proposed alternative activities of Hemopexin
Protein Science, 2011Co-Authors: Marcia R. Mauk, Ann Smith, A. Grant MaukAbstract:Hemopexin is a plasma protein that plays a well-established biological role in sequestering heme that is released into the plasma from hemoglobin and myoglobin as the result of intravascular or extravascular hemolysis as well as from skeletal muscle trauma or neuromuscular disease. In recent years, a variety of additional biological activities have been attributed to Hemopexin, for example, hyaluronidase activity, serine protease activity, pro-inflammatory and anti-inflammatory activity as well as suppression of lymphocyte necrosis, inhibition of cellular adhesion, and binding of divalent metal ions. This review examines the challenges involved in the purification of Hemopexin from plasma and in the recombinant expression of Hemopexin and evaluates the questions that these challenges and the characteristics of Hemopexin raise concerning the validity of many of the new activities proposed for this protein. As well, an homology model of the three-dimensional structure of human Hemopexin is used to reveal that the protein lacks the catalytic triad that is characteristic of many serine proteases but that Hemopexin possesses two highly exposed Arg-Gly-Glu sequences that may promote interaction with cell surfaces.
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Effects of Reduction and Ligation of Heme Iron on the Thermal Stability of Heme-Hemopexin Complexes
Journal of Protein Chemistry, 2001Co-Authors: Natalia V. Shipulina, Ann Smith, William T. MorganAbstract:Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (Kd < 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on Hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-Hemopexin unfolds with a Tm of 52°C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (Tm 63°C). Bis-histidyl heme coordination by Hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-Hemopexin (Tm 55.5°C) to the ferro-heme form (Tm 48°C), and NaCl stabilizes both complexes by 10-15°C. CO binding to ferro-heme-Hemopexin restores complex stability (Tm 67°C). Upon cooling, unfolded apo- and ferri-heme-Hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the Tm of heme complexes by 5-12°C. The stability of the apo-N-terminal domain (Tm 53°C) is similar to that of intact Hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have Tm values of 53°C, 33°C, and 75°C, respectively.
Nathan Brinkman - One of the best experts on this subject based on the ideXlab platform.
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Prevention of Heme-Induced Human Endothelial Cell Activation By Hemopexin in Vitro
Blood, 2020Co-Authors: Jacqueline Adam, Nathan Brinkman, Thomas Gentinetta, Svetlana Diditchenko, Alexander Schaub, Gregory J Kato, Adrian ZuercherAbstract:Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein Hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to Hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of Hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different Hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with Hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of Hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of Hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, Hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:Hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that Hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and Hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, Hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of Hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, Hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of Hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of Hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of Hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.
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haptoglobin and Hemopexin inhibit vaso occlusion and inflammation in murine sickle cell disease role of heme oxygenase 1 induction
PLOS ONE, 2018Co-Authors: John D Belcher, Chunsheng Chen, Julia Nguyen, Fuad Abdulla, Ping Zhang, Hao Nguyen, Phong Nguyen, Trevor Killeen, Sylvia Miescher, Nathan BrinkmanAbstract:During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and Hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and Hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and Hemopexin supplementation. A single infusion of haptoglobin or Hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or Hemopexin. Haptoglobin and Hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and Hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and Hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.
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haptoglobin and Hemopexin infusion efficiently activates the nrf2 ho 1 axis and inhibits inflammation and vaso occlusion in murine sickle cell disease
Blood, 2016Co-Authors: John D Belcher, Nathan Brinkman, Chunsheng Chen, Julia Nguyen, Fuad Abdulla, Ping Zhang, Hao Nguyen, Phong Nguyen, Gregory M VercellottiAbstract:Free hemoglobin and hemin, released by red blood cells during intravascular hemolysis, promote vasculopathy, inflammation, thrombosis, and renal injury. Plasma haptoglobin and Hemopexin tightly bind free hemoglobin and hemin, respectively, thwarting these clinical sequelae. In sickle cell disease (SCD), chronic hemolysis can deplete plasma haptoglobin and Hemopexin in humans and mice. To explore mechanisms mediating this protection and provide a basis for supplementation in SCD patients, dorsal skin fold chambers were implanted onto Townes-SS mice and microvascular stasis (% non-flowing venules) was measured in response to a hemoglobin challenge. Human haptoglobin, Hemopexin, or albumin was co-infused with hemoglobin or 1 hour after hemoglobin at equimolar concentrations. Sickle mice co-infused with hemoglobin/haptoglobin, hemoglobin/Hemopexin or hemoglobin/haptoglobin/Hemopexin had less stasis 1 to 4 hours after infusion, compared to albumin- and saline-treated mice (*p Disclosures Belcher:CSL-Behring: Research Funding; Imara: Research Funding. Chen:Imara: Research Funding. Brinkman:CSL-Behring: Employment. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.
