RESUMO
Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and ß = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.
Assuntos
Calcificação Fisiológica , Proteínas de Ligação ao Cálcio , Pinctada , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cristalização , Pinctada/genética , Pinctada/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.
Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ferro/metabolismo , Citrato de Sódio/farmacologia , Vibrio parahaemolyticus/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinamatos/química , Cinamatos/metabolismo , Depsídeos/química , Depsídeos/metabolismo , Sinergismo Farmacológico , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Ligação Proteica , Vibrio parahaemolyticus/metabolismo , Ácido RosmarínicoRESUMO
Lysine N-pyrrolation, converting lysine residues to Nϵ-pyrrole-l-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNA-binding molecules such as anti-DNA autoantibodies. The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of Nϵ-pyrrole-l-lysine-containing proteins. In this study, we used microbeads coupled to pyrrolated or nonpyrrolated protein to screen for binding activities of human serum-resident nonimmunoglobin proteins to the pyrrolated proteins. This screen identified apolipoprotein E (apoE) as a protein that innately binds the DNA-mimicking proteins in serum. Using an array of biochemical assays, we observed that the pyrrolated proteins bind to the N-terminal domain of apoE and that oligomeric apoE binds these proteins better than does monomeric apoE. Employing surface plasmon resonance and confocal microscopy, we further observed that apoE deficiency leads to significant accumulation of pyrrolated serum albumin and is associated with an enhanced immune response. These results, along with the observation that apoE facilitates the binding of pyrrolated proteins to cells, suggest that apoE may contribute to the clearance of pyrrolated serum proteins. Our findings uncover apoE as a binding target of pyrrolated proteins, providing a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.
Assuntos
Apolipoproteínas E/metabolismo , Mimetismo Molecular/fisiologia , Pirróis/metabolismo , Adulto , Sequência de Aminoácidos/genética , Animais , Apolipoproteína E3/sangue , Apolipoproteína E3/metabolismo , Apolipoproteína E4/sangue , Apolipoproteína E4/metabolismo , Apolipoproteínas E/sangue , Apolipoproteínas E/fisiologia , Fenômenos Biofísicos , DNA/genética , DNA/metabolismo , Feminino , Humanos , Hiperlipidemias/metabolismo , Cinética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína/fisiologia , Proteínas/metabolismo , Pirróis/químicaRESUMO
Alginate oligosaccharides (AOs) are linear oligosaccharides with alternating sequences of mannuronic acid (M) and guluronic acid (G) residues. AOs can be used as a safe elicitor to induce glyceollins, which have many human health benefits, in soybean seeds. In this research, four AO fractions with different chemical structures and molecular weights were separated, purified, and then characterized by NMR spectroscopy and ESI-MS. With a 4,5-unsaturated hexuronic acid residue (â³) at the non-reducing terminus, the structures of these four AO fractions were â³G, â³MG, â³GMG and â³MGGG, which exhibited glyceollin-inducing activities of 1.2339, 0.3472, 0.6494 and 1.0611 (mg/g dry weight) in soybean seeds, respectively. The results demonstrated that a larger molecular weight or a higher G/M ratio might correlate with a higher glyceollin-inducing activity. Moreover, the alginate disaccharide â³G could be introduced as relatively safe and efficient elicitor of high glyceollin content in soybeans.
RESUMO
Bee pollen, a beehive product collected from flowers by honeybees, contains over 250 biological substances, and has attracted increasing attention as a functional food. However, commercial bee pollen products are often multifloral, and samples from different countries vary significantly. There is no universal standard for objective quality assessment of bee pollen based on its chemical composition. Here, we report metabolomic analysis of 11 bee pollen samples from Spain, China, and Australia for quality control. The characteristics of the samples depend on the sucrose, nucleoside, amino acid, and flavanol concentrations. Bee pollen samples from Spain and Australia had higher sucrose and adenosine concentrations, whereas those from China had higher trigonelline, uridine, and cytidine concentrations. Interestingly, acetic acid was only detected in samples from China. These components can be used to identify the country of origin. The obtained profiles of the samples will contribute to universal standard development for bee pollen products.
