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1.
Sci Rep ; 12(1): 7152, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35504999

RESUMO

Heme internalization by pathogenic bacteria inside a human host to accomplish the requirement of iron for important cellular processes is of paramount importance. Despite this, the mechanism of heme import by the ATP-binding-cassette (ABC) transporter HutCD in Vibrio cholerae remains unexplored. We have performed biochemical studies on ATPase HutD and its mutants, along with molecular modelling, docking and unbiased all-atom MD simulations on lipid-solvated models of permease-ATPase complex HutCD. The results demonstrated mechanisms of ATP binding/hydrolysis and trapped transient and global conformational changes in HutCD, necessary for heme internalization. ATPase HutD forms a dimer, independent of the permease HutC. Each HutD monomer canonically binds ATP in a 1:1 stoichiometry. MD simulations demonstrated that a rotational motion of HutC dimer occurs synchronously with the inter-dimeric D-loop interactions of HutDs. F151 of TM4-TM5 loop of HutC, packs with ATP and Y15 of HutD, initiating 'cytoplasmic gate opening' which mimics an 'outward-facing' to 'inward-facing' conformational switching upon ATP hydrolysis. The simulation on 'inward-facing' HutCD culminates to an 'occluded' state. The simulation on heme-docked HutCD indicated that the event of heme release occurs in ATP-free 'inward-facing' state. Gradual conformational changes of the TM5 helices of HutC towards the 'occluded' state facilitate ejection of heme.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias/metabolismo , Heme , Vibrio cholerae , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Humanos , Vibrio cholerae/metabolismo
2.
Anal Methods ; 14(17): 1739-1746, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35468173

RESUMO

ß-Amyloid (Aß) peptides can bind both Cu2+ and heme cofactors simultaneously to form heme-Cu2+-Aß complexes, which are proposed to generate toxic partially reduced oxygen species (PROS, e.g., H2O2) and play a vital role in Alzheimer's disease (AD). In this paper, a competitive dual-mechanism-driven electrochemiluminescence (ECL) aptasensor integrating the synergistic enhancement and steric hindrance effect was described for Aß detection. Specifically, graphite carbon nitride (g-C3N4) as an effective ECL luminescent substrate and Au nanoparticles were sequentially assembled on the Au electrode surface, and then a thiol-modified aptamer for capturing Aß peptide was attached to the surface of the electrode through the Au-S bond. Aß peptides were simultaneously incubated with heme and Cu2+, and the forming heme-Cu2+-Aß complexes were subsequently anchored on the electrode through the specific recognition between the target Aß and the aptamer. When the concentration of the target Aß is low, the synergistic enhancement effect arising from K2S2O8 with in situ generated H2O2 is predominant, resulting in an increase in the ECL signal of g-C3N4. In contrast, when the concentration of Aß is high, the steric hindrance effect generated from heme-Cu2+-Aß complexes is dominant, leading to a decrease in the ECL signal. The present sensor exhibits a favorable linear response for the detection of Aß with a relatively low detection limit of 0.24 pM, and provides a more sensitive and selective platform for bioanalysis.


Assuntos
Peptídeos beta-Amiloides , Nanopartículas Metálicas , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Ouro/química , Heme/química , Heme/metabolismo , Peróxido de Hidrogênio , Nanopartículas Metálicas/química
3.
Int J Mol Sci ; 23(7)2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-35408976

RESUMO

The cytochrome P450 superfamily are heme-thiolate enzymes able to carry out monooxygenase reactions. Several studies have demonstrated the feasibility of using a soluble bacterial reductase from Bacillus megaterium, BMR, as an artificial electron transfer partner fused to the human P450 domain in a single polypeptide chain in an approach known as 'molecular Lego'. The 3A4-BMR chimera has been deeply characterized biochemically for its activity, coupling efficiency, and flexibility by many different biophysical techniques leading to the conclusion that an extension of five glycines in the loop that connects the two domains improves all the catalytic parameters due to improved flexibility of the system. In this work, we extend the characterization of 3A4-BMR chimeras using differential scanning calorimetry to evaluate stabilizing role of BMR. We apply the 'molecular Lego' approach also to CYP19A1 (aromatase) and the data show that the activity of the chimeras is very low (<0.003 min-1) for all the constructs tested with a different linker loop length: ARO-BMR, ARO-BMR-3GLY, and ARO-BMR-5GLY. Nevertheless, the fusion to BMR shows a remarkable effect on thermal stability studied by differential scanning calorimetry as indicated by the increase in Tonset by 10 °C and the presence of a cooperative unfolding process driven by the BMR protein domain. Previously characterized 3A4-BMR constructs show the same behavior of ARO-BMR constructs in terms of thermal stabilization but a higher activity as a function of the loop length. A comparison of the ARO-BMR system to 3A4-BMR indicates that the design of each P450-BMR chimera should be carefully evaluated not only in terms of electron transfer, but also for the biophysical constraints that cannot always be overcome by chimerization.