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Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis
Molecular Medicine, 2016Co-Authors: Feng Huang, Tjitske S R Engelen, Sujatha R Thundivalappil, Frank E Riley, Alexander L Watters, Nathan Brinkman, Michael Super, Donald E. Ingber, Ann Smith, H Shaw WarrenAbstract:Infusion of the heme-binding protein Hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma Hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-Hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived Hemopexin and recombinant Fc-Hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between Hemopexins. Recombinant Fc-Hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any Hemopexin preparation on chemotaxis, and purified Hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and Hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that Hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.
John D Belcher - One of the best experts on this subject based on the ideXlab platform.
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haptoglobin and Hemopexin inhibit vaso occlusion and inflammation in murine sickle cell disease role of heme oxygenase 1 induction
PLOS ONE, 2018Co-Authors: John D Belcher, Chunsheng Chen, Julia Nguyen, Fuad Abdulla, Ping Zhang, Hao Nguyen, Phong Nguyen, Trevor Killeen, Sylvia Miescher, Nathan BrinkmanAbstract:During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and Hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and Hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and Hemopexin supplementation. A single infusion of haptoglobin or Hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or Hemopexin. Haptoglobin and Hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and Hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and Hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.
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haptoglobin and Hemopexin infusion efficiently activates the nrf2 ho 1 axis and inhibits inflammation and vaso occlusion in murine sickle cell disease
Blood, 2016Co-Authors: John D Belcher, Nathan Brinkman, Chunsheng Chen, Julia Nguyen, Fuad Abdulla, Ping Zhang, Hao Nguyen, Phong Nguyen, Gregory M VercellottiAbstract:Free hemoglobin and hemin, released by red blood cells during intravascular hemolysis, promote vasculopathy, inflammation, thrombosis, and renal injury. Plasma haptoglobin and Hemopexin tightly bind free hemoglobin and hemin, respectively, thwarting these clinical sequelae. In sickle cell disease (SCD), chronic hemolysis can deplete plasma haptoglobin and Hemopexin in humans and mice. To explore mechanisms mediating this protection and provide a basis for supplementation in SCD patients, dorsal skin fold chambers were implanted onto Townes-SS mice and microvascular stasis (% non-flowing venules) was measured in response to a hemoglobin challenge. Human haptoglobin, Hemopexin, or albumin was co-infused with hemoglobin or 1 hour after hemoglobin at equimolar concentrations. Sickle mice co-infused with hemoglobin/haptoglobin, hemoglobin/Hemopexin or hemoglobin/haptoglobin/Hemopexin had less stasis 1 to 4 hours after infusion, compared to albumin- and saline-treated mice (*p Disclosures Belcher:CSL-Behring: Research Funding; Imara: Research Funding. Chen:Imara: Research Funding. Brinkman:CSL-Behring: Employment. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.
A. Smith - One of the best experts on this subject based on the ideXlab platform.
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Safe coordinated trafficking of heme and iron with copper maintain cell homeostasis: modules from the Hemopexin system
BioMetals, 2019Co-Authors: Roberto M Vanacore, Jeffrey D. Eskew, L. Sung, T. Davis, A. SmithAbstract:Studies with patients, animal models of human disease and Hemopexin null mice have shown that the heme-binding protein Hemopexin is vital for the protection of a variety of cell types and tissues against heme toxicity. The presence of Hemopexin in all biological fluids examined to date indicates wide roles in abrogating heme toxicity in human tissues; and, thus, is clinically relevant. Heme-Hemopexin endocytosis leads to coordinated trafficking of heme, iron and copper as heme traffics from endosomes to heme oxygenases (HOs) in the smooth endoplasmic reticulum and to the nucleus. This is safe redox-metal trafficking, without oxidative stress, as iron released from heme catabolism by HOs as well as copper taken up with heme-Hemopexin move through the cell. To our knowledge, this coordinated metal trafficking has been described only for the Hemopexin system and differs from the cell’s response to non-protein bound heme, which can be toxic. We propose that defining how cells respond to heme-Hemopexin endocytosis, a natural cytoprotective system, will aid our understanding of how cells adapt as they safely respond to increases in heme, Fe(II) and copper. This is relevant for many genetic hemolytic diseases and conditions, stroke and hemorrhage as well as neurodegeneration. Such analyses will help to define a pattern of events that can be utilized to characterize how dysfunctional redox and transition metal handling is linked to the development of pathology in disease states such as Alzheimer’s disease when metal homeostasis is not restored; and potentially provide novel targets and approaches to improve therapies.
