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1.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445381

RESUMO

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.


Assuntos
Hemina/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Lacase/metabolismo , Albumina Sérica Humana/química , Tirosina/química , Sítios de Ligação , Biocatálise , Química Click , Sistemas de Liberação de Medicamentos , Humanos , Ibuprofeno/química , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Albumina Sérica Humana/metabolismo
2.
Biomolecules ; 11(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34439841

RESUMO

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mitochondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5-10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site.


Assuntos
Encéfalo/metabolismo , Hemina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Ligação Proteica , Proteolipídeos/metabolismo , Ratos
3.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360665

RESUMO

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K+ or NH4+ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.


Assuntos
Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , DNA Catalítico/metabolismo , Fibrinogênio/metabolismo , Quadruplex G , Hemina/metabolismo , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Humanos , Modelos Moleculares
4.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443603

RESUMO

Abnormal levels of reduced glutathione (GSH) and glutathione reductase (GR) are usually related to a variety of diseases, so it is of great significance to determine the GSH concentration and GR activity. We herein develop a smartphone-assisted colorimetric biosensor for the detection of GSH and GR activity in human serum and mouse liver using hemin/G-quadruplex DNAzyme. Firstly, an obvious color change from colorless to green can be observed, owing to the high peroxidase-like activity of hemin/G-quadruplex DNAzyme toward 2,2'-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS). With the addition of GSH or GR, the H2O2-mediated oxidation of ABTS catalyzed by hemin/G-quadruplex DNAzyme is significantly inhibited, resulting in remarkable color fading. Therefore, the detection of GSH and GR activity can be achieved by observing the color transition or measuring the absorbance at 420 nm. The detection limit was estimated to be as low as 0.1 µM and 10 µU/mL for GSH and GR, respectively. More interestingly, the RGB values of the sensing system can be identified by the smartphone application (APP, color collect), which makes it an ideal format for on-site determination and point-of-care testing (POCT). In addition, the proposed method shows excellent selectivity and acceptable applicability for the determination of GSH concentration and GR activity in human serum samples and mouse liver tissues, which might hold great application potential in clinical diagnosis and drug screening.


Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , Glutationa Redutase/sangue , Glutationa/sangue , Hemina/metabolismo , Fígado/metabolismo , Smartphone , Animais , Colorimetria , DNA Catalítico/química , Quadruplex G , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Camundongos , Oxirredução
5.
Int J Mol Sci ; 22(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34445741

RESUMO

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.


Assuntos
COVID-19/etiologia , Hemoglobinas/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , COVID-19/sangue , Hemina/metabolismo , Hemoglobinas/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteômica , Protoporfirinas/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Estruturais Virais/ultraestrutura , Ligação Viral , Replicação Viral
6.
Appl Environ Microbiol ; 87(15): e0036721, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-33990314

RESUMO

Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer, a Gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes hemoglobin-derived hemin as an iron source; however, the mechanism by which this bacterium acquires hemin from hemoglobin is largely unknown. Here, rhuA disruption was shown to impair iron utilization from duck hemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane hemin-binding protein, but it could not extract hemin from duck hemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A, and RhuAH149A lost hemin-binding ability, suggesting that amino acid sites at tyrosine 144 (Y144), Y177, and histidine 149 (H149) are crucial for hemin binding. Furthermore, rhuR, the gene adjacent to rhuA, encodes a TonB2-dependent hemin transporter. The function of rhuA in duck hemoglobin utilization was abolished in the rhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1 ΔrhuA rather than R. anatipestifer CH-1 ΔrhuR ΔrhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA hemin utilization locus exhibits no similarity to those of characterized hemin transport systems. Thus, this locus is a novel hemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, hemin from hemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain hemin from hemoglobin, but the mechanisms of hemin acquisition from hemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of hemin acquisition from mammalian hemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in hemin acquisition from duck hemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane hemin-binding protein, and RhuR was identified as a TonB2-dependent hemin transporter. Moreover, the function of RhuA in hemoglobin utilization is RhuR dependent and not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel hemin transportation system of Gram-negative bacteria. This study not only was important for understanding hemin uptake in R. anatipestifer but also enriched the knowledge about the hemin transportation pathway in Gram-negative bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Hemina/metabolismo , Proteínas de Membrana/metabolismo , Riemerella/metabolismo , Animais , Proteínas de Bactérias/genética , Patos , Escherichia coli/genética , Hemoglobinas/metabolismo , Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas Recombinantes/metabolismo
7.
J Mater Chem B ; 9(16): 3509-3514, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33909733

RESUMO

Early glucose detection is important in both healthy people and diabetic patients. The glucose colorimetric detection techniques usually consist of multiple steps and their preparation processes are time consuming. In this work, we fabricate a GOX-hemin nanogel (GHN) that could be used for one-step colorimetry detection of glucose. The GHN was prepared by carrying out polymerization on the surface of GOX. Each GOX-hemin nanogel consists of a single GOX encapsulated with a thin polymer network containing hemin. The proximity of hemin to GOX facilitates two reactions, i.e. the oxidation of glucose catalysed by GOX to yield H2O2, and the subsequent 3,3',5,5'-tetramethylbenzidine (TMB) oxidation reaction catalysed by hemin to yield the blue colored product. These processes work in tandem, which greatly enhances the efficacy, sensitivity and stability of the detection system. The limit of detection in our system was determined to be as low as 4 µM. Furthermore, the glucose detection activity still maintained more than 70% even after being incubated at 55 °C for 30 minutes, or in 20% (v/v) aqueous solution of DMF, CH3CN or THF for 25 minutes at room temperature. It is anticipated that this work can provide a method for developing diverse functional materials based on proteins.


Assuntos
Glucose Oxidase/química , Glucose/análise , Hemina/química , Nanogéis/química , Glucose Oxidase/metabolismo , Hemina/metabolismo , Humanos , Estrutura Molecular , Tamanho da Partícula
8.
Eur J Med Chem ; 215: 113271, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33596489

RESUMO

Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was implemented in the present study in an attempt to produce compound candidates to treat malarial infections by CQ-resistant strains. Two groups of molecules were produced from the 4-aminoquinoline ring in conjugation to hydrazones (HQ) and imines (IQ). Physicochemical and pharmacokinetic properties were found to be favorable when analyzed in silico and cytotoxicity and antiplasmodial activity were assayed in vitro and in vivo showing low cytotoxicity and selectiveness to the parasites. Candidates IQ5 and IQ6 showed important values of parasite growth inhibition in vivo on the 5th day after infection (IQ5 15 mg/kg = 72.64% and IQ6 15 mg/kg = 71.15% and 25 mg/kg = 93.7%). IQ6 also showed interaction with ferriprotoporphyrin IX similarly to CQ. The process of applying condensation reactions to yield imines is promising and capable of producing molecules with antiplasmodial activity.


Assuntos
Antimaláricos/uso terapêutico , Malária/tratamento farmacológico , Quinolinas/uso terapêutico , Animais , Antimaláricos/síntese química , Antimaláricos/toxicidade , Linhagem Celular , Eritrócitos/efeitos dos fármacos , Feminino , Hemeproteínas/metabolismo , Hemina/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Quinolinas/síntese química , Quinolinas/toxicidade
9.
J Chem Theory Comput ; 17(3): 1883-1899, 2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33533244

RESUMO

Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicit-solvent molecular dynamics (MD) simulations to scrutinize the G4/hemin interaction. We find that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. The simulations do not support the catalytic mechanism relying on a wobbling guanine. Similarly, the catalytic role of the iron-bound water molecule is not in line with our results; however, given the simulation limitations, this observation should be considered with some caution. The simulations rather suggest tentative mechanisms in which the external G-quartet itself could be responsible for the unique H2O2-promoted biocatalytic properties of the G4/hemin complexes. Once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD technique are briefly discussed.


Assuntos
DNA Catalítico/química , Hemina/química , Simulação de Dinâmica Molecular , Biocatálise , DNA Catalítico/metabolismo , Quadruplex G , Hemina/metabolismo
10.
Food Chem ; 343: 128428, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33131955

RESUMO

The antioxidant effect of porcine pancreatic phospholipase A2 (PLA2) was previously demonstrated. Understanding how PLA2 inhibits lipid oxidation promoted by hemoglobin (Hb) is important for its applications in muscle foods. Effects of enzyme dose, pH, and calcium ion on the ability of PLA2 to inhibit trout hemoglobin-mediated lipid oxidation were investigated in washed cod muscle (WCM). Results indicated that PLA2 required calcium ion for both the hydrolyzing activity and the antioxidant effect. The abilities of PLA2 to inhibit lipid oxidation and suppress oxidation of Hb to form methemoglobin and ferryl hemoglobin were pH-dependent. The lag phase before lipid oxidation enters the exponential phase reciprocally shortened as more hemin was bound to the insoluble matrix of WCM. However, PLA2 was able to inhibit lipid oxidation without preventing the interaction between hemin and the insoluble matrix of the washed muscle.


Assuntos
Hemina/metabolismo , Hemoglobinas/metabolismo , Fosfolipases A2/química , Truta/metabolismo , Animais , Antioxidantes/metabolismo , Produtos Pesqueiros , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Hemina/química , Hemoglobinas/química , Concentração de Íons de Hidrogênio , Metabolismo dos Lipídeos , Músculo Esquelético/metabolismo , Oxirredução , Fosfolipases A2/metabolismo , Suínos
11.
Med Hypotheses ; 144: 110242, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33254548

RESUMO

The outbreak of coronavirus disease 2019 (COVID-19) requires urgent need for effective treatment. Severe COVID-19 is characterized by a cytokine storm syndrome with subsequent multiple organ failure (MOF) and acute respiratory distress syndrome (ARDS), which may lead to intensive care unit and increased risk of death. While awaiting a vaccine, targeting COVID-19-induced cytokine storm syndrome appears currently as the efficient strategy to reduce the mortality of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The stress-responsive enzyme, heme oxygenase-1 (HO-1) is largely known to protect against inflammatory response in animal models. HO-1 is induced by hemin, a well-tolerated molecule, used for decades in the treatment of acute intermittent porphyria. Experimental studies showed that hemin-induced HO-1 mitigates cytokine storm and lung injury in mouse models of sepsis and renal ischemia-reperfusion injury. Furthermore, HO-1 may also control numerous viral infections by inhibiting virus replication. In this context, we suggest the hypothesis that HO-1 cytoprotective pathway might be a promising target to control SARS-CoV-2 infection and mitigate COVID-19-induced cytokine storm and subsequent ARDS.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/metabolismo , Síndrome da Liberação de Citocina/tratamento farmacológico , Heme Oxigenase-1/metabolismo , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Vacinas contra COVID-19 , Cuidados Críticos , Síndrome da Liberação de Citocina/prevenção & controle , Citocinas/metabolismo , Hemina/metabolismo , Humanos , Inflamação , Interleucina-6/metabolismo , Modelos Teóricos , Polimorfismo Genético , Síndrome do Desconforto Respiratório/virologia
12.
Chem Commun (Camb) ; 56(78): 11641-11644, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33000777

RESUMO

Peroxidase-proximity protein labeling was performed using a hemin-parallel G-quadruplex (G4) complex. A tyrosine labeling reaction using an N-methyl luminol derivative was accelerated in close proximity to the hemin with enhanced peroxidase activity by binding to parallel G4. The TERRA-hemin complex activated the labeling of many RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins, in a HeLa cell lysate.


Assuntos
Quadruplex G , Hemina/química , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Hemina/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/química , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Luminol/química , Mutagênese Sítio-Dirigida , Peroxidase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
13.
Cell Death Dis ; 11(9): 787, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968051