Assuntos
Bacillus megaterium , Heme , Proteínas de Bactérias/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/metabolismo , Humanos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas Recombinantes de Fusão/genética
4.
Commun Biol ; 5(1): 355, 2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35418619

RESUMO

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB's are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.


Assuntos
Proteínas de Escherichia coli , Serratia marcescens , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Ligação Proteica , Serratia marcescens/química , Serratia marcescens/genética , Serratia marcescens/metabolismo
5.
Arch Biochem Biophys ; 723: 109220, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35430214

RESUMO

Electroactive Geobacter bacteria can perform extracellular electron transfer and present a wide metabolic versatility. These bacteria reduce organic, toxic and radioactive compounds, and produce electric current while interacting with electrodes, making them interesting targets for numerous biotechnological applications. Their global electrochemical responses rely on an efficient interface between the inside and the cell's exterior, which is driven by the highly abundant periplasmic triheme PpcA-family cytochromes. The functional features of these cytochromes have been studied in G. sulfurreducens and G. metallireducens, and although they share a high degree of structural homology and sequence identity, their properties are quite distinct. In this work, the heme axial ligand geometries and the magnetic properties of PpcF from G. metallireducens were determined. The data obtained constitute important constraints for the determination of its solution structure in the oxidized state and indicate that the (i) heme core architecture; (ii) axial ligands geometries and (iii) magnetic properties of the cytochrome are conserved compared to the other members of the PpcA-families. Furthermore, the results also indicate that the heme arrangement is crucial to maintain an intrinsic regulation of the protein's redox properties and hence its electron transfer efficiency and functionality.


Assuntos
Geobacter , Proteínas de Bactérias/química , Citocromos/química , Heme/metabolismo , Humanos , Ligantes , Oxirredução
6.
Mol Pharm ; 19(5): 1434-1448, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35416046

RESUMO

One of the factors determining efficient antimicrobial photodynamic inactivation (aPDI) is the accumulation of a light-activated compound, namely, a photosensitizer (PS). Targeted PS recognition is the approach based on the interaction between the membrane receptor on the bacterial surface and the PS, whereas the compound is efficiently accumulated by the same mechanism as the natural ligand. In this study, we showed that gallium mesoporphyrin IX (Ga3+MPIX) provided dual functionality─iron metabolism disruption and PS properties in aPDI. Ga3+MPIX induced efficient (>5log10 reduction in CFU/mL) bacterial photodestruction with excitation in the area of Q band absorption with relatively low eukaryotic cytotoxicity and phototoxicity. The Ga3+MPIX is recognized by the same systems as haem by the iron-regulated surface determinant (Isd). However, the impairment in the ATPase of the haem detoxification efflux pump was the most sensitive to the Ga3+MPIX-mediated aPDI phenotype. This indicates that changes within the metalloporphyrin structure (vinyl vs ethyl groups) did not significantly alter the properties of recognition of the compound but influenced its biophysical properties.


Assuntos
Anti-Infecciosos , Gálio , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Gálio/farmacologia , Heme/metabolismo , Humanos , Mesoporfirinas , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Staphylococcus aureus
7.
Biomolecules ; 12(4)2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35454139

RESUMO

Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems.


Assuntos
Citocromos c , Liases , Citocromos c/metabolismo , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Heme/metabolismo , Liases/metabolismo
8.
Redox Biol ; 52: 102316, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35489241

RESUMO

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.


