RESUMO
ConspectusCentral to the quest of understanding the emergence of life is to uncover the role of metals, particularly iron, in shaping prebiotic chemistry. Iron, as the most abundant of the accessible transition metals on the prebiotic Earth, played a pivotal role in early biochemical processes and continues to be indispensable to modern biology. Here, we discuss our recent contributions to probing the plausibility of prebiotic complexes with iron, including heme and iron-sulfur clusters, in mediating chemistry beneficial to a protocell. Laboratory experiments and spectroscopic findings suggest plausible pathways, often facilitated by UV light, for the synthesis of heme and iron-sulfur clusters. Once formed, heme displays catalytic, peroxidase-like activity when complexed with amphiphiles. This activity could have been beneficial in two ways. First, heme could have catalytically removed a molecule (H2O2) that could have had degradative effects on a protocell. Second, heme could have helped in the synthesis of the building blocks of life by coupling the reduction of H2O2 with the oxidation of organic substrates. The necessity of amphiphiles to avoid the formation of inactive complexes of heme is telling, as the modern-day electron transport chain possesses heme embedded within a lipid membrane. Conversely, prebiotic iron-sulfur peptides have yet to be reported to partition into lipid membranes, nor have simple iron-sulfur peptides been found to be capable of participating in the synthesis of organic molecules. Instead, iron-sulfur peptides span a wide range of reduction potentials complementary to the reduction potentials of hemes. The reduction potential of iron-sulfur peptides can be tuned by the type of iron-sulfur cluster formed, e.g., [2Fe-2S] versus [4Fe-4S], or by the substitution of ligands to the metal center. Since iron-sulfur clusters easily form upon stochastic encounters between iron ions, hydrosulfide, and small organic molecules possessing a thiolate, including peptides, the likelihood of soluble iron-sulfur clusters seems to be high. What remains challenging to determine is if iron-sulfur peptides participated in early prebiotic chemistry or were recruited later when protocellular membranes evolved that were compatible with the exploitation of electron transfer for the storage of energy as a proton gradient. This problem mirrors in some ways the difficulty in deciphering the origins of metabolism as a whole. Chemistry that resembles some facets of extant metabolism must have transpired on the prebiotic Earth, but there are few clues as to how and when such chemistry was harnessed to support a (proto)cell. Ultimately, unraveling the roles of hemes and iron-sulfur clusters in prebiotic chemistry promises to deepen our understanding of the origins of life on Earth and aids the search for life elsewhere in the universe.
Assuntos
Heme , Heme/química , Heme/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Ferro/química , Ferro/metabolismo , Enxofre/química , Células Artificiais/química , Células Artificiais/metabolismoRESUMO
Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis to cause dysbiosis also in the gut microbiome.
Assuntos
Microbioma Gastrointestinal , Porphyromonas gingivalis , Humanos , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Disbiose , Heme/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Developing artificial enzymes based on organic molecules or polymers for reactive oxygen species (ROS)-related catalysis has broad applicability. Herein, inspired by porphyrin-based heme mimics, we report the synthesis of polyphthalocyanine-based conjugated polymers (Fe-PPc-AE) as a new porphyrin-evolving structure to serve as efficient and versatile artificial enzymes for augmented reactive oxygen catalysis. Owing to the structural advantages, such as enhanced π-conjugation networks and π-electron delocalization, promoted electron transfer, and unique Fe-N coordination centers, Fe-PPc-AE showed more efficient ROS-production activity in terms of Vmax and turnover numbers as compared with porphyrin-based conjugated polymers (Fe-PPor-AE), which also surpassed reported state-of-the-art artificial enzymes in their activity. More interestingly, by changing the reaction medium and substrates, Fe-PPc-AE also revealed significantly improved activity and environmental adaptivity in many other ROS-related biocatalytic processes, validating the potential of Fe-PPc-AE to replace conventional (poly)porphyrin-based heme mimics for ROS-related catalysis, biosensors, or biotherapeutics. It is suggested that this study will offer essential guidance for designing artificial enzymes based on organic molecules or polymers.
