RESUMO
Engineered hemoproteins can selectively incorporate nitrogen from nitrene precursors like hydroxylamine, O-substituted hydroxylamines, and organic azides into organic molecules. Although iron-nitrenoids are often invoked as the reactive intermediates in these reactions, their innate reactivity and transient nature have made their characterization challenging. Here we characterize an iron-nitrosyl intermediate generated from NH2OH within a protoglobin active site that can undergo nitrogen-group transfer catalysis, using UV-vis, electron paramagnetic resonance (EPR) spectroscopy, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) techniques. The mechanistic insights gained led to the discovery of aminating reagentsânitrite (NO2-), nitric oxide (NO), and nitroxyl (HNO)âthat are new to both nature and synthetic chemistry. Based on the findings, we propose a catalytic cycle for C-H amination inspired by the nitrite reductase pathway. This study highlights the potential of engineered hemoproteins to access natural nitrogen sources for sustainable chemical synthesis and offers a new perspective on the use of biological nitrogen cycle intermediates in biocatalysis.
Assuntos
Hemeproteínas , Aminação , Hemeproteínas/química , Espectroscopia de Ressonância de Spin Eletrônica , Óxido Nítrico/química , Espectrometria de Massas por Ionização por Electrospray , BiocatáliseRESUMO
The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (µs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) "locked off", Fe(II)-CO "on", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.
Assuntos
Proteínas de Bactérias , Monóxido de Carbono , Espectroscopia de Ressonância de Spin Eletrônica , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Heme/química , Heme/metabolismo , Ligação Proteica , Conformação Proteica , Regulação Alostérica , Hemeproteínas , TransativadoresRESUMO
Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase ß (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.
Assuntos
1-Fosfatidilinositol 4-Quinase , Antimaláricos , Hemeproteínas , Naftiridinas , Plasmodium falciparum , Peixe-Zebra , Plasmodium falciparum/efeitos dos fármacos , Animais , Naftiridinas/farmacologia , Naftiridinas/química , Naftiridinas/síntese química , Naftiridinas/uso terapêutico , Antimaláricos/farmacologia , Antimaláricos/química , Antimaláricos/síntese química , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/metabolismo , Humanos , Relação Estrutura-Atividade , Hemeproteínas/antagonistas & inibidores , Hemeproteínas/metabolismo , Camundongos , Ratos , Malária Falciparum/tratamento farmacológico , Masculino , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese químicaRESUMO
The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins.
Assuntos
Técnicas Biossensoriais , Heme , Hemeproteínas , Heme/biossíntese , Heme/genética , Heme/metabolismo , Hemeproteínas/genética , Hemeproteínas/metabolismo , Hemeproteínas/biossíntese , Transcriptoma/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Animais , Sistemas CRISPR-Cas , Engenharia Metabólica , Regiões Promotoras GenéticasRESUMO
Hemozoin is a natural biomarker formed during the hemoglobin metabolism of Plasmodium parasites, the causative agents of malaria. The rotating-crystal magneto-optical detection (RMOD) has been developed for its rapid and sensitive detection both in cell cultures and patient samples. In the current article we demonstrate that, besides quantifying the overall concentration of hemozoin produced by the parasites, RMOD can also track the size distribution of the hemozoin crystals. We establish the relations between the magneto-optical signal, the mean parasite age and the median crystal size throughout one erythrocytic cycle of Plasmodium falciparum parasites, where the latter two are determined by optical and scanning electron microscopy, respectively. The significant correlation between the magneto-optical signal and the stage distribution of the parasites indicates that the RMOD method can be utilized for species-specific malaria diagnosis and for the quick assessment of drug efficacy.