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Affinity, conservation, and surface exposure of Hemopexin-binding proteins in Haemophilus influenzae.
Infection and immunity, 1995Co-Authors: J. C. Y. Wong, A. Smith, R. Patel, D. Kendall, P. W. Whitby, J Holland, Paul WilliamsAbstract:Haemophilus influenzae can acquire heme from Hemopexin for use as a source of both essential porphyrin and iron. In classical ligand-binding studies, we observed time-dependent, saturable, and displaceable binding of human 125I-labelled Hemopexin to intact cells of H. influenzae type b (Hib) strain 760705 grown in an iron-restricted medium. From these experiments, which demonstrate that Hemopexin associates with a single class of binding site, the affinities (Kds) and receptor numbers were calculated for heme-Hemopexin (Kd, 205 nM; 3,200 receptors per cell) and apoHemopexin (Kd, 392 nM; 4,400 receptors per cell). Thus, Hib expresses a specific Hemopexin receptor which shows some preference for the heme-protein complex. Affinity chromatography on Hemopexin-Sepharose 4B of detergent-solubilized membranes from Hib strain 760705 results in the copurification of three proteins with molecular masses of 57, 38, and 29 kDa. Trypsinization of whole cells of Hib 760705 abolishes Hemopexin binding and correlates with the disappearance of the 57-kDa Hemopexin-binding protein and appearance of a 52-kDa species which does not bind either Hemopexin in ligand blot assays or a monoclonal antibody (MAbT11-30) raised against the 57-kDa protein. From immunoblotting assays and NH2-terminal amino acid sequence analysis, the 38-kDa protein isolated following Hemopexin affinity chromatography was identified as the porin protein P2. These data, taken together with the receptor-binding studies which support a single class of Hemopexin-binding site, suggest that P2 and the 29-kDa protein function as accessory proteins to the 57-kDa Hemopexin-binding protein to facilitate the uptake of heme from receptor-bound Hemopexin. To determine whether Hemopexin binding and the 57-kDa protein are conserved in Haemophilus strains, whole-cell dot blots and immunoblots of the outer membrane proteins prepared from strains belonging to each of 21 different Hib outer membrane protein subtypes, six nontypeable strains, and five Haemophilus parainfluenzae strains were probed with either Hemopexin or MAbT11-30. Only the H. parainfluenzae strains which lack the 57-kDa protein do not bind Hemopexin. Since H. influenzae has also been shown to produce a soluble 100-kDa Hemopexin-binding protein, cell-free culture supernatants were also examined for the presence of this protein. Apart from Hib 760705 and H. parainfluenzae, the 100-kDa Hemopexin-binding protein was detected in all the other Haemophilus strains. The abilities of Hib 760705 to both bind and acquire heme from Hemopexin without expressing a 100-kDa soluble Hemopexin-binding protein show that in strain 760705, this 100-kDa protein is not essential for the utilization of heme from Hemopexin.
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Identification and characterization of an iron-regulated Hemopexin receptor in Haemophilus influenzae type b.
Infection and Immunity, 1994Co-Authors: J. C. Y. Wong, A. Smith, J Holland, T. Parsons, Paul WilliamsAbstract:Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to Hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-Hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from Hemopexin for use as an iron source. The ability of human and rabbit Hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated Hemopexin as ligand, H. influenzae bound heme-Hemopexin and apo-Hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-Hemopexin and apo-Hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of Hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for Hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa Hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated Hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-Hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound Hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa Hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa Hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two Hemopexin receptors, the expression of which is determined by the prevailing growth environment.
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Heme-Hemopexin-mediated induction of metallothionein gene expression.