Assuntos
Heme , Porfirinas , Heme/química , Oxigênio/química , Espécies Reativas de Oxigênio , Porfirinas/química , Catálise , PolímerosRESUMO
Dye-decolorizing peroxidases (DyPs), a type of heme-containing oxidoreductase enzymes, catalyze the peroxide-dependent oxidation of various industrial dyes as well as lignin and lignin model compounds. In our previous work, we have recently reported the crystal structures of class A-type DyP from Bacillus subtilis at pH 7.0 (BsDyP7), exposing the location of three binding sites for small substrates and high redox-potential substrates. The biochemical studies revealed the optimum acidic pH for enzyme activity. In the present study, the crystal structure of BsDyP at acidic pH (BsDyP4) reveals two-monomer units stabilized by intermolecular salt bridges and a hydrogen bond network in a homo-dimeric unit. Based on the monomeric structural comparison of BsDyP4 and BsDyP7, minor differences were observed in the loop regions, that is, LI (Ala64-Gln71), LII (Glu96-Lys108), LIII (Pro117-Leu124), and LIV (Leu295-Asp303). Despite these differences, BsDyP4 adopts similar heme architecture as well as three substrate-binding sites to BsDyP7. In BsDyP4, a shift in Asp187, heme pocket residue discloses the plausible reason for optimal acidic pH for BsDyP activity. This study provides insight into the structural changes in BsDyP at acidic pH, where BsDyP is biologically active.
Assuntos
Bacillus subtilis , Peroxidase , Peroxidase/metabolismo , Corantes/metabolismo , Lignina/química , Peroxidases/química , Peroxidases/metabolismo , Concentração de Íons de Hidrogênio , Heme/metabolismoRESUMO
Genetically modified microorganisms (GMMs) can enable a wide range of important applications including environmental sensing and responsive engineered living materials. However, containment of GMMs to prevent environmental escape and satisfy regulatory requirements is a bottleneck for real-world use. While current biochemical strategies restrict unwanted growth of GMMs in the environment, there is a need for deployable physical containment technologies to achieve redundant, multi-layered and robust containment. We developed a hydrogel-based encapsulation system that incorporates a biocompatible multilayer tough shell and an alginate-based core. This deployable physical containment strategy (DEPCOS) allows no detectable GMM escape, bacteria to be protected against environmental insults including antibiotics and low pH, controllable lifespan and easy retrieval of genomically recoded bacteria. To highlight the versatility of DEPCOS, we demonstrated that robustly encapsulated cells can execute useful functions, including performing cell-cell communication with other encapsulated bacteria and sensing heavy metals in water samples from the Charles River.
Assuntos
Bactérias/efeitos dos fármacos , Hidrogéis/farmacologia , Alginatos/química , Antibacterianos/farmacologia , Bactérias/genética , Materiais Biocompatíveis , Bioengenharia , DNA Bacteriano/química , DNA Bacteriano/genética , Monitoramento Ambiental , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Heme/química , Metais Pesados/química , Organismos Geneticamente Modificados , Percepção de Quorum , Rios , Poluentes da Água/químicaRESUMO
The binding of nitric oxide (NO) to heme in the ß1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1ß1 and α2ß1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and ß1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and ß1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the ß1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.
Assuntos
Polpa Dentária , Guanilato Ciclase , Humanos , Guanilil Ciclase Solúvel/genética , Guanilil Ciclase Solúvel/metabolismo , Guanilato Ciclase/metabolismo , Polpa Dentária/metabolismo , Óxido Nítrico/metabolismo , Inflamação , Heme , GMP Cíclico/metabolismoRESUMO
Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD.
Assuntos
Anemia Falciforme , Malária , MicroRNAs , Humanos , MicroRNAs/metabolismo , Hemólise , Lipossomos/metabolismo , Heme/metabolismo , Células Endoteliais/metabolismo , Hemopexina/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Proteínas 14-3-3/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Inflamação/genética , Inflamação/metabolismo , Anti-Inflamatórios/metabolismo , Malária/metabolismoRESUMO
A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N3- readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 µM. The enzyme is also stimulated to varying degrees by α-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl- 6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism. © 1985 Academic Press, Inc.
Assuntos
Lignina , Phanerochaete , Corantes , Difosfatos , Heme , Peroxidase do Rábano Silvestre , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , NAD , Peroxidase , Peroxidases/metabolismoRESUMO
Cell-free heme, which was previously shown to have adverse effects on the innate immune system, does not induce inflammation when bound to a protein carrier via overexpression of the enzyme heme-oxygenase 1 (HO-1). Studies in mouse macrophage cell culture and human endothelial cells have confirmed HO-1 catalyzed breakdown of protein bound heme into biliverdin, iron, and carbon monoxide (CO), which elicits anti-inflammatory effects. However, to fully realize the anti-inflammatory therapeutic effects of heme, a colloidally stable heme protein carrier must be developed. To accomplish this goal, we incorporated multiple heme molecules into human serum albumin (HSA) via partial unfolding of HSA at basic pH followed by refolding at neutral pH, and subsequently conjugated the surface of the heme-HSA complex with polyethylene glycol (PEG) to stabilize heme-HSA. Quantification studies confirmed that a maximum of 5-6 hemes could be bound to HSA without precipitation or degradation of heme-HSA. Dynamic light scattering, size exclusion-high performance liquid chromatography (SEC-HPLC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the increase in hydrodynamic diameter and molecular weight (MW), respectively, upon PEGylation of heme-HSA. Furthermore, PEG-heme-HSA was stable upon exposure to different pH environments, freeze-thaw cycles, and storage at 4°C. Taken together, we devised a synthesis and purification platform for the production of PEGylated heme-incorporated HSA that can be used to test the potential anti-inflammatory effects of heme in vivo.
Assuntos
Heme , Albumina Sérica , Humanos , Camundongos , Animais , Heme/metabolismo , Albumina Sérica/química , Células Endoteliais/metabolismo , Polietilenoglicóis/química , Anti-InflamatóriosRESUMO
To acquire heme, Porphyromonas gingivalis uses a hemophore-like protein (HmuY). HmuY sequesters heme from host hemoproteins or heme-binding proteins produced by cohabiting bacteria, and delivers it to the TonB-dependent outer-membrane receptor (HmuR). Although three-dimensional protein structures of members of the novel HmuY family are overall similar, significant differences exist in their heme-binding pockets. Histidines (H134 and H166) coordinating the heme iron in P. gingivalis HmuY are unique and poorly conserved in the majority of its homologs, which utilize methionines. To examine whether changes observed in the evolution of these proteins in the Bacteroidetes phylum might result in improved heme binding ability of HmuY over its homologs, we substituted histidine residues with methionine residues. Compared to the native HmuY, site-directed mutagenesis variants bound Fe(III)heme with lower ability in a similar manner to Bacteroides vulgatus Bvu and Tannerella forsythia Tfo. However, a mixed histidine-methionine couple in the HmuY was sufficient to bind Fe(II)heme, similarly to T. forsythia Tfo, Prevotella intermedia PinO and PinA. Double substitution resulted in abolished heme binding. The structure of HmuY heme-binding pocket may have been subjected to evolution, allowing for P. gingivalis to gain an advantage in heme acquisition regardless of environmental redox conditions.
Assuntos
Hemeproteínas , Porphyromonas gingivalis , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Heme/química , Hemeproteínas/química , Hemeproteínas/genética , Porphyromonas gingivalis/químicaRESUMO
One of the most widely used molecules used for photodynamic therapy (PDT) is 5-aminolevulinic acid (5-ALA), a precursor in the synthesis of tetrapyrroles such as chlorophyll and heme. The 5-ALA skin permeation is considerably reduced due to its hydrophilic characteristics, decreasing its local bioavailability and therapeutic effect. For this reason, five different systems containing polymeric particles of poly [D, L-lactic-co-glycolic acid (PLGA)] were developed to encapsulate 5-ALA based on single and double emulsions methodology. All systems were standardized (according to the volume of reagents and mass of pharmaceutical ingredients) and compared in terms of laboratory scaling up, particle formation and stability over time. UV-VIS spectroscopy revealed that particle absorption/adsorption of 5-ALA was dependent on the method of synthesis. Different size distribution was observed by DLS and NTA techniques, revealing that 5-ALA increased the particle size. The contact angle evaluation showed that the system hydrophobicity was dependent on the surfactant and the 5-ALA contribution. The FTIR results indicated that the type of emulsion influenced the particle formation, as well as allowing PEG functionalization and interaction with 5-ALA. According to the 1H-NMR results, the 5-ALA reduced the T1 values of polyvinyl alcohol (PVA) and PLGA in the double emulsion systems due to the decrease in molecular packing in the hydrophobic region. The results indicated that the system formed by single emulsion containing the combination PVA-PEG presented greater stability with less influence from 5-ALA. This system is a promising candidate to successfully encapsulate 5-ALA and achieve good performance and specificity for in vitro skin cancer treatment.