Assuntos
Hemeproteínas , Plasmodium falciparum , Hemeproteínas/metabolismo , Hemeproteínas/química , Plasmodium falciparum/crescimento & desenvolvimento , Humanos , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Malária Falciparum/diagnóstico , Microscopia Eletrônica de Varredura/métodosRESUMO
Recent structural and biophysical studies of O2-sensing FixL, NO-sensing soluble guanylate cyclase, and other biological heme-based sensing proteins have begun to reveal the details of their molecular mechanisms and shed light on how nature regulates important biological processes such as nitrogen fixation, blood pressure, neurotransmission, photosynthesis and circadian rhythm. The O2-sensing FixL protein from S. meliloti, the eukaryotic NO-sensing protein sGC, and the CO-sensing CooA protein from R. rubrum transmit their biological signals through gas-binding to the heme domain of these proteins, which inhibits or activates the regulatory, enzymatic domain. These proteins appear to propagate their signal by specific structural changes in the heme sensor domain initiated by the appropriate gas binding to the heme, which is then propagated through a coiled-coil linker or other domain to the regulatory, enzymatic domain that sends out the biological signal. The current understanding of the signal transduction mechanisms of O2-sensing FixL, NO-sensing sGC, CO-sensing CooA and other biological heme-based gas sensing proteins and their mechanistic themes are discussed, with recommendations for future work to further understand this rapidly growing area of biological heme-based gas sensors.
Assuntos
Heme , Hemeproteínas , Oxigênio , Transdução de Sinais , Oxigênio/metabolismo , Oxigênio/química , Hemeproteínas/metabolismo , Hemeproteínas/química , Heme/metabolismo , Heme/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Humanos , Histidina QuinaseRESUMO
Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.
Assuntos
Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Ácido Peroxinitroso , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Mycobacterium tuberculosis/química , Hemeproteínas/química , Hemeproteínas/metabolismo , Compostos Férricos/química , Compostos Férricos/metabolismo , TermodinâmicaRESUMO
PURPOSE: Artemisinin combination therapies, the first-line antimalarials in Nigeria, have reportedly suffered multiple failures in malaria treatment, hence the search for novel combination of other compounds. Methyl gallate and palmatine have been reported to exhibit antiplasmodial activities but the antimalarial activity of their combination has not been evaluated. Therefore, the evaluation of the combination of methyl gallate and palmatine for antimalarial activity in vitro and in vivo in the presence of piperine was carried out. MATERIALS AND METHODS: The inhibitory potential of methyl gallate and palmatine combination on ß-hematin (hemozoin) formation was studied in vitro. Also, the antimalarial activity of methyl gallate and palmatine combination with/without a bioenhancer (piperine) was evaluated in Plasmodium berghei NK65-infected mice. RESULTS: Methyl gallate and palmatine in the ratio 3:2 acted synergistically in vitro and had the highest inhibitory effect (IC50 = 0.73 µg/mL) on ß-hematin (hemozoin) formation. The 3:2 combination of methyl gallate and palmatine exhibited no antimalarial activity in vivo in the absence of piperine but caused reduction in parasitemia that exceeded 40% in the presence of piperine at the dose of 25 mg/kg body weight on days 6 and 8 post-inoculation in mice. CONCLUSION: The 3:2 combination of methyl gallate and palmatine in the presence of piperine exhibited antimalarial activity in vivo, possibly by synergistic inhibition of hemozoin formation which may cause accumulation of haem within the food vacuole of Plasmodium spp. and its death.