The Journal of biological chemistry, 1992Co-Authors: J Alam, A. SmithAbstract:Abstract Hemopexin-mediated heme transport into mouse hepatoma (Hepa) cells and human promyelocytic (HL-60) cells stimulates the expression of heme oxygenase via transcriptional activation (Alam, J., and Smith, A. (1989) J. Biol. Chem. 264, 17637-17640). Incubation of both these cell types in serum-free medium containing heme-Hemopexin is shown here also to increase the steady-state level of metallothionein (MT) mRNA in a time- and dose-dependent manner. Heme-Hemopexin is a far more effective inducer (12-fold) of the MT isozyme 1 (MT-1) in Hepa cells than nonprotein-bound heme (4-fold). ApoHemopexin has no effect on MT-1 expression, and incubation with heme-Hemopexin of mouse L fibroblasts that lack Hemopexin receptors does not affect MT-1 expression. Thus, an interaction between the heme-Hemopexin complex and its receptor is necessary for increased accumulation of MT-1 transcripts. In vitro nuclear "run-on" analysis indicates that the heme-Hemopexin-mediated accumulation of MT-1 mRNA is regulated primarily at the level of initiation of transcription. A highly labile protein is required for constitutive MT-1 gene expression and acts to repress transcription. Transcriptional activation by heme or metals may require decreased concentrations or inactivation of the repressor as well as an additional inducer-specific trans-acting factor. Inhibition of protein synthesis augments the heme-Hemopexin-mediated accumulation of MT-1 mRNA. Activation of heme oxygenase (HO) gene transcription by heme requires the synthesis of one (or more) heme-inducible proteins that are labile or become labile upon cycloheximide-sensitive processing or activation. Our comparison of MT and HO points to significant differences in the mechanisms of gene regulation by heme. The concomitant regulation of gene expression of MT-1 and HO in response to heme-Hemopexin appears to be a concerted adaptive response of the cells, mediated at the level of the plasma membrane Hemopexin receptor, and may relate to the proposed role of MT as an intracellular antioxidant or to a need to sequester zinc which otherwise would compete with iron and occupy sites on regulatory proteins such as the iron-responsive elements.
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Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems
European journal of cell biology, 1990Co-Authors: A. Smith, R. C. HuntAbstract:Receptor-mediated transport of heme by Hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apoHemopexin recycles from cells. However, until now, the intracellular transport of Hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that Hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, Hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that Hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]Hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]Hemopexin showed the presence of Hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of Hemopexin. Colloidal gold-conjugated Hemopexin and electron microscopy showed that Hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, Hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of Hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated Hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-Hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, Hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.
Jeffrey D. Eskew - One of the best experts on this subject based on the ideXlab platform.
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Safe coordinated trafficking of heme and iron with copper maintain cell homeostasis: modules from the Hemopexin system
BioMetals, 2019Co-Authors: Roberto M Vanacore, Jeffrey D. Eskew, L. Sung, T. Davis, A. SmithAbstract:Studies with patients, animal models of human disease and Hemopexin null mice have shown that the heme-binding protein Hemopexin is vital for the protection of a variety of cell types and tissues against heme toxicity. The presence of Hemopexin in all biological fluids examined to date indicates wide roles in abrogating heme toxicity in human tissues; and, thus, is clinically relevant. Heme-Hemopexin endocytosis leads to coordinated trafficking of heme, iron and copper as heme traffics from endosomes to heme oxygenases (HOs) in the smooth endoplasmic reticulum and to the nucleus. This is safe redox-metal trafficking, without oxidative stress, as iron released from heme catabolism by HOs as well as copper taken up with heme-Hemopexin move through the cell. To our knowledge, this coordinated metal trafficking has been described only for the Hemopexin system and differs from the cell’s response to non-protein bound heme, which can be toxic. We propose that defining how cells respond to heme-Hemopexin endocytosis, a natural cytoprotective system, will aid our understanding of how cells adapt as they safely respond to increases in heme, Fe(II) and copper. This is relevant for many genetic hemolytic diseases and conditions, stroke and hemorrhage as well as neurodegeneration. Such analyses will help to define a pattern of events that can be utilized to characterize how dysfunctional redox and transition metal handling is linked to the development of pathology in disease states such as Alzheimer’s disease when metal homeostasis is not restored; and potentially provide novel targets and approaches to improve therapies.
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Role for Copper in Transient Oxidation and Nuclear Translocation of MTF-1, but Not of NF-κB, by the Heme–Hemopexin Transport System
Antioxidants & Redox Signaling, 2000Co-Authors: Roberto M Vanacore, Jeffrey D. Eskew, Pedro J. Morales, Lokman Sung, Ann SmithAbstract:ABSTRACT Heme–Hemopexin (2–10 μM) is used as a model for intravenous heme released in trauma, stroke, and ischemia-reperfusion. A transient increase in cellular protein oxidation occurs during receptor-mediated heme transport from Hemopexin which is inhibited by the nonpermeable Cu(I) chelator, bathocuproinedisulfonate. Thus, participation of surface redox process involving Cu(I) generation are proposed to be linked to the induction of the protective proteins heme oxygenase-1 (HO-1) and metallothionein-1 (MT-1) by heme–Hemopexin. The region (–153 to –42) in the proximal promoter of the mouse MT-1 gene responds to heme– and CoPP–Hemopexin in transient transfection assays and contains metal-responsive elements for MTF-1 and an antioxidant-responsive element (ARE) overlapping a GC-rich E-box to which USF-1 and -2 bind. No decreases in DNA binding of the diamide-oxidation sensitive USF-1 and -2 occur upon exposure of cells to heme–Hemopexin. MTF-1 and the ARE-binding proteins are relatively resistant to diami...
