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1.
J Biol Chem ; 296: 100275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33428928

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.


Assuntos
Heme Oxigenase (Desciclizante)/genética , Proteínas Ligantes de Grupo Heme/genética , Hemeproteínas/genética , Pseudomonas aeruginosa/genética , Regulação Bacteriana da Expressão Gênica , Heme/genética , Homeostase/genética , Humanos , Ferro/metabolismo , Pseudomonas aeruginosa/patogenicidade , Shigella dysenteriae/genética , Shigella dysenteriae/patogenicidade , Virulência/genética
2.
Front Immunol ; 11: 1964, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32983129

RESUMO

Toll-like receptors (TLRs), also known as pattern recognition receptors, respond to exogenous pathogens and to intrinsic danger signals released from damaged cells and tissues. The tetrapyrrole heme has been suggested to be an agonist for TLR4, the receptor for the pro-inflammatory bacterial component lipopolysaccharide (LPS), synonymous with endotoxin. Heme is a double-edged sword with contradictory functions. On the one hand, it has vital cellular functions as the prosthetic group of hemoproteins including hemoglobin, myoglobin, and cytochromes. On the other hand, if released from destabilized hemoproteins, non-protein bound or "free" heme can have pro-oxidant and pro-inflammatory effects, the mechanisms of which are not fully understood. In this review, the complex interactions between heme and TLR4 are discussed with a particular focus on the role of heme-binding serum proteins in handling extracellular heme and its impact on TLR4 signaling. Moreover, the role of heme as a direct and indirect trigger of TLR4 activation and species-specific differences in the regulation of heme-dependent TLR4 signaling are highlighted.


Assuntos
Proteínas Ligantes de Grupo Heme/metabolismo , Heme/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Microdomínios da Membrana/metabolismo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo
3.
Elife ; 92020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32780017

RESUMO

Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII hemoprotein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.


Assuntos
Proteínas Ligantes de Grupo Heme/química , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas Ligantes de Grupo Heme/genética , Proteínas de Transferência de Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais
4.
Biosci Rep ; 40(9)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32830860

RESUMO

In recent decades, many genome-wide association studies on insomnia have reported numerous genes harboring multiple risk variants. Nevertheless, the molecular functions of these risk variants conveying risk to insomnia are still ill-studied. In the present study, we integrated GWAS summary statistics (N=386,533) with two independent brain expression quantitative trait loci (eQTL) datasets (N=329) to determine whether expression-associated SNPs convey risk to insomnia. Furthermore, we applied numerous bioinformatics analyses to highlight promising genes associated with insomnia risk. By using Sherlock integrative analysis, we detected 449 significant insomnia-associated genes in the discovery stage. These identified genes were significantly overrepresented in six biological pathways including Huntington's disease (P=5.58 × 10-5), Alzheimer's disease (P=5.58 × 10-5), Parkinson's disease (P=6.34 × 10-5), spliceosome (P=1.17 × 10-4), oxidative phosphorylation (P=1.09 × 10-4), and wnt signaling pathways (P=2.07 × 10-4). Further, five of these identified genes were replicated in an independent brain eQTL dataset. Through a PPI network analysis, we found that there existed highly functional interactions among these five identified genes. Three genes of LDHA (P=0.044), DALRD3 (P=5.0 × 10-5), and HEBP2 (P=0.032) showed significantly lower expression level in brain tissues of insomnic patients than that in controls. In addition, the expression levels of these five genes showed prominently dynamic changes across different time points between behavioral states of sleep and sleep deprivation in mice brain cortex. Together, the evidence of the present study strongly suggested that these five identified genes may represent candidate genes and contributed risk to the etiology of insomnia.


Assuntos
Predisposição Genética para Doença , Locos de Características Quantitativas , Privação do Sono/genética , Distúrbios do Início e da Manutenção do Sono/genética , Animais , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Genômica , Proteínas Ligantes de Grupo Heme/genética , Humanos , L-Lactato Desidrogenase/genética , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , Córtex Pré-Frontal/patologia , Proteínas da Gravidez/genética , Prevalência , Mapeamento de Interação de Proteínas , Privação do Sono/patologia , Distúrbios do Início e da Manutenção do Sono/epidemiologia , Distúrbios do Início e da Manutenção do Sono/patologia , tRNA Metiltransferases/genética
5.
Sci Rep ; 10(1): 12021, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694623

RESUMO

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.


Assuntos
Antibacterianos/biossíntese , Bambermicinas/biossíntese , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Fósforo-Oxigênio Liases/metabolismo , Streptomyces/enzimologia , Streptomyces/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Ligantes de Grupo Heme/genética , Proteínas Ligantes de Grupo Heme/metabolismo , Morfogênese/genética , Fósforo-Oxigênio Liases/genética , Regiões Promotoras Genéticas , Sistemas do Segundo Mensageiro/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Philos Trans R Soc Lond B Biol Sci ; 375(1801): 20190488, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32362261

RESUMO

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae. Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.


Assuntos
Arabidopsis/metabolismo , Proteínas Ligantes de Grupo Heme/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Rodófitas/metabolismo , Proteínas de Algas/metabolismo , Núcleo Celular/metabolismo , Proteômica
7.
Funct Integr Genomics ; 20(4): 609-619, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32285226

RESUMO

The myofibrillar fragmentation index (MFI) is an indicative trait for meat tenderness. Longissimus thoracis muscle samples from the 20 most extreme bulls (out of 80 bulls set) for MFI (high (n = 10) and low (n = 10) groups) trait were used to perform transcriptomic analysis, using RNA Sequencing (RNA-Seq). An average of 24.616 genes was expressed in the Nellore muscle transcriptome analysis. A total of 96 genes were differentially expressed (p value ≤ 0.001) between the two groups of divergent bulls for MFI. The HEBP2 and BDH1 genes were overexpressed in animals with high MFI. The MYBPH and MYL6, myosin encoders, were identified. The differentially expressed genes were related to increase mitochondria efficiency, especially in cells under oxidative stress conditions, and these also were related to zinc and calcium binding, membrane transport, and muscle constituent proteins, such as actin and myosin. Most of those genes were involved in metabolic pathways of oxidation-reduction, transport of lactate in the plasma membrane, and muscle contraction. This is the first study applying MFI phenotypes in transcriptomic studies to identify and understand differentially expressed genes for beef tenderness. These results suggest that differences detected in gene expression between high and low MFI animals are related to reactive mechanisms and structural components of oxidative fibers under the condition of cellular stress. Some genes may be selected as positional candidate genes to beef tenderness, MYL6, MYBPH, TRIM63, TRIM55, TRIOBP, and CHRNG genes. The use of MFI phenotypes could enhance results of meat tenderness studies.


Assuntos
Bovinos/genética , Músculo Esquelético/metabolismo , Característica Quantitativa Herdável , Carne Vermelha/normas , Transcriptoma , Animais , Bovinos/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Perfilação da Expressão Gênica , Proteínas Ligantes de Grupo Heme/genética , Proteínas Ligantes de Grupo Heme/metabolismo , Masculino , Miosinas/genética , Miosinas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo
8.
Nanotechnology ; 31(31): 314002, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32259806

RESUMO

Biological electron transfer (ET) is one of the most studied biochemical processes due to its immense importance in biology. For many years, biological ET was explained using the classical incoherent transport mechanism, i.e. sequential hopping. One of the relatively recent major observations in this field is long-range extracellular ET (EET), where some bacteria were shown to mediate electrons for extremely long distances on the micrometer length scales across individual nanowires. This fascinating finding has resulted in several suggested mechanisms that might explain this intriguing EET. More recently, the structure of a conductive G. sulfurreducens nanowire has been solved, which showed a highly ordered quasi-1D wire of a hexaheme cytochrome protein, named OmcS. Based on this new structure, we suggest here several electron diffusion models for EET, involving either purely hopping or several degrees of mixed hopping and coherent transport, in which the coherent part is due to a local rigidification of the protein structure, associated with a decrease in the local reorganization energy. The effect is demonstrated for two closely packed heme sites as well as for longer chains containing up to several dozens porphyrins. We show that the pure hopping model probably cannot explain the reported conductivity values of the G. sulfurreducens nanowire using conventional values of reorganization energy and electronic coupling. On the other hand, we show that for a wide range of the latter energy values, the mixed hopping-coherent model results in superior electron diffusion compared to the pure hopping model, and especially for long-range coherent transport, involving multiple porphyrin sites.