Assuntos
Alcaloides , Antimaláricos , Benzodioxóis , Alcaloides de Berberina , Sinergismo Farmacológico , Ácido Gálico , Malária , Piperidinas , Plasmodium berghei , Alcamidas Poli-Insaturadas , Animais , Alcamidas Poli-Insaturadas/farmacologia , Antimaláricos/farmacologia , Benzodioxóis/farmacologia , Piperidinas/farmacologia , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Ácido Gálico/farmacologia , Ácido Gálico/análogos & derivados , Alcaloides/farmacologia , Plasmodium berghei/efeitos dos fármacos , Alcaloides de Berberina/farmacologia , Parasitemia/tratamento farmacológico , Concentração Inibidora 50 , HemeproteínasRESUMO
Nitric oxide (ËNO) is a free radical that induces nitrosative stress, which can jeopardize cell viability. Yeasts have evolved diverse detoxification mechanisms to effectively counteract ËNO-mediated cytotoxicity. One mechanism relies on the flavohemoglobin Yhb1, whereas a second one requires the S-nitrosoglutathione reductase Fmd2. To investigate heme-dependent activation of Yhb1 in response to ËNO, we use hem1Δ-derivative Schizosaccharomyces pombe strains lacking the initial enzyme in heme biosynthesis, forcing cells to assimilate heme from external sources. Under these conditions, yhb1+ mRNA levels are repressed in the presence of iron through a mechanism involving the GATA-type transcriptional repressor Fep1. In contrast, when iron levels are low, the transcription of yhb1+ is derepressed and further induced in the presence of the ËNO donor DETANONOate. Cells lacking Yhb1 or expressing inactive forms of Yhb1 fail to grow in a hemin-dependent manner when exposed to DETANONOate. Similarly, the loss of function of the heme transporter Str3 phenocopies the effects of Yhb1 disruption by causing hypersensitivity to DETANONOate under hemin-dependent culture conditions. Coimmunoprecipitation and bimolecular fluorescence complementation assays demonstrate the interaction between Yhb1 and the heme transporter Str3. Collectively, our findings unveil a novel pathway for activating Yhb1, fortifying yeast cells against nitrosative stress.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Fatores de Transcrição GATA , Regulação Fúngica da Expressão Gênica , Heme/metabolismo , Hemeproteínas/metabolismo , Hemeproteínas/genética , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Óxido Nítrico/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Hemozoin is a crystal synthesized by Plasmodium parasites during hemoglobin digestion in the erythrocytic stage. The hemozoin released when the parasites egress from the red blood cell, which is complexed with parasite DNA, is cleared from the circulation by circulating and tissue-resident monocytes and macrophages, respectively. Recently, we reported that intravenous administration of purified hemozoin complexed with Plasmodium berghei DNA (HzPbDNA) resulted in an innate immune response that blocked liver stage development of sporozoites that was dose-dependent and time-limited. Here, we further characterize the organismal, cellular, and molecular events associated with this protective innate response in the liver and report that a large proportion of the IV administered HzPbDNA localized to F4/80+ cells in the liver and that the rapid and strong protection against liver-stage development waned quickly such that by 1 week post-HzPbDNA treatment animals were fully susceptible to infection. RNAseq of the liver after IV administration of HzPbDNA demonstrated that the rapid and robust induction of genes associated with the acute phase response, innate immune activation, cellular recruitment, and IFN-γ signaling observed at day 1 was largely absent at day 7. RNAseq analysis implicated NK cells as the major cellular source of IFN-γ. In vivo cell depletion and IFN-γ neutralization experiments supported the hypothesis that tissue-resident macrophages and NK cells are major contributors to the protective response and the NK cell-derived IFN-γ is key to induction of the mechanisms that block sporozoite development in the liver. These findings advance our understanding of the innate immune responses that prevent liver stage malaria infection.
Assuntos
Hemeproteínas , Imunidade Inata , Interferon gama , Fígado , Malária , Plasmodium berghei , Esporozoítos , Animais , Plasmodium berghei/imunologia , Esporozoítos/imunologia , Malária/imunologia , Malária/prevenção & controle , Malária/parasitologia , Hemeproteínas/imunologia , Camundongos , Fígado/parasitologia , Fígado/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Macrófagos/imunologia , Macrófagos/parasitologia , DNA de Protozoário/genética , FemininoRESUMO
Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes.