RESUMO

The consumption of red meat is probably carcinogenic to humans and is associated with an increased risk to develop colorectal cancer (CRC). Red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. In this study, we investigated the genotoxic and cytotoxic effects of heme iron (i.e., hemin) versus inorganic iron in human colonic epithelial cells (HCEC), human CRC cell lines and murine intestinal organoids. Hemin catalyzed the formation of reactive oxygen species (ROS) and induced oxidative DNA damage as well as DNA strand breaks in both HCEC and CRC cells. In contrast, inorganic iron hardly affected ROS levels and only slightly increased DNA damage. Hemin, but not inorganic iron, caused cell death and reduced cell viability. This occurred preferentially in non-malignant HCEC, which was corroborated in intestinal organoids. Both hemin and inorganic iron were taken up into HCEC and CRC cells, however with differential kinetics and efficiency. Hemin caused stabilization and nuclear translocation of Nrf2, which induced heme oxygenase-1 (HO-1) and ferritin heavy chain (FtH). This was not observed after inorganic iron treatment. Chemical inhibition or genetic knockdown of HO-1 potentiated hemin-triggered ROS generation and oxidative DNA damage preferentially in HCEC. Furthermore, HO-1 abrogation strongly augmented the cytotoxic effects of hemin in HCEC, revealing its pivotal function in colonocytes and highlighting the toxicity of free intracellular heme iron. Taken together, this study demonstrated that hemin, but not inorganic iron, induces ROS and DNA damage, resulting in a preferential cytotoxicity in non-malignant intestinal epithelial cells. Importantly, HO-1 conferred protection against the detrimental effects of hemin.


Assuntos
Dano ao DNA/efeitos dos fármacos , Heme Oxigenase-1/farmacologia , Ferro/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/metabolismo , Heme Oxigenase-1/metabolismo , Hemina/metabolismo , Humanos , Ferro/metabolismo , Substâncias Protetoras/farmacologia
14.
Anal Methods ; 12(18): 2391-2397, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32930265

RESUMO

A method for the aptamer-based determination of chloramphenicol (CAP) was developed by exploiting the peroxidase mimicking activity of hemin. The method includes two hemin-modified DNA probes termed P1 and P2. P1, which was modified at its 5' end with one hemin monomer, contains the CAP-binding sequence. The hybridization between P1 and P2 brings the two hemin monomers in close proximity, resulting in the formation of a hemin dimer with low peroxidase mimicking activity. The duplex structure was dehybridized in the presence of CAP. The formed hemin monomer featured a strong peroxidase mimicking activity and catalyzed the conversion of non-fluorescent tyramine into fluorescent dityramine by hydrogen peroxide. Fluorescence (with an excitation/emission maxima at 320 and 410 nm, respectively) increased linearly in the 0.1 ng mL-1 to 10 ng mL-1 CAP concentration range. The detection limit based on the 3σ/k criterion reached 0.07 ng mL-1. The proposed assay was successfully employed for CAP detection in (spiked) honey samples with recoveries of 94.3-117.2%. Given its high sensitivity and good stability, this method shows potential in providing a platform for antibiotic detection.


Assuntos
Biomimética , Técnicas de Química Analítica , Cloranfenicol , Hemina , Peroxidase , Aptâmeros de Nucleotídeos , Técnicas de Química Analítica/métodos , Cloranfenicol/análise , Fluorometria , Análise de Alimentos/métodos , Hemina/metabolismo , Mel/análise , Limite de Detecção , Peroxidase/metabolismo
15.
Commun Biol ; 3(1): 462, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826945

RESUMO

The human myelogenous leukemic cell line, K562 undergoes erythroid differentiation by exposure to hemin. Here, we uncovered NSD2 as an innate erythroid differentiation-related factor through a genome-wide CRISPR library screen and explored the regulatory role of NSD2 during myeloid leukemia cell differentiation. We found that NSD2 stability was disrupted by poly-ubiquitination in differentiated K562 cells. Proteomic analysis revealed an interaction between NSD2 and an E3 ubiquitin ligase, BRCA1, which ubiquitylates NSD on K292. Depletion of BRCA1 stabilized NSD2 protein and suppressed K562 cell differentiation. Furthermore, BRCA1 protein level was decreased in bone marrow tumor, while NSD2 level was elevated. Surprisingly, among BRCA1 mutation(s) discovered in lymphoma patients, BRCA1 K1183R prevented its translocation into the nucleus, failed to reduce NSD2 protein levels in hemin-treated K562 cells and eventually disrupted cell differentiation. Our results indicate the regulation of NSD2 stability by BRCA1-mediated ubiquitination as a potential therapeutic target process in multiple myeloma.


