Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 13.913
Filtrar
1.
J Chem Theory Comput ; 15(8): 4602-4614, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31268700

RESUMO

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg2+/Ca2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.


Assuntos
Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Metaloproteínas/metabolismo , Animais , Cálcio/química , Cátions Bivalentes/química , Bases de Dados de Proteínas , Humanos , Magnésio/química , Metaloproteínas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Software , Eletricidade Estática , Termodinâmica
2.
Comput Biol Chem ; 80: 498-511, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176140

RESUMO

Magnesium (Mg) is an important micronutrient for various physiological processes in plants. In this study, putative Magnesium Transporter (MGT) genes have been identified in Solanum lycopersicum, Solanum tuberosum, Brachypodium distachyon, Fagaria vesca, Brassica juncea and were classified into 5 distinct groups based on their sequence homology. MGT genes are very diverse and possess very low sequence identity within its family. However, the Gly-Met-Asn (GMN) signature motif is present in most of the genes which are believed to be essential for Mg2+ recognition. In S. lycopersicum, different physiological root growth pattern was observed in both Mg excess and deficient conditions. Quantitative RT-PCR gene expression study shows that most of the SlMGT genes were upregulated in response to Mg deficient condition.


Assuntos
Proteínas de Transporte de Cátions/genética , Genoma , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Magnésio/metabolismo , Magnoliopsida/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Reação em Cadeia da Polimerase em Tempo Real , Sementes/genética , Homologia de Sequência , Estresse Fisiológico/genética , Regulação para Cima
3.
Nat Commun ; 10(1): 2413, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160571

RESUMO

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. Here we report the cryo-electron microscopy structure of the Syt1-SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg2+)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the 'primary' interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.


Assuntos
Membrana Celular/ultraestrutura , Lipídeos de Membrana/metabolismo , Proteínas SNARE/ultraestrutura , Sinaptotagmