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Iqbal Hamza - One of the best experts on this subject based on the ideXlab platform.
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regulation of intracellular Heme trafficking revealed by subcellular reporters
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Xiaojing Yuan, Amit R Reddi, Iqbal Hamza, Nicole Rietzschel, Hanna Kwon, Ana Beatriz Walter Nuno, David A Hanna, Emma Lloyd RavenAbstract:Abstract Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether Heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous Heme contribute to cellular labile Heme and that extracellular Heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile Heme. Restoration of reporter activity by Heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous Heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental Heme influences labile Heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest Heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous Heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on Heme trafficking in cellular and whole-animal models.
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Heme uptake by leishmania amazonensis is mediated by the transmembrane protein lhr1
PLOS Pathogens, 2012Co-Authors: Iqbal Hamza, Xiaojing Yuan, Danilo C Miguel, Norma W Andrews, Rebecca L Renberg, Olga Protchenko, Caroline C Philpott, Chau HuynhAbstract:Trypanosomatid protozoan parasites lack a functional Heme biosynthetic pathway, so must acquire Heme from the environment to survive. However, the molecular pathway responsible for Heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane Heme transporter HRG-4, functions in Heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent Heme analog ZnMP, and increased the total intracellular Heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular Heme pool by approximately 50%, indicating that LHR1 is a major Heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import Heme from the environment, and rescued growth of a strain deficient in Heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.
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Lack of Heme synthesis in a free-living eukaryote.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Anita U. Rao, Lynn K. Carta, Iqbal Hamza, Emmanuel LesuisseAbstract:In most free-living eukaryotes studied thus far, Heme is synthesized from a series of intermediates through a well defined evolutionarily conserved pathway. We found that free-living worms, including the model genetic organism Caenorhabditis elegans, and parasitic helminths are unable to synthesize Heme de novo, even though these animals contain hemoproteins that function in key biological processes. Radioisotope, fluorescence labeling, and Heme analog studies suggest that C. elegans acquires Heme from exogenous sources. Iron-deprived worms were unable to grow in the presence of adequate Heme unless rescued by increasing Heme levels in the growth medium. These data indicate that although worms use dietary Heme for incorporation into hemoproteins, ingested Heme is also used as an iron source when iron is limiting. Our results provide a biochemical basis for the dependence of worm growth and development on Heme, and they suggest that pharmacologic targeting of Heme transport pathways in worms could be an important control measure for helminthic infections.
Annabelle Fernandez - One of the best experts on this subject based on the ideXlab platform.
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discovery of intracellular Heme binding protein hrtr which controls Heme efflux by the conserved hrtb hrta transporter in lactococcus lactis
Journal of Biological Chemistry, 2012Co-Authors: Delphine Lechardeur, Benedicte Cesselin, Ursula Liebl, Marten H Vos, Annabelle FernandezAbstract:Most commensal and food bacteria lack Heme biosynthesis genes. For several of these, the capture of environmental Heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that Heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (Heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a Heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by Heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular Heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular Heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls Heme toxicity by its direct and specific efflux. The use of an intracellular Heme sensor to control Heme efflux constitutes a novel paradigm for bacterial Heme homeostasis.
Amit R Reddi - One of the best experts on this subject based on the ideXlab platform.
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glyceraldehyde 3 phosphate dehydrogenase is a chaperone that allocates labile Heme in cells
Journal of Biological Chemistry, 2018Co-Authors: Elizabeth A Sweeny, Anuradha Bharara Singh, Ritu Chakravarti, Osiris Martinezguzman, Arushi Saini, Mohammad Mahfuzul Haque, Greer Garee, Pablo D Dans, Luciana Hannibal, Amit R ReddiAbstract:Cellular Heme is thought to be distributed between a pool of sequestered Heme that is tightly bound within Hemeproteins and a labile Heme pool required for signaling and transfer into proteins. A Heme chaperone that can hold and allocate labile Heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable Heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust Heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH Heme binding, antagonized Heme delivery to both cytosolic and nuclear Hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile Heme to rise dramatically. We conclude that by virtue of its Heme-binding property, GAPDH binds and chaperones labile Heme to create a Heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring Heme homeostasis in health and disease.
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regulation of intracellular Heme trafficking revealed by subcellular reporters
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Xiaojing Yuan, Amit R Reddi, Iqbal Hamza, Nicole Rietzschel, Hanna Kwon, Ana Beatriz Walter Nuno, David A Hanna, Emma Lloyd RavenAbstract:Abstract Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether Heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous Heme contribute to cellular labile Heme and that extracellular Heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile Heme. Restoration of reporter activity by Heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous Heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental Heme influences labile Heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest Heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous Heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on Heme trafficking in cellular and whole-animal models.
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thermodynamic investigation into the mechanisms of proton coupled electron transfer events in Heme protein maquettes
Biochemistry, 2007Co-Authors: Amit R Reddi, Charles J Reedy, Steven Mui, Brian R GibneyAbstract:To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed Heme proteins are elucidated. Both Heme protein maquettes chosen, Heme b−[H10A24]2 and Heme b−[Δ7-His]2, are four-α-helix bundles that display pH-dependent Heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous Heme b with these maquettes allow the individual contributions of Heme−protein association, iron−histidine ligation, and Heme−protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds Heme b in both oxidation states tighter than the smaller and more well-structured [Δ7-His]2 due to a stronger porphyrin−protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their Heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous Heme in [H10A2...
Toru Shimizu - One of the best experts on this subject based on the ideXlab platform.
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soul in mouse eyes is a new hexameric Heme binding protein with characteristic optical absorption resonance raman spectral and Heme binding properties
Biochemistry, 2004Co-Authors: Takeshi Uchida, Ikuko Sagami, Toru Shimizu, Akira Sato, Teizo Kitagawa, Jotaro Igarashi, Emiko SatoAbstract:SOUL is specifically expressed in the retina and pineal gland and displays more than 40% sequence homology with p22HBP, a Heme protein ubiquitously expressed in numerous tissues. SOUL was purified as a dimer in the absence of Heme from the Escherichia coli expression system but displayed a hexameric structure upon Heme binding. Heme-bound SOUL displayed optical absorption and resonance Raman spectra typical of 6-coordinate low-spin Heme protein, with one Heme per monomeric unit for both the Fe(III) and Fe(II) complexes. Spectral data additionally suggest that one of the axial ligands of the Fe(III) Heme complex is His. Mutation of His42 (the only His of SOUL) to Ala resulted in loss of Heme binding, confirming that this residue is an axial ligand of SOUL. The Kd value of Heme for SOUL was estimated as 4.8 × 10-9 M from the association and dissociation rate constants, suggesting high binding affinity. On the other hand, p22HBP was obtained as a monomer containing one Heme per subunit, with a Kd value of 2....
Delphine Lechardeur - One of the best experts on this subject based on the ideXlab platform.
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discovery of intracellular Heme binding protein hrtr which controls Heme efflux by the conserved hrtb hrta transporter in lactococcus lactis
Journal of Biological Chemistry, 2012Co-Authors: Delphine Lechardeur, Benedicte Cesselin, Ursula Liebl, Marten H Vos, Annabelle FernandezAbstract:Most commensal and food bacteria lack Heme biosynthesis genes. For several of these, the capture of environmental Heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that Heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (Heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a Heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by Heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular Heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular Heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls Heme toxicity by its direct and specific efflux. The use of an intracellular Heme sensor to control Heme efflux constitutes a novel paradigm for bacterial Heme homeostasis.