Assuntos
Proteína BRCA1/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Leucemia/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Biomarcadores , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Hemina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Humanos , Células K562 , Leucemia/etiologia , Leucemia/patologia , Gradação de Tumores , Ligação Proteica , Proteólise , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
16.
Front Immunol ; 11: 1488, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32765515

RESUMO

The excessive release of heme during hemolysis contributes to the severity of sickle cell anemia (SCA) by exacerbating hemoglobin S (HbS) autoxidation, inflammation and systemic tissue damage. The present study investigated the effect of hydroxyurea (HU) on free radical neutralization and its stimulation of antioxidant genes in human peripheral blood mononuclear cells (PBMC) and human umbilical vein endothelial cells (HUVEC) in the presence or absence of hemin. HU (100 and 200 µM) significantly reduced the production of intracellular reactive oxygen species (ROS) induced by hemin at 70 µM in HUVEC. HUVECs treated with HU+hemin presented significant increases in nitric oxide (NO) production in culture supernatants. HU alone or in combination with hemin promoted the induction of superoxide dismutase-1 (SOD1) and glutathione disulfide-reductase (GSR) in HUVECs and PBMCs, and glutathione peroxidase (GPX1) in PBMCs. Microarray analysis performed in HUVECs indicated that HU induces increased expression of genes involved in the antioxidant response system: SOD2, GSR, microsomal glutathione S-transferase (MGST1), glutathione S-transferase mu 2 (GSTM2), carbonyl reductase 1 (CBR1) and klotho B (KLB). Significant increases in expression were observed in genes with kinase activity: protein kinase C beta (PRKCB), zeta (PRKCZ) and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B). HU also induced a significant increase in expression of the gene p62/sequestosome (p62/SQSTM1) and a significant decrease in the expression of the transcriptional factor BACH1 in HUVECs. Upstream analysis predicted the activation of Jun, miR-155-5p and mir-141-3p. These results suggest that HU directly scavenges free radicals and induces the expression of antioxidant genes via induction of the Nrf2 signaling pathway.


Assuntos
Anemia Falciforme/metabolismo , Endotélio Vascular/metabolismo , Hemoglobina Falciforme/metabolismo , Hidroxiureia/metabolismo , Leucócitos Mononucleares/metabolismo , Antioxidantes/metabolismo , Regulação da Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Hemina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
17.
Bioorg Med Chem ; 28(17): 115641, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32773092

RESUMO

G-quadruplex DNA plays a very important role in clinical diagnosis and fluorescence analysis has attracted extensive attention. A class of carbazole-based fluorescent probes for the detection of G-quadruplex DNA was established in this work. In this system, the installation of an oligo(ethylene glycol) chain on the scaffold will improve the water-solubility and biocompatibility. The presence of styrene-like different side groups could tune the selectivity toward G-quadruplex DNA binding. Results revealed that the substitution pattern and position gave a great influence on the ability for the discrimination of the G-quadruplex from other DNA structures. Especially, probe E1 bound to G-quadruplex DNA with superior selectivity, which exhibiting almost no fluorescence response in the presence of non-G-quadruplex DNA structures. Comprehensive analyses revealed that E1 could bind both ends of the G-quadruplex, resulting in a significant increase of fluorescence emission intensity. Cellular uptake assay suggested that E1 could pass through membrane and enter living cells with low cytotoxicity.


Assuntos
Carbazóis/química , Corantes Fluorescentes/farmacologia , Quadruplex G/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Hemina/antagonistas & inibidores , Hemina/metabolismo , Humanos , Microscopia Confocal , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Proteínas Proto-Oncogênicas c-myc/genética , Espectrometria de Fluorescência
18.
ACS Appl Mater Interfaces ; 12(38): 42604-42611, 2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32852185