Assuntos
Proteínas de Bactérias/química , Geobacter/metabolismo , Proteínas Ligantes de Grupo Heme/química , Porfirinas/metabolismo , Condutividade Elétrica , Transporte de Elétrons , Geobacter/química , Modelos Moleculares , Nanofios
9.
Am J Physiol Heart Circ Physiol ; 318(3): H671-H681, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004074

RESUMO

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2-/- mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin-/- mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs.NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.


Assuntos
Heme Oxigenase-1/metabolismo , Proteínas Ligantes de Grupo Heme/metabolismo , Regulação para Cima , Trombose Venosa/metabolismo , Animais , Modelos Animais de Doenças , Heme Oxigenase-1/genética , Proteínas Ligantes de Grupo Heme/genética , Hemina/farmacologia , Camundongos , Camundongos Knockout , Trombose Venosa/genética
10.
Mol Microbiol ; 113(2): 381-398, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31742788

RESUMO

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.


Assuntos
Haemophilus influenzae/efeitos dos fármacos , Haemophilus/metabolismo , Proteínas Ligantes de Grupo Heme , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/crescimento & desenvolvimento , Heme/metabolismo , Proteínas Ligantes de Grupo Heme/química , Proteínas Ligantes de Grupo Heme/isolamento & purificação , Proteínas Ligantes de Grupo Heme/farmacologia , Humanos
11.
Antioxid Redox Signal ; 32(6): 378-395, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31559835

RESUMO

Aims: Structural and functional characterization of the globin-coupled sensors (GCSs) from Azotobacter vinelandii (AvGReg) and Bordetella pertussis (BpeGReg). Results: Ultraviolet/visible and resonance Raman spectroscopies confirm the presence in AvGReg and BpeGReg of a globin domain capable of reversible gaseous ligand binding. In AvGReg, an influence of the transmitter domain on the heme proximal region of the globin domain can be seen, and k'CO is higher than for other GCSs. The O2 binding kinetics suggests the presence of an open and a closed conformation. As for BpeGReg, the fully oxygenated AvGReg show a very high diguanylate cyclase activity. The carbon monoxide rebinding to BpeGReg indicates that intra- and intermolecular interactions influence the ligand binding. The globin domains of both proteins (AvGReg globin domain and BpeGRegGb with cysteines (Cys16, 45, 114, 154) mutated to serines [BpeGReg-Gb*]) share the same GCS fold, a similar proximal but a different distal side structure. They homodimerize through a G-H helical bundle as in other GCSs. However, BpeGReg-Gb* shows also a second dimerization mode. Innovation: This article extends our knowledge on the GCS proteins and contributes to a better understanding of the GCSs role in the formation of bacterial biofilms. Conclusions: AvGReg and BpeGReg conform to the GCS family, share a similar overall structure, but they have different properties in terms of the ligand binding. In particular, AvGReg shows an open and a closed conformation that in the latter form will very tightly bind oxygen. BpeGReg has only one closed conformation. In both proteins, it is the fully oxygenated GCS form that catalyzes the production of the second messenger.