Assuntos
Ácido Aminolevulínico , Ácido Poliglicólico , Clorofila , Emulsões , Heme , Ácido Láctico/química , Tamanho da Partícula , Polietilenoglicóis/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Álcool de Polivinil/química , Tensoativos , TetrapirróisRESUMO
Heme enzymes, with the pentacoordinate heme iron active sites, possess high catalytic activity and selectivity in biosensing applications. However, they are still subject to limited catalytic stability in the complex environment and high cost for broad applications in electrochemical sensing. It is meaningful to develop a novel substitute that has a similar structure to some heme enzymes and mimics their enzyme activities. One emerging strategy is to design the Fe-N-C based single-atomic site catalysts (SASCs). The obtained atomically dispersed Fe-Nx active sites can mimic the active sites of heme enzymes effectively. In this work, a SASC (Fe-SASC/NW) is synthesized by doping single iron atoms in polypyrrole (PPy) derived carbon nanowire via a zinc-atom-assisted method. The proposed Fe-SASC/NW shows high heme enzyme-like catalytic performance for hydrogen peroxide (H2 O2 ) with a specific activity of 42.8 U mg-1 . An electrochemical sensor based on Fe-SASC/NW is developed for the detection of H2 O2 . This sensor exhibits a wide detection concentration range from 5.0 × 10-10 m to 0.5 m and an excellent limit of detection (LOD) of 46.35 × 10-9 m. Such excellent catalytic activity and electrochemical sensing sensitivity are attributed to the isolated Fe-Nx active sites and their structural similarity with natural metalloproteases.
Assuntos
Heme , Peróxido de Hidrogênio , Domínio Catalítico , Polímeros , PirróisRESUMO
As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.
Assuntos
Heme/genética , Hemeproteínas/genética , Periodontite/genética , Prevotella intermedia/genética , Regulação Bacteriana da Expressão Gênica/genética , Heme/química , Hemeproteínas/química , Humanos , Ferro/metabolismo , Periodontite/microbiologia , Periodontite/patologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/patogenicidade , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
The underlying mechanisms of microRNAs (miRNAs) in regulating nanoplastic toxicity are still largely unclear in organisms. In nanopolystyrene (NPS) exposed Caenorhabditis elegans, the expression of mir-76 (a neuronal miRNA) was significantly decreased, and the mir-76 mutant was resistant to the toxicity of NPS. The aim of this study was to determine the molecular basis of mir-76 in controlling NPS toxicity in nematodes. The mir-76 mutation increased expression of glb-10 encoding a globin protein in NPS (1 µg/L) exposed nematodes. Exposure to NPS (1-100 µg/L) increased the glb-10 expression, and the glb-10(RNAi) worm was susceptible to NPS toxicity in inducing reactive oxygen species (ROS) production and in decreasing locomotion behavior. Using ROS production and locomotion behavior as endpoints, mutation of glb-10 inhibited resistance of mir-76 mutant to NPS toxicity, and neuronal overexpression of mir-76 inhibited the resistance to NPS toxicity in nematodes overexpressing neuronal glb-10 containing 3' untranslated region (3'UTR). Thus, GLB-10 functioned as a target of mir-76 in the neurons to regulate the NPS toxicity. Moreover, a signaling cascade of HRG-7-HRG-5 required for the control of heme homeostasis was identified to function downstream of neuronal GLB-10 to regulate the NPS toxicity. In this signaling cascade, the neuronal HRG-7 regulated the NPS toxicity by antagonizing function of intestinal HRG-5. Furthermore, in the intestine, HRG-5 controlled NPS toxicity by inhibiting functions of hypoxia-inducible transcriptional factor HIF-1 and transcriptional factor ELT-2. Our results highlight the crucial function of heme homeostasis related signaling in regulating the NPS toxicity in organisms.