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cellular protection mechanisms against extracellular heme heme Hemopexin but not free heme activates the n terminal c jun kinase
Journal of Biological Chemistry, 1999Co-Authors: Jeffrey D. Eskew, Pedro J. Morales, Lokman Sung, Roberto M Vanacore, Ann SmithAbstract:Abstract Hemopexin protects cells lacking Hemopexin receptors by tightly binding heme abrogating its deleterious effects and preventing nonspecific heme uptake, whereas cells with Hemopexin receptors undergo a series of cellular events upon encountering heme-Hemopexin. The biochemical responses to heme-Hemopexin depend on its extracellular concentration and range from stimulation of cell growth at low levels to cell survival at otherwise toxic levels of heme. High (2–10 μm) but not low (0.01–1 μm) concentrations of heme-Hemopexin increase, albeit transiently, the protein carbonyl content of mouse hepatoma (Hepa) cells. This is due to events associated with heme transport since cobalt-protoporphyrin IX-Hemopexin, which binds to the receptor and activates signaling pathways without tetrapyrrole transport, does not increase carbonyl content. The N-terminal c-Jun kinase (JNK) is rapidly activated by 2–10 μm heme-Hemopexin, yet the increased intracellular heme levels are neither toxic nor apoptotic. After 24 h exposure to 10 μm heme-Hemopexin, Hepa cells become refractory to the growth stimulation seen with 0.1–0.75 μm heme-Hemopexin but HO-1 remains responsive to induction by heme-Hemopexin. Since free heme does not induce JNK, the signaling events, like phosphorylation of c-Jun via activation of JNK as well as the nuclear translocation of NFκB, G2/M arrest, and increased expression of p53 and of the cell cycle inhibitor p21WAF1/CIP1/SDI1 generated by heme-Hemopexin appear to be of paramount importance in cellular protection by heme-Hemopexin.
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Role of heme-Hemopexin in human T-lymphocyte proliferation.
Experimental Cell Research, 1997Co-Authors: Ann Smith, Jeffrey D. Eskew, Corina M. Borza, Michael Pendrak, Richard C. HuntAbstract:Abstract Heme–Hemopexin supports and stimulates proliferation of human acute T-lymphoblastic (MOLT-3) cells, suggesting the participation of heme in cell growth and division. MOLT-3 cells express approximately 58,000 Hemopexin receptors per cell (apparent K d 20 n M ), of which about 20% are on the cell surface. Binding is dose- and temperature-dependent, and growth in serum-free IMDM medium is stimulated by 100–1000 n M heme–Hemopexin, consistent with the high affinity of the receptor for Hemopexin, and maximal growth is seen in response to 500 n M complex. Growth was similar in defined minimal medium supplemented with either low concentrations of heme–Hemopexin or iron-transferrin, and either of these complexes were about 80% as effective as a serum supplement. Heme–Hemopexin, but not apo–Hemopexin, reversed the growth inhibition caused by desferrioxamine showing that heme–iron derived from heme catabolism is used for cell growth. Cobalt-protoporphyrin (CoPP)–Hemopexin, which binds to the receptor but is not transported intracellularly [Smith et al., (1993) J. Biol. Chem. 268, 7365], also stimulated cell proliferation in serum-free IMDM but did not “rescue” the cells from desferrioxamine. Furthermore, CoPP–Hemopexin effectively competed for the Hemopexin receptor with heme–Hemopexin and diminished its growth stimulatory effects. In addition, protein kinase C (PKC) is translocated to the plasma membrane within 5 min after heme–Hemopexin is added to the medium, reaches maximum activity within 5–10 min, and declines to unstimulated levels by 30 min. Heme–Hemopexin and CoPP-Hemopexin both augmented MOLT-3 cell growth stimulated by serum. Thus, heme–Hemopexin not only functions as an iron source for T-cells but occupancy of the Hemopexin receptor itself triggers signaling pathway(s) involved in the regulation of cell growth. The stimulation of growth of human T-lymphocytes by heme–Hemopexin is likely to be a physiologically relevant mechanism at sites of injury, infection, and inflammation.