Assuntos
Escherichia coli O157 , Heme Oxigenase (Desciclizante) , Proteínas Ligantes de Grupo Heme , Heme , Escherichia coli O157/metabolismo , Escherichia coli O157/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Heme/metabolismo , Proteínas Ligantes de Grupo Heme/metabolismo , Hemeproteínas/metabolismo , Hemeproteínas/genética , Peróxido de Hidrogênio/metabolismo , Oxirredução , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Andrographis paniculata (AP) ((Burm f.) Wall. ex Nees) is a medicinal plant, documented for its folkloric use in the treatment of malaria. AIM: This study was designed to determine the potency of extract and fractions of A. paniculata (AP) as a curative, both for susceptible and resistant malaria and to also determine the plant's mechanism of action. This study was also designed to determine whether AP extract and its most potent fraction will mitigate infection-mediated mitochondrial dysfunction, and to assess the phytochemical constituents of the most potent fraction. MATERIALS AND METHODS: n-Hexane, dichloromethane, ethylacetate and methanol were used to partition the methanol extract of A. paniculata. Graded doses of these extract and fractions were used to treat mice infected with chloroquine-sensitive strain of P. berghei in a curative model. The most potent fraction was used to treat mice infected with resistant (ANKA strain) P. berghei. Inhibition of hemozoin formation, reversal of mitochondrial dysfunction and antiinflammatory potentials were determined. A combination of ultraperformance liquid chromatography-quadrupole time of flight-mass spectrometry and nuclear magnetic resonance spectroscopy were used for chemical analysis. RESULTS: Microscopy revealed that the dichloromethane fraction decreased the parasite burden the most, and inhibition of the hemozoin formation is one of its mechanisms of action. The dichloromethane fraction reversed parasite-induced mitochondrial pore opening in the host, enzyme-dependent ATP hydrolysis and peroxidation of host mitochondrial membrane phospholipids as well as its antiinflammatory potentials. The UPLC-qTOF-MS report and NMR fingerprints of the dichloromethane fraction of A. paniculata yielded fourteen compounds of which sibiricinone C was identified from the plant for the first time. CONCLUSION: Fractions of A. paniculata possess antiplasmodial effects with the dichloromethane fraction having the highest potency. The potent effect of this fraction may be attributed to the phytochemicals present because it contains terpenes implicated with antimalarial and antiinflammatory activities.
Assuntos
Andrographis , Antimaláricos , Malária , Extratos Vegetais , Plasmodium berghei , Animais , Plasmodium berghei/efeitos dos fármacos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Malária/tratamento farmacológico , Malária/parasitologia , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Camundongos , Andrographis/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Masculino , Hemeproteínas/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , FemininoRESUMO
The process of heme binding to a protein is prevalent in almost all forms of life to control many important biological properties, such as O2-binding, electron transfer, gas sensing or to build catalytic power. In these cases, heme typically binds tightly (irreversibly) to a protein in a discrete heme binding pocket, with one or two heme ligands provided most commonly to the heme iron by His, Cys or Tyr residues. Heme binding can also be used as a regulatory mechanism, for example in transcriptional regulation or ion channel control. When used as a regulator, heme binds more weakly, with different heme ligations and without the need for a discrete heme pocket. This makes the characterization of heme regulatory proteins difficult, and new approaches are needed to predict and understand the heme-protein interactions. We apply a modified version of the ProFunc bioinformatics tool to identify heme-binding sites in a test set of heme-dependent regulatory proteins taken from the Protein Data Bank and AlphaFold models. The potential heme binding sites identified can be easily visualized in PyMol and, if necessary, optimized with RosettaDOCK. We demonstrate that the methodology can be used to identify heme-binding sites in proteins, including in cases where there is no crystal structure available, but the methodology is more accurate when the quality of the structural information is high. The ProFunc tool, with the modification used in this work, is publicly available at https://www.ebi.ac.uk/thornton-srv/databases/profunc and can be readily adopted for the examination of new heme binding targets.
Assuntos
Heme , Ligação Proteica , Humanos , Sítios de Ligação , Biologia Computacional/métodos , Simulação por Computador , Bases de Dados de Proteínas , Heme/metabolismo , Heme/química , Hemeproteínas/metabolismo , Hemeproteínas/química , Hemeproteínas/genética , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.