Assuntos
Gasotransmissores , Mycobacterium tuberculosis , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Dioctil Sulfossuccínico/metabolismo , Gasotransmissores/metabolismo , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Ferro/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Protamina Quinase/química , Protamina Quinase/genética , Protamina Quinase/metabolismo , Regulon
9.
Cells ; 11(7)2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35406740

RESUMO

The oncogenic expression or mutation of tumor suppressors drives metabolic alteration, causing cancer cells to utilize diverse nutrients. Lactate is a known substrate for cancer cells, yet the regulatory mechanisms of lactate catabolism are limited. Here, we show that a heme-binding transcription factor, BACH1, negatively regulates lactate catabolic pathways in triple-negative breast cancer (TNBC) cells. BACH1 suppresses the transcriptional expression of monocarboxylate transporter 1 (MCT1) and lactate dehydrogenase B, inhibiting lactate-mediated mitochondrial metabolism. In our studies, the depletion of BACH1 either genetically or pharmacologically increased the lactate use of TNBC cells, increasing their sensitivity to MCT1 inhibition. Thus, small inhibitory molecules (SR13800 and AZD3965) blocking MCT1 better suppressed the growth of BACH1-depleted TNBC cells than did the controls. Particularly, hemin treatment degrading BACH1 proteins induced lactate catabolism in TNBC cells, generating synthetic lethality with MCT1 inhibition. Our data indicates that targeting BACH1 generates metabolic vulnerability and increases sensitivity to lactate transporter inhibition, suggesting a potential novel combination therapy for cancer patients with TNBC.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Ácido Láctico , Neoplasias de Mama Triplo Negativas , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Heme/metabolismo , Humanos , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo
10.
Int J Mol Sci ; 23(7)2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35409149

RESUMO

Hemopexin (Hx) is a plasma glycoprotein that scavenges heme (Fe(III) protoporphyrin IX). Hx has important implications in hemolytic disorders and hemorrhagic conditions because releasing hemoglobin increases the labile heme, which is potentially toxic, thus producing oxidative stress. Therefore, Hx has been considered for therapeutic use and diagnostics. In this work, we analyzed and mapped the interaction sequences of Hx with hemin and hemoglobin. The spot-synthesis technique was used to map human hemopexin (P02790) binding to hemin and human hemoglobin. A library of 15 amino acid peptides with a 10-amino acid overlap was designed to represent the entire coding region (aa 1-462) of hemopexin and synthesized onto cellulose membranes. An in silico approach was taken to analyze the amino acid frequency in the identified interaction regions, and molecular docking was applied to assess the protein-protein interaction. Seven linear peptide sequences in Hx were identified to bind hemin (H1-H7), and five were described for Hb (Hb1-Hb5) interaction, with just two sequences shared between hemin and Hb. The amino acid composition of the identified sequences demonstrated that histidine residues are relevant for heme binding. H105, H293, H373, H400, H429, and H462 were distributed in the H1-H7 peptide sequences, but other residues may also play an important role. Molecular docking analysis demonstrated Hx's association with the ß-chain of Hb, with several hotspot amino acids that coordinated the interaction. This study provides new insights into Hx-hemin binding motifs and protein-protein interactions with Hb. The identified binding sequences and specific peptides can be used for therapeutic purposes and diagnostics as hemopexin is under investigation to treat different diseases and there is an urgent need for diagnostics using labile heme when monitoring hemolysis.


Assuntos
Hemina , Hemopexina , Compostos Férricos , Heme/metabolismo , Hemina/metabolismo , Hemoglobinas/metabolismo , Hemólise , Hemopexina/metabolismo , Histidina , Humanos , Simulação de Acoplamento Molecular
11.
PLoS One ; 17(3): e0265512, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35294485

RESUMO

The heme component of myoglobin plays a crucial role in the pathogenesis of rhabdomyolysis-associated acute kidney injury (RM-AKI). Heme oxiganenase-1 (HO-1) is the rate-limiting enzyme of heme catabolism, and its metabolites, iron, biliverdin, and carbon monoxide, have antioxidant properties. Tin chloride (SnCl2) is a kidney specific HO-1 inducer. In this study, we examined whether the induction of HO-1 in the kidney by SnCl2 pretreatment ameliorates RM-AKI in rats and if the effect is due to the degradation of excess renal free heme. We developed an RM-AKI rat (male Sprague-Dawley rats) model by injecting glycerol (Gly) in the hind limbs. RM-AKI rats were pretreated with saline or SnCl2 or additional SnMP (tin mesoporphyrin, a specific HO inhibitor) followed by Gly treatment. Serum blood urea nitrogen (BUN) and creatinine (Crea) were measured as indicators of renal function. Renal free heme level was assessed based on the levels of δ-aminolevulinate synthase (ALAS1), a heme biosynthetic enzyme, and nuclear BTB and CNC homology 1 (Bach1), an inhibitory transcription factor of HO-1. Elevated free heme levels lead to decreases in ALAS1 and nuclear Bach1. After 24 h of Gly injection, serum BUN and Crea levels in saline-pretreated rats were significantly higher than those in untreated control rats. In contrast, SnCl2-pretreated rats showed no significant increase in the indices. However, additional treatment of SnMP abolished the beneficial effect of SnCl2. Renal ALAS1 mRNA levels and renal nuclear Bach1 protein levels in the saline pretreated rats were significantly lower than those in control rats 3 h after Gly injection. In contrast, the levels in SnCl2-pretreated rats were not altered. The findings indicate that SnCl2 pretreatment confers protection against RM-AKI by virtue of HO-1 induction in the renal system, at least in part through excess free heme degradation.


Assuntos
Injúria Renal Aguda , Rabdomiólise , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Feminino , Heme/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Rim/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Rabdomiólise/metabolismo , Compostos de Estanho
12.
Biomolecules ; 12(3)2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35327558

RESUMO

Ferritins are iron storage proteins assembled from 24 subunits into a spherical and hollow structure. The genomes of many bacteria harbor genes encoding two types of ferritin-like proteins, the bacterial ferritins (Ftn) and the bacterioferritins (Bfr), which bind heme. The genome of P. aeruginosa PAO1 (like the genomes of many bacteria) contains genes coding for two different types of ferritin-like molecules, ftnA (PA4235) and bfrB (PA3531). The reasons for requiring the presence of two distinct types of iron storage protein in bacterial cells have remained largely unexplained. Attempts to understand this issue in P. aeruginosa through the recombinant expression of the ftnA and bfrB genes in E. coli host cells, coupled to the biochemical and structural characterization of the recombinant 24-mer FtnA and 24-mer BfrB molecules, have shown that each of the recombinant molecules can form an Fe3+-mineral core. These observations led to the suggestion that 24-mer FtnA and 24-mer BfrB molecules coexist in P. aeruginosa cells where they share iron storage responsibilities. Herein, we demonstrate that P. aeruginosa utilizes a single heterooligomeric 24-mer Bfr assembled from FtnA and BfrB subunits. The relative content of the FtnA and BfrB subunits in Bfr depends on the O2 availability during cell culture, such that Bfr isolated from aerobically cultured P. aeruginosa is assembled from a majority of BfrB subunits. In contrast, when the cells are cultured in O2-limiting conditions, the proportion of FtnA subunits in the isolated Bfr increases significantly and can become the most abundant subunit. Despite the variability in the subunit composition of Bfr, the 24-mer assembly is consistently arranged from FtnA subunit dimers devoid of heme and BfrB subunit dimers each containing a heme molecule.


Assuntos
Escherichia coli , Pseudomonas aeruginosa , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b , Escherichia coli/genética , Escherichia coli/metabolismo , Ferritinas/metabolismo , Heme/metabolismo , Ferro/metabolismo , Oxigênio/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
13.
J Bacteriol ; 204(4): e0056721, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35311542

RESUMO

In this study, we provide the first characterization of a chemoreceptor from Leptospira interrogans, the cause of leptospirosis. This receptor is related to the Aer2 receptors that have been studied in other bacteria. In those organisms, Aer2 is a soluble receptor with one or two PAS-heme domains and signals in response to O2 binding. In contrast, L. interrogans Aer2 (LiAer2) is an unusual membrane-bound Aer2 with a periplasmic domain and three cytoplasmic PAS-heme domains. Each of the three PAS domains bound b-type heme via conserved Eη-His residues. They also bound O2 and CO with similar affinities to each other and other PAS-heme domains. However, all three PAS domains were uniquely hexacoordinate in the deoxy-heme state, whereas other Aer2-PAS domains are pentacoordinate. Similar to other Aer2 receptors, LiAer2 could hijack the E. coli chemotaxis pathway but only when it was expressed with an E. coli high-abundance chemoreceptor. Unexpectedly, the response was inverted relative to classic Aer2 receptors. That is, LiAer2 caused E. coli to tumble (it was signal-on) in the absence of O2 and to stop tumbling in its presence. Thus, an endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2, and its displacement for gas-binding turned signaling off. This response also occurred in a soluble version of LiAer2 lacking the periplasmic domain, transmembrane (TM) region, and first two PAS domains, meaning that PAS3 alone was sufficient for O2-mediated control. Future studies are needed to understand the unique signaling mechanisms of this unusual Aer2 receptor. IMPORTANCE Leptospira interrogans, the cause of the zoonotic infection leptospirosis, is found in soil and water contaminated with animal urine. L. interrogans survives in complex environments with the aid of 12 chemoreceptors, none of which has been explicitly studied. In this study, we characterized the first L. interrogans chemoreceptor, LiAer2, and reported its unique characteristics. LiAer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b-type heme and O2 turned LiAer2 signaling off. An endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2 and its displacement for O2-binding turned signaling off. Our study corroborated previous findings that Aer2 receptors are O2 sensors, but also demonstrated that they do not all function the same way.


Assuntos
Leptospira interrogans , Leptospirose , Animais , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Heme/metabolismo , Leptospira interrogans/genética , Ligantes , Oxigênio/metabolismo
14.
Mar Biotechnol (NY) ; 24(2): 263-275, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35275290

RESUMO

Color polymorphism is frequently observed in molluscan shellfish, while the molecular regulation of shell pigmentation is not well understood. Peroxidase is a key enzyme involved in melanogenesis. Here, we identified a heme-peroxidase 2 gene (CgHPX2), and characterized the expression patterns and transcriptional regulation of CgHPX2 in the Pacific oyster Crassostrea gigas. Tissues expression analysis showed that CgHPX2 was a mantle-specific gene and primarily expressed in the edge mantle in black shell color oyster compared with white shell oyster. In situ hybridization showed that strong signals for CgHPX2 were detected in the both inner and outer surface of the outer fold of mantle in the black shell color oyster, whereas positive signals in white shell oyster were mainly localized in the outer surface of the outer fold of mantle. In the embryos and larvae, a high expression level of CgHPX2 was detected in the trochophore stage in both black and white shell color oysters. The temporal localization of CgHPX2 was mainly detected in the shell gland and edge mantle of trochophore and calcified shell larvae, respectively. In addition, a 2227 bp of 5' flanking region sequence of CgHPX2 was cloned, which contained a presumed core promoter region and many potential transcription factor binding sites. Further luciferase assay experiment confirmed that POU domain, class 2, transcription factor 1 (POU2F1), and SRY-box transcription factor 5 (SOX5) were involved in transcriptional regulation of CgHPX2 gene through binding to its specific promoter region. After CgPOU2F1 and CgSOX5 RNA interference, the CgHPX2 gene expression was significantly decreased. These results suggested that CgPOU2F1 and CgSOX5 might be two important transcription factors that positively regulated the expression of CgHPX2 gene, improving our understanding of the transcriptional regulation of molluscan shell pigmentation.


Assuntos
Crassostrea , Exoesqueleto/metabolismo , Animais , Crassostrea/genética , Crassostrea/metabolismo , Heme/metabolismo , Larva , Peroxidase/metabolismo , Pigmentação/genética , Fatores de Transcrição/metabolismo
15.
Int J Mol Sci ; 23(5)2022 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-35269706