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/efeitos dos fármacos , Heme/metabolismo , Homeostase , MicroRNAs/genética , Nanoestruturas/toxicidade , Poliestirenos/toxicidade , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Intestinos/efeitos dos fármacos , Nanoestruturas/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Poliestirenos/química , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Human oral and gut microbiomes are crucial for maintenance of homeostasis in the human body. Porphyromonas gingivalis, the key etiologic agent of chronic periodontitis, can cause dysbiosis in the mouth and gut, which results in local and systemic infectious inflammatory diseases. Our previous work resulted in extensive biochemical and functional characterization of one of the major P. gingivalis heme acquisition systems (Hmu), with the leading role played by the HmuY hemophore-like protein. We continued our studies on the homologous heme acquisition protein (Bvu) expressed by Bacteroides vulgatus, the dominant species of the gut microbiome. Results from spectrophotometric experiments showed that Bvu binds heme preferentially under reducing conditions using Met145 and Met172 as heme iron-coordinating ligands. Bvu captures heme bound to human serum albumin and only under reducing conditions. Importantly, HmuY is able to sequester heme complexed to Bvu. This is the first study demonstrating that B. vulgatus expresses a heme-binding hemophore-like protein, thus increasing the number of members of a novel HmuY-like family. Data gained in this study confirm the importance of HmuY in the context of P. gingivalis survival in regard to its ability to cause dysbiosis also in the gut microbiome.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides/metabolismo , Heme/metabolismo , Porphyromonas gingivalis/metabolismo , Humanos , Ligação ProteicaRESUMO
Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Heme/química , Mutação , Peroxidase/química , Peroxidase/metabolismo , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Corantes/química , Corantes/metabolismo , Estabilidade Enzimática , Heme/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Peroxidase/genética , Conformação ProteicaRESUMO
The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency (kcat/Km) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-ß-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.
Assuntos
Corantes/química , Lignina/metabolismo , Mioglobina/química , Peroxidase/metabolismo , Engenharia de Proteínas , Animais , Cromatografia Líquida de Alta Pressão , Cor , Guaifenesina/análogos & derivados , Heme/química , Peróxido de Hidrogênio/metabolismo , Cinética , Oxirredução , Polimerização , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , CachaloteRESUMO
The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV-visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.
Assuntos
Infecções por Bacteroidaceae/metabolismo , Complicações do Diabetes/fisiopatologia , Eritrócitos/metabolismo , Heme/metabolismo , Hemoglobinas/metabolismo , Periodontite/microbiologia , Porphyromonas gingivalis/patogenicidade , Animais , Infecções por Bacteroidaceae/microbiologia , Infecções por Bacteroidaceae/patologia , Glicosilação , Hemeproteínas/química , Hemoglobinas/química , Cavalos , Periodontite/patologia , Porphyromonas gingivalis/isolamento & purificação , Porphyromonas gingivalis/metabolismoRESUMO
Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α1- and ß1-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α1- and ß1-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α1- and ß1-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.
Assuntos
Inflamação/genética , Óxido Nítrico/genética , Periodonto/metabolismo , Guanilil Ciclase Solúvel/genética , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , GMP Cíclico/genética , Regulação da Expressão Gênica/genética , Heme/genética , Humanos , Inflamação/patologia , Ferro/metabolismo , Osteoclastos/metabolismo , Oxirredução/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Ligamento Periodontal/patologia , Periodonto/patologiaRESUMO
A recently characterized cytochrome P450 isozyme GcoA activates lignin components through a selective O-demethylation or alternatively an acetal formation reaction. These are important reactions in biotechnology and, because lignin is readily available; it being the main component in plant cell walls. In this work we present a density functional theory study on a large active site model of GcoA to investigate syringol activation by an iron(IV)-oxo heme cation radical oxidant (Compoundâ I) leading to hemiacetal and acetal products. Several substrate-binding positions were tested and full energy landscapes calculated. The study shows that substrate positioning determines the product distributions. Thus, with the phenol group pointing away from the heme, an O-demethylation is predicted, whereas an initial hydrogen-atom abstraction of the weak phenolic O-H group would trigger a pathway leading to ring-closure to form acetal products. Predictions on how to engineer P450 GcoA to get more selective product distributions are given.