Assuntos
Hemeproteínas , Synechocystis , Heme , Zinco , Histidina , Hemeproteínas/genética , Synechocystis/genética , Carbono , FerroRESUMO
Cellular life relies on enzymes that require metals, which must be acquired from extracellular sources. Bacteria utilize surface and secreted proteins to acquire such valuable nutrients from their environment. These include the cargo proteins of the type eleven secretion system (T11SS), which have been connected to host specificity, metal homeostasis, and nutritional immunity evasion. This Sec-dependent, Gram-negative secretion system is encoded by organisms throughout the phylum Proteobacteria, including human pathogens Neisseria meningitidis, Proteus mirabilis, Acinetobacter baumannii, and Haemophilus influenzae. Experimentally verified T11SS-dependent cargo include transferrin-binding protein B (TbpB), the hemophilin homologs heme receptor protein C (HrpC), hemophilin A (HphA), the immune evasion protein factor-H binding protein (fHbp), and the host symbiosis factor nematode intestinal localization protein C (NilC). Here, we examined the specificity of T11SS systems for their cognate cargo proteins using taxonomically distributed homolog pairs of T11SS and hemophilin cargo and explored the ligand binding ability of those hemophilin cargo homologs. In vivo expression in Escherichia coli of hemophilin homologs revealed that each is secreted in a specific manner by its cognate T11SS protein. Sequence analysis and structural modeling suggest that all hemophilin homologs share an N-terminal ligand-binding domain with the same topology as the ligand-binding domains of the Haemophilus haemolyticus heme binding protein (Hpl) and HphA. We term this signature feature of this group of proteins the hemophilin ligand-binding domain. Network analysis of hemophilin homologs revealed five subclusters and representatives from four of these showed variable heme-binding activities, which, combined with sequence-structure variation, suggests that hemophilins are diversifying in function.IMPORTANCEThe secreted protein hemophilin and its homologs contribute to the survival of several bacterial symbionts within their respective host environments. Here, we compared taxonomically diverse hemophilin homologs and their paired Type 11 secretion systems (T11SS) to determine if heme binding and T11SS secretion are conserved characteristics of this family. We establish the existence of divergent hemophilin sub-families and describe structural features that contribute to distinct ligand-binding behaviors. Furthermore, we demonstrate that T11SS are specific for their cognate hemophilin family cargo proteins. Our work establishes that hemophilin homolog-T11SS pairs are diverging from each other, potentially evolving into novel ligand acquisition systems that provide competitive benefits in host niches.
Assuntos
Proteínas de Bactérias , Heme , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Heme/metabolismo , Proteínas Ligantes de Grupo Heme/metabolismo , Hemeproteínas/metabolismo , Hemeproteínas/genética , Hemeproteínas/química , Ligação Proteica , Proteobactérias/metabolismo , Proteobactérias/genéticaRESUMO
In Gram-positive bacteria, sophisticated machineries to acquire the heme group of hemoglobin (Hb) have evolved to extract the precious iron atom contained in it. In the human pathogen Streptococcus pyogenes, the Shr protein is a key component of this machinery. Herein we present the crystal structure of hemoglobin-interacting domain 2 (HID2) of Shr bound to Hb. HID2 interacts with both, the protein and heme portions of Hb, explaining the specificity of HID2 for the heme-bound form of Hb, but not its heme-depleted form. Further mutational analysis shows little tolerance of HID2 to interfacial mutations, suggesting that its interaction surface with Hb could be a suitable candidate to develop efficient inhibitors abrogating the binding of Shr to Hb.
Assuntos
Hemeproteínas , Humanos , Hemeproteínas/genética , Streptococcus pyogenes/genética , Heme , Reconhecimento Psicológico , FerroRESUMO
Heme is an iron-containing prosthetic group necessary for the function of several proteins termed "hemoproteins." Erythrocytes contain most of the body's heme in the form of hemoglobin and contain high concentrations of free heme. In nonerythroid cells, where cytosolic heme concentrations are 2 to 3 orders of magnitude lower, heme plays an essential and often overlooked role in a variety of cellular processes. Indeed, hemoproteins are found in almost every subcellular compartment and are integral in cellular operations such as oxidative phosphorylation, amino acid metabolism, xenobiotic metabolism, and transcriptional regulation. Growing evidence reveals the participation of heme in dynamic processes such as circadian rhythms, NO signaling, and the modulation of enzyme activity. This dynamic view of heme biology uncovers exciting possibilities as to how hemoproteins may participate in a range of physiologic systems. Here, we discuss how heme is regulated at the level of its synthesis, availability, redox state, transport, and degradation and highlight the implications for cellular function and whole organism physiology.
Assuntos
Fenômenos Fisiológicos Celulares , Heme , Animais , Humanos , Ritmo Circadiano/fisiologia , Heme/metabolismo , Hemeproteínas/metabolismo , Oxirredução , Transdução de Sinais , Espaço Intracelular/metabolismo , Fenômenos Fisiológicos Celulares/fisiologiaRESUMO
Heme proteins and their derivatives play important roles in inducing lipid oxidation to produce volatile compounds during bacon drying. This study investigated the effects of heme proteins and their derivatives (hemoglobin, myoglobin, nitrosylmyoglobin, hemin, Fe2+, and Fe3+) on lipid and volatiles profiles in the washed pig muscle (WPM) model. The results of the study indicated that the inducers primarily caused the oxidation of glycerophospholipids. Furthermore, hemoglobin and myoglobin had the most significant impact, and their potential substrates may include PE (O-18:2/20:4), PE (O-18:1/20:4), PC (16:0/18:1), and PE (O-18:2/18:2). Nitrosomyoglobin has limited ability to promote lipid oxidation and may protect ether phospholipids from oxidation. The analysis of the volatiles in the model revealed that heme proteins and their derivatives have the ability to induce the production of key aroma compounds. The descending order of effectiveness in inducing the production of aroma compounds is as follows: hemoglobin, myoglobin, hemin, and nitrosylmyoglobin. The effectiveness of Fe2+ and Fe3+ is similar to that of nitrosylmyoglobin.
Assuntos
Hemeproteínas , Lipídeos , Animais , Suínos , Hemeproteínas/química , Hemeproteínas/metabolismo , Lipídeos/química , Produtos da Carne/análise , Compostos Orgânicos Voláteis/química , Temperatura Alta , Odorantes/análise , Oxirredução , DessecaçãoRESUMO
Malaria is a severe disease caused by cytozoic parasites of the genus Plasmodium, which infiltrate and infect red blood cells. Several drugs have been developed to combat the devastating effects of malaria. Antimalarials based on quinolines inhibit the crystallization of hematin into hemozoin within the parasite, ultimately leading to its demise. Despite the frequent use of these agents, there are unanswered questions about their mechanisms of action. In the present study, the quinoline chloroquine and its interaction with the target structure hematin was investigated using an advanced, highly parallelized Raman difference spectroscopy (RDS) setup. Simultaneous recording of the spectra of hematin and chloroquine mixtures with varying compositions enabled the observation of changes in peak heights and positions based on the altered molecular structure resulting from their interaction. A shift of (-1.12 ± 0.05) cm-1 was observed in the core-size marker band ν(CαCm)asym peak position of the 1:1 chloroquine-hematin mixture compared to pure hematin. The oxidation-state marker band ν(pyrrole half-ring)sym exhibited a shift by (+0.93 ± 0.13) cm-1. These results were supported by density functional theory (DFT) calculations, indicating a hydrogen bond between the quinolinyl moiety of chloroquine and the oxygen atom of ferric protoporphyrin IX hydroxide (Fe(III)PPIX-OH). The consequence is a reduced electron density within the porphyrin moiety and an increase in its core size. This hypothesis provided further insights into the mechanism of hemozoin inhibition, suggesting chloroquine binding to the monomeric form of hematin, thereby preventing its further crystallization to hemozoin.