RESUMO

Coproheme decarboxylase (ChdC) is an important enzyme in the coproporphyrin-dependent pathway (CPD) of Gram-positive bacteria that decarboxylates coproheme on two propionates at position 2 and position 4 sequentially to generate heme b by using H2O2 as an oxidant. This work focused on the ChdC from Geobacillus stearothermophilus (GsChdC) to elucidate the mechanism of its sequential two-step decarboxylation of coproheme. The models of GsChdC in a complex with substrate and reaction intermediate were built to investigate the reorienting mechanism of harderoheme. Targeted molecular dynamics simulations on these models validated that harderoheme is able to rotate in the active site of GsChdC with a 19.06-kcal·mol-1 energy barrier after the first step of decarboxylation to bring the propionate at position 4 in proximity of Tyr145 to continue the second decarboxylation step. The harderoheme rotation mechanism is confirmed to be much easier than the release-rebinding mechanism. In the active site of GsChdC, Trp157 and Trp198 comprise a "gate" construction to regulate the clockwise rotation of the harderoheme. Lys149 plays a critical role in the rotation mechanism, which not only keeps the Trp157-Trp198 "gate" from being closed but also guides the propionate at position 4 through the gap between Trp157 and Trp198 through a salt bridge interaction.


Assuntos
Carboxiliases , Carboxiliases/metabolismo , Descarboxilação , Geobacillus stearothermophilus , Heme/metabolismo , Peróxido de Hidrogênio/metabolismo , Propionatos/química
16.
PLoS One ; 17(3): e0265008, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35271636

RESUMO

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.


Assuntos
Coturnix , Casca de Ovo , Ácido Aminolevulínico , Animais , Coturnix/genética , Casca de Ovo/fisiologia , Ovos , Feminino , Heme/metabolismo , Codorniz/metabolismo , Coelhos , Útero/metabolismo
17.
Sci Rep ; 12(1): 3589, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246566

RESUMO

Soluble guanylate cyclase (sGC) requires a heme-group bound in order to produce cGMP, a second messenger involved in memory formation, while heme-free sGC is inactive. Two compound classes can increase sGC activity: sGC stimulators acting on heme-bound sGC, and sGC activators acting on heme-free sGC. In this rodent study, we investigated the potential of the novel brain-penetrant sGC stimulator BAY-747 and sGC activator runcaciguat to enhance long-term memory and attenuate short-term memory deficits induced by the NOS-inhibitor L-NAME. Furthermore, hippocampal plasticity mechanisms were investigated. In vivo, oral administration of BAY-747 and runcaciguat to male Wistar rats enhanced memory acquisition in the object location task (OLT), while only BAY-747 reversed L-NAME induced memory impairments in the OLT. Ex vivo, both BAY-747 and runcaciguat enhanced hippocampal GluA1-containing AMPA receptor (AMPAR) trafficking in a chemical LTP model for memory acquisition using acute mouse hippocampal slices. In vivo only runcaciguat acted on the glutamatergic AMPAR system in hippocampal memory acquisition processes, while for BAY-747 the effects on the neurotrophic system were more pronounced as measured in male mice using western blot. Altogether this study shows that sGC stimulators and activators have potential as cognition enhancers, while the underlying plasticity mechanisms may determine disease-specific effectiveness.


Assuntos
GMP Cíclico , Guanilato Ciclase , Animais , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Heme/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Guanilil Ciclase Solúvel/metabolismo , Vasodilatadores
18.
Molecules ; 27(6)2022 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-35335120

RESUMO

The rich source of heme within malarial parasites has been considered to underly the action specificity of artemisinin. We reasoned that increasing intraparasitic free heme levels might further sensitize the parasites to artemisinin. Various means, such as modulating heme synthesis, degradation, polymerization, or hemoglobin digestion, were tried to boost intracellular heme levels, and under several scenarios, free heme levels were significantly augmented. Interestingly, all results arrived at the same conclusion, i.e., elevating heme acted in a strongly negative way, impacting the antimalarial action of artemisinin, but exerted no effect on several other antimalarial drugs. Suppression of the elevated free heme level by introducing heme oxygenase expression effectively restored artemisinin potency. Consistently, zinc protoporphyrin IX/zinc mesoporphyrin, as analogues of heme, drastically increased free heme levels and, concomitantly, the EC50 values of artemisinin. We were unable to effectively mitigate free heme levels, possibly due to an unknown compensating heme uptake pathway, as evidenced by our observation of efficient uptake of a fluorescent heme homologue by the parasite. Our results thus indicate the existence of an effective and mutually compensating heme homeostasis network in the parasites, including an uncharacterized heme uptake pathway, to maintain a certain level of free heme and that augmentation of the free heme level negatively impacts the antimalarial action of artemisinin. Importance: It is commonly believed that heme is critical in activating the antimalarial action of artemisinins. In this work, we show that elevating free heme levels in the malarial parasites surprisingly negatively impacts the action of artemisinin. We tried to boost free heme levels with various means, such as by modulating heme synthesis, heme polymerization, hemoglobin degradation and using heme analogues. Whenever we saw elevation of free heme levels, reduction in artemisinin potency was also observed. The homeostasis of heme appears to be complex, as there exists an unidentified heme uptake pathway in the parasites, nullifying our attempts to effectively reduce intraparasitic free heme levels. Our results thus indicate that too much heme is not good for the antimalarial action of artemisinins. This research can help us better understand the biological properties of this mysterious drug.