RESUMO
Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a crucial regulator that produces multiple physiological benefits, such as the prevention of cancer and age-related diseases. SIRT1 is activated by sirtuin-activating compounds (STACs). Here, we report that quercetin 3,5,7,3',4'-pentamethyl ether (KPMF-8), a natural STAC from Thai black ginger Kaempferia parviflora, interacts with SIRT1 directly and stimulates SIRT1 activity by enhancing the binding affinity of SIRT1 with Ac-p53 peptide, a native substrate peptide without a fluorogenic moiety. The binding affinity between SIRT1 and Ac-p53 peptide was enhanced 8.2-fold by KPMF-8 but only 1.4-fold by resveratrol. The specific binding sites of KPMF-8 to SIRT1 were mainly localized to the helix2-turn-helix3 motif in the N-terminal domain of SIRT1. Intracellular deacetylase activity in MCF-7 cells was promoted 1.7-fold by KPMF-8 supplemented in the cell medium but only 1.2-fold by resveratrol. This work reveals that KPMF-8 activates SIRT1 more effectively than resveratrol does.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Quercetina/farmacologia , Sirtuína 1/metabolismo , Zingiberaceae , Regulação Alostérica , Antineoplásicos Fitogênicos/isolamento & purificação , Sítios de Ligação , Neoplasias da Mama/enzimologia , Ativação Enzimática , Ativadores de Enzimas/isolamento & purificação , Feminino , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Quercetina/análogos & derivados , Quercetina/isolamento & purificação , Resveratrol/farmacologia , Zingiberaceae/químicaRESUMO
Post-fermented tea (dark tea) is produced from enzyme-inactivated fresh tea leaves by microbial fermentation. Batabata tea and Awaban tea are two major dark teas fermented under aerobic and anaerobic conditions, respectively. However, how their chemical compositions and functionalities change during different post-fermentation processes remains unclear. Nuclear magnetic resonance (NMR)-based analyses showed that (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) decreased in Batabata tea during post-fermentation with aerobic molds. In contrast, EGC and EC increased, and pyrogallol was produced in Awaban tea during post-fermentation with lactic acid bacteria (LAB). The anti-glycation activities of two dark teas were investigated using an in vitro assay system with human serum albumin (HSA). The anti-glycation activity decreased in Batabata tea, but it was retained in Awaban tea during post-fermentation. Our results showed that post-fermentation with LAB was an efficient way to enhance phenol content and that pyrogallol contributed to anti-glycation activity of Awaban tea.
Assuntos
Chá/química , Bactérias/crescimento & desenvolvimento , Reatores Biológicos , Catequina/análogos & derivados , Catequina/metabolismo , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Folhas de Planta/química , Folhas de Planta/metabolismo , Análise de Componente Principal , Albumina Sérica/metabolismo , Chá/metabolismoRESUMO
The ferric ion binding protein A of Thermus thermophilus HB8 (TtFbpA) is the periplasmic subunit of an ABC-type iron transporter. Two Fe3+-bound crystal structures at pH 5.5 and pH 7.5 and one apo structure have been reported for TtFbpA. In addition to three Tyr residues, TtFbpA coordinates with Fe3+ using two monodentate HCO3- and one H2O to form a six-coordinated mode at pH 5.5 or one bidentate CO32- to form a five-coordinated mode at pH 7.5. We investigated the biological significance of these Fe3+-bound forms of TtFbpA and the synergistic anions (HCO3- and CO32-). Quantum mechanical calculations in silico indicated that only these coordination modes were plausible out of six possibilities. Comparison of the crystal structures revealed a key motif, RZX1X2L(I/V), that would couple the Fe3+ coordination mode and the TtFbpA protein conformation. Both gel filtration chromatography and isothermal titration calorimetry showed that TtFbpA could bind Fe3+ at pH 7.5 but not at pH 5.5. Isothermal titration calorimetry also revealed that the binding at pH 7.5 was a three-step endothermic reaction that required NaHCO3. These results indicate that the holo structure at pH 5.5 is unstable in solution and may correspond to a transition state of TtFbpA-Fe3+ binding at pH 7.5 because HCO3- is much more abundant than CO32- at both pH values. Reorganisation of the synergistic ions and coupled protein conformational change will occur to form the stable TtFbpA-Fe3+ complex at pH 7.5, but not at pH 5.5. Identification of such a transition state will contribute to molecular design of novel FbpA inhibitors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Periplasma/metabolismo , Thermus thermophilus/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Ferro/química , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Roasted chicory root (Cichorium intybus) has been widely accepted as the most important coffee substitute. In this study, a nuclear magnetic resonance (NMR)-based comprehensive analysis was performed to monitor the substantial changes in the composition of chicory root during the roasting process. A detailed signal assignment of dried raw and roasted chicory roots was carried out using 1H, 13C, 1H-1H DQF-COSY, 1H-13C edited-HSQC, 1H-13C CT-HMBC, and 1H-13C HSQC-TOCSY NMR spectra. On the basis of the signal assignments, 36 NMR-visible components were monitored simultaneously during roasting. Inulins, sucrose, and most of the amino acids were largely degraded during the roasting process, whereas monosaccharides decreased at the beginning and then increased until the dark roasting stage. Acetamide, 5-hydroxymethylfurfural, di-d-fructose dianhydride, and norfuraneol were newly formed during roasting. Furthermore, a principal component analysis score plot indicated that similar chemical composition profiles could be achieved by roasting the chicory root either at a higher firepower for a shorter time or at a lower firepower for a longer time.