Assuntos
Azotobacter vinelandii/química , Proteínas de Bactérias/química , Bordetella pertussis/química , Globinas/química , Sítios de Ligação/fisiologia , Proteínas Ligantes de Grupo Heme/química , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Relação Estrutura-Atividade
12.
J Microbiol Immunol Infect ; 53(1): 87-93, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29807723

RESUMO

BACKGROUND/PURPOSE: HtsA (Streptococcus heme transporter A) is the lipoprotein component of the streptococcal heme ABC transporter (HtsABC). The aim of this study is to investigate whether the HtsA protein has immunoprotective effect against group A Streptococcus (GAS) infection in mice. METHODS: The HtsA protein was purified by sequential chromatography on Ni-sepharose, DEAE-sepharose and Phenyl-sepharose, CD-1 mice were actively immunized with ALUM (control) or HtsA/ALUM, and passively immunized with control or anti-HtsA serum. Mice were challenged with GAS after immunization, and the survival rate, skin lesion size and systemic GAS dissemination were determined. RESULTS: The HtsA gene was cloned, and the recombinant protein HtsA was successfully purified. HtsA has a strong antigenicity, and active immunization with the HtsA protein significantly protected mice against lethal subcutaneous GAS infection, inhibited invasion of the skin by GAS, and reduced GAS systemic dissemination in blood and organs. In addition, passive immunization with anti-HtsA serum also significantly protected mice against subcutaneous GAS infection, and inhibited invasion of the skin by GAS. CONCLUSION: The results showed that both active and passive immunization with the HtsA protein protected mice against subcutaneous GAS infection, suggesting that HtsA may be a candidate of GAS vaccine to protect against GAS infection.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas Ligantes de Grupo Heme/imunologia , Imunização Passiva , Lipoproteínas/imunologia , Infecções Estreptocócicas/prevenção & controle , Vacinação , Animais , Proteínas de Bactérias/administração & dosagem , Feminino , Proteínas Ligantes de Grupo Heme/administração & dosagem , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lipoproteínas/administração & dosagem , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Infecções Estreptocócicas/imunologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-31767723

RESUMO

Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. bovis katG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INH-resistance mechanisms, and suggest paths to overcome them.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Isoniazida/farmacologia , Mycobacterium/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Catalase/genética , Ativação Enzimática , Proteínas Ligantes de Grupo Heme/genética , Proteínas Ligantes de Grupo Heme/metabolismo , Mycobacterium/enzimologia , Mycobacterium/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/enzimologia , Micobactérias não Tuberculosas/genética , Filogenia , Multimerização Proteica , Alinhamento de Sequência , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
14.
Res Microbiol ; 170(8): 345-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31678562

RESUMO

Iron acquisition is an essential aspect of cell physiology for most bacteria. Although much is known about how bacteria initially recognize the various iron sources they can encounter, whether siderophore, heme, host iron/heme binding proteins, much less is known about how the iron containing compounds (Fe2+, Fe3+, Fe3+-siderophore complex or heme) are transported across the cytoplasmic membrane. This last transport step is powered by specific ABC (ATP-Binding-Cassette) transporters, made up of a substrate binding protein (SBP) that delivers its cargo to the TMD (TransMembrane Domain) of the ABC transporter triggering the entry of the substrate inside the cytoplasm upon catalytic activity of the ABC module. This review focuses on structural aspects of the functioning of such ABC transporters with the most part devoted to the substrate binding proteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bactérias/metabolismo , Compostos de Ferro/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Heme/metabolismo , Proteínas Ligantes de Grupo Heme/metabolismo , Modelos Moleculares , Sideróforos/metabolismo
15.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31453805

RESUMO

Alzheimer's disease is the most prevalent neurodegenerative disorder leading to progressive cognitive decline. Despite decades of research, understanding AD progression at the molecular level, especially at its early stages, remains elusive. Here, we identified several presymptomatic AD markers by investigating brain proteome changes over the course of neurodegeneration in a transgenic mouse model of AD (3×Tg-AD). We show that one of these markers, heme-binding protein 1 (Hebp1), is elevated in the brains of both 3×Tg-AD mice and patients affected by rapidly-progressing forms of AD. Hebp1, predominantly expressed in neurons, interacts with the mitochondrial contact site complex (MICOS) and exhibits a perimitochondrial localization. Strikingly, wildtype, but not Hebp1-deficient, neurons showed elevated cytotoxicity in response to heme-induced apoptosis. Increased survivability in Hebp1-deficient neurons is conferred by blocking the activation of the mitochondrial-associated caspase signaling pathway. Taken together, our data highlight a role of Hebp1 in progressive neuronal loss during AD progression.


Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Expressão Gênica , Proteínas Ligantes de Grupo Heme/biossíntese , Animais , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Proteoma/análise
16.
Virulence ; 10(1): 315-333, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30973092

RESUMO

Nutrient iron sequestration is the most significant form of nutritional immunity and causes bacterial pathogens to evolve strategies of host iron scavenging. Cigarette smoking contains iron particulates altering lung and systemic iron homeostasis, which may enhance colonization in the lungs of patients suffering chronic obstructive pulmonary disease (COPD) by opportunistic pathogens such as nontypeable. NTHi is a heme auxotroph, and the NTHi genome contains multiple heme acquisition systems whose role in pulmonary infection requires a global understanding. In this study, we determined the relative contribution to NTHi airway infection of the four heme-acquisition systems HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF that are located at the bacterial outer membrane or the periplasm. Our computational studies provided plausible 3D models for HbpA, SapA, PE, and HxuA interactions with heme. Generation and characterization of single mutants in the hxuCBA, hpe, sapA, and hbpA genes provided evidence for participation in heme binding-storage and inter-bacterial donation. The hxuA, sapA, hbpA, and hpe genes showed differential expression and responded to heme. Moreover, HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF presented moonlighting properties related to resistance to antimicrobial peptides or glutathione import, together likely contributing to the NTHi-host airway interplay, as observed upon cultured airway epithelia and in vivo lung infection. The observed multi-functionality was shown to be system-specific, thus limiting redundancy. Together, we provide evidence for heme uptake systems as bacterial factors that act in a coordinated and multi-functional manner to subvert nutritional- and other sources of host innate immunity during NTHi airway infection.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Haemophilus influenzae/patogenicidade , Heme/metabolismo , Interações Hospedeiro-Patógeno , Pulmão/microbiologia , Infecções Respiratórias/microbiologia , Células A549 , Animais , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Simulação por Computador , Feminino , Proteínas Ligantes de Grupo Heme/genética , Proteínas Ligantes de Grupo Heme/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular
17.
J Microbiol ; 57(2): 138-142, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30706342

RESUMO

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.


Assuntos
Oxigênio/metabolismo , Thermococcus/enzimologia , Thermococcus/crescimento & desenvolvimento , Thermococcus/genética , Antioxidantes , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citocromos/genética , Citocromos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica em Archaea , Genes Arqueais/genética , Proteínas Ligantes de Grupo Heme , Hemeproteínas/genética , Hemeproteínas/metabolismo , Hemeritrina/genética , Hemeritrina/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Rubredoxinas/genética , Rubredoxinas/metabolismo , Thermococcus/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Transcriptoma , Regulação para Cima
18.
Pathol Oncol Res ; 25(1): 279-288, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29103201

RESUMO

The aim of the present study was to investigate the relationship between the intensity of biomarker expression and the response to radiochemotherapy in patients with advanced esophageal squamous cell cancer (ESCC). Ninety-two patients with locally advanced ESCC were examined retrospectively. Pre-treatment tumor samples were stained for proteins SOUL, Hsp 16.2, Growth Hormone-Releasing Hormone Receptor (GHRH-R) and p-Akt using immunhistochemistry methods. Kaplan-Meier curves were used to show the relationship between intensity of expression of biomarkers and clinical parameters and 3-year OS. A significant correlation was found between high intensity staining for Hsp 16.2, p-Akt and SOUL and poor response to NRCT. Application of a higher dose of radiation and higher dose of cisplatin resulted in better clinical and histopathological responses, respectively. Among the clinical parameters, the localization of the tumor in the upper-third of the esophagus and less than 10% weight loss were independent prognostic factors for increased 3-year OS. Hsp16.2, p-Akt and SOUL are predictors of negative response to NRCT, therefore these biomarkers may become promising targets for therapy. Furthermore, level of expression of p-Akt, weight loss and the localization of the tumor are significant factors in the prediction of OS in ESCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Quimiorradioterapia/mortalidade , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/terapia , Feminino , Seguimentos , Proteínas Ligantes de Grupo Heme , Hemeproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas da Gravidez/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estudos Retrospectivos , Taxa de Sobrevida
19.
Int J Mol Sci ; 19(12)2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30501057