Assuntos
Antimaláricos , Artemisininas , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Artemisininas/metabolismo , Artemisininas/farmacologia , Heme/metabolismo , Plasmodium falciparum
19.
Cells ; 11(6)2022 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-35326427

RESUMO

The maturation of hemeprotein dictates that they incorporate heme and become active, but knowledge of this essential cellular process remains incomplete. Studies on chaperon Hsp90 has revealed that it drives functional heme maturation of inducible nitric oxide synthase (iNOS), soluble guanylate cyclase (sGC) hemoglobin (Hb) and myoglobin (Mb) along with other proteins including GAPDH, while globin heme maturations also need an active sGC. In all these cases, Hsp90 interacts with the heme-free or apo-protein and then drives the heme maturation by an ATP dependent process before dissociating from the heme-replete proteins, suggesting that it is a key player in such heme-insertion processes. As the studies on globin maturation also need an active sGC, it connects the globin maturation to the NO-sGC (Nitric oxide-sGC) signal pathway, thereby constituting a novel NO-sGC-Globin axis. Since many aggressive cancer cells make Hbß/Mb to survive, the dependence of the globin maturation of cancer cells places the NO-sGC signal pathway in a new light for therapeutic intervention. Given the ATPase function of Hsp90 in heme-maturation of client hemeproteins, Hsp90 inhibitors often cause serious side effects and this can encourage the alternate use of sGC activators/stimulators in combination with specific Hsp90 inhibitors for better therapeutic intervention.


Assuntos
Proteínas de Choque Térmico HSP90 , Heme , Proteínas de Choque Térmico HSP90/metabolismo , Heme/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Mioglobina/metabolismo , Óxido Nítrico/metabolismo , Guanilil Ciclase Solúvel/metabolismo
20.
Front Immunol ; 13: 842858, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35281042

RESUMO

Iron metabolism is vital for the survival of both humans and microorganisms. Heme oxygenase-1 (HO-1) is an essential stress-response enzyme highly expressed in the lungs, and catabolizes heme into ferrous iron, carbon monoxide (CO), and biliverdin (BV)/bilirubin (BR), especially in pathological conditions which cause oxidative stress and inflammation. Ferrous iron (Fe2+) is an important raw material for the synthesis of hemoglobin in red blood cells, and patients with iron deficiency are often associated with decreased cellular immunity. CO and BR can inhibit oxidative stress and inflammation. Thus, HO-1 is regarded as a cytoprotective molecule during the infection process. However, recent study has unveiled new information regarding HO-1. Being a highly infectious pathogenic bacterium, Mycobacterium tuberculosis (MTB) infection causes acute oxidative stress, and increases the expression of HO-1, which may in turn facilitate MTB survival and growth due to increased iron availability. Moreover, in severe cases of MTB infection, excessive reactive oxygen species (ROS) and free iron (Fe2+) due to high levels of HO-1 can lead to lipid peroxidation and ferroptosis, which may promote further MTB dissemination from cells undergoing ferroptosis. Therefore, it is important to understand and illustrate the dual role of HO-1 in tuberculosis. Herein, we critically review the interplay among HO-1, tuberculosis, and the host, thus paving the way for development of potential strategies for modulating HO-1 and iron metabolism.


Assuntos
Heme Oxigenase-1 , Tuberculose , Bilirrubina , Heme/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Inflamação , Ferro/metabolismo
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