RESUMO

A novel photoelectrochemical (PEC) aptasensor was fabricated for DNA detection based on the coupling of cosensitization and peroxidase-like catalytic activity. Specifically, the surfaces of branched-TiO2 nanorods (B-TiO2 NRs) were modified with Cd2+ and S2+ to obtain B-TiO2 NRs/CdS hybrid structures, which were subsequently used as matrices to immobilize hairpin DNA (hDNA) probes. CdTe/TCPP (TCPP = meso-tetra(4-carboxyphenyl)-porphine) used for signal amplification was labeled on the terminal of the hDNA probe. Without the target DNA (tDNA) presence, the immobilized hDNA probe with CdTe/TCPP possessed a hairpin form and was located near the B-TiO2 NRs/CdS electrode surface, forming a cosensitized structure formation and then generating strong photocurrent with H2O2 as the electron donor. During detection, the specific recognition of tDNA by the sensing hDNA probe triggered the formation of the G-quadruplex/hemin DNAzyme, which effectively catalyzed the decomposition of H2O2. Meanwhile, cosensitization disappeared when the hDNA probe hybridized with tDNA, further reducing the photocurrent. With a double-signal amplification strategy, the sensing platform designed in this work demonstrated a linear detection ability in the 0.5 fM-5 nM range with a detection limit equal to 0.14 fM. Notably, through encoding in the base sequences of the hDNA and marking it, a versatile PEC platform could be structured for the detection of various DNA targets, which could promise applications in point-of-care diagnostic fields.


Assuntos
Compostos de Cádmio/química , DNA Catalítico/química , DNA/análise , Hemina/química , Nanocompostos/química , Porfirinas/química , Sulfetos/química , Titânio/química , Biocatálise , Sondas de DNA/química , DNA Catalítico/metabolismo , Eletrodos , Quadruplex G , Hemina/metabolismo , Humanos , Tamanho da Partícula , Propriedades de Superfície
19.
Metabolism ; 110: 154306, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32621820

RESUMO

BACKGROUND: Iron is finely regulated due to its vital roles in organisms and the peroxidase reactivity if excess. Solute Carrier Family 46 Member 1 (SLC46A1), also named PCFT or HCP1, is the main importer of heme­iron in the intestine, but has a high abundance in the liver. Since the liver has a central role in iron homeostasis, whether SLC46A1 regulates hepatic iron metabolism is of interest to be identified. METHODS: The recombinant adeno-associated virus vectors were used to hepatic-specifically inhibit SLC46A1 expression to observe its effects on hepatic iron metabolism. Then the abilities of SLC46A1 in importing heme and folate, and consequent alterations of iron content in hepatocytes were determined. Furthermore, effects of iron on SLC46A1 expression were investigated both in vitro and in vivo. RESULTS: The hepatocyte-specific inhibition of SLC46A1 decreases iron content in the liver and increases iron content in serum. Expressions of iron-related molecules, transferrin receptor 1, hepcidin and ferroportin, are correspondingly altered. Interestingly, free heme concentration in serum is increased, indicating a decreased import of heme by the liver. In hepatocytes, SLC46A1 is capable of importing hemin, increasing intracellular iron content. The import of hemin by SLC46A1 is unaffected by its other substrate, folate. Instead, hemin treatment decreases SLC46A1 expression, reducing the import of folate. In addition, SLC46A1 itself shows to be iron-responsive both in vivo and in vitro, making it available for regulating iron metabolism. CONCLUSION: The results elucidate that SLC46A1 regulates iron metabolism in the liver through a folate-independent manner of importing heme. The iron-responsive characters of SLC46A1 give us a new clue to link heme or iron overload with folate deficiency diseases.


Assuntos
Heme/metabolismo , Ferro/metabolismo , Fígado/metabolismo , Transportador de Folato Acoplado a Próton/fisiologia , Animais , Células Cultivadas , Hemina/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transportador de Folato Acoplado a Próton/antagonistas & inibidores
20.
Pflugers Arch ; 472(5): 551-560, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32388729

RESUMO

N-type inactivation of voltage-gated K+ channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary ß subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K+ channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvß1.1, Kvß1.2, and Kvß3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvß1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC50 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary ß subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation.


Assuntos
Hemina/metabolismo , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Sítios de Ligação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Células HEK293 , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Ligação Proteica , Ratos , Xenopus
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