RESUMO

SOUL, a heme-binding protein-2 (HEBP-2), interacts with apoptosis-linked gene 2 protein (ALG-2) in a Ca2+-dependent manner. To investigate the properties of the interaction of SOUL with ALG-2, we generated several mutants of SOUL and ALG-2 and analyzed the recombinant proteins using pulldown assay and isothermal titration calorimetry. The interaction between SOUL and ALG-2 (delta3-23ALG-2) was an exothermic reaction, with 1:1 stoichiometry and high affinity (Kd = 32.4 nM) in the presence of Ca2+. The heat capacity change (ΔCp) of the reaction showed a large negative value (-390 cal/K·mol), which suggested the burial of a significant nonpolar surface area or disruption of a hydrogen bond network that was induced by the interaction (or both). One-point mutation of SOUL Phe100 or ALG-2 Trp57 resulted in complete loss of heat change, supporting the essential roles of these residues for the interaction. Nevertheless, a truncated mutant of SOUL1-143 that deleted the domain required for the interaction with ALG-2 Trp57 still showed 1:1 binding to ALG-2 with an endothermic reaction. These results provide a better understanding of the target recognition mechanism and conformational change of SOUL in the interaction with ALG-2.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Hemeproteínas/metabolismo , Proteínas da Gravidez/metabolismo , Termodinâmica , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Calorimetria , Cromatografia em Gel , Dicroísmo Circular , Proteínas Ligantes de Grupo Heme , Hemeproteínas/genética , Camundongos , Proteínas da Gravidez/genética , Ligação Proteica
20.
Rev. biol. trop ; 66(4): 1412-1420, oct.-dic. 2018. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-1003334

RESUMO

Resumen Nuevos agentes antimaláricos a partir de plantas son estudiados como alternativas en el tratamiento de la malaria. Los principales antimaláricos como la cloroquina tienen varios mecanismos de acción contra parásitos, uno de ellos es la inhibición de polimerización del grupo hemo, modelo que ha permitido el diseño de nuevos candidatos antimaláricos. En este sentido, el objetivo de este trabajo fue evaluar extractos de plantas de género Piper y Calophyllum sobre la capacidad de inhibición de la β-hematina. Se informa las concentraciones inhibitorias de la formación de β-hematina por parte de 40 extractos de diferente polaridad obtenidos a partir de las especies P. piedecuestanum, C. brasiliense, C. longinforium, y Calophyllum. sp. 19 extractos mostraron un mayor potencial para inhibir la formación de β−hematina con CI50 < 3mg / ml. Estas actividades respaldan principalmente, futuros estudios con el género Calophyllum, en el desarrollo y descubrimiento de nuevas sustancias antiplasmodiales con modos de acción conocido.(AU)


Abstract New antimalarial agents from plants are studied as alternatives in the treatment of malaria. The main antimalarials such as chloroquine have several mechanisms of action against parasites, one of which is the inhibition of polymerization of the heme group, a model that has allowed the design of new antimalarial candidates. In this sense the objective of this work was to evaluate extracts of genus Piper and Calophyllum plants on the inhibition capacity of β-hematin. Inhibitory concentrations of β-hematin are reported from 40 extracts of different polarity obtained from the species P. piedecuestanum, C. brasiliense, C. longinforium, and Calophyllum. sp. 19 extracts showed a greater potential to inhibit β-hematin with IC50 < 3 mg/ml. These activities mainly support future studies with the genus Calophyllum in the development and discovery of new antiplasmodial substances with known modes of action.(AU)


Assuntos
Cloroquina/farmacologia , Polimerização/efeitos dos fármacos , Proteínas Ligantes de Grupo Heme , Malária/tratamento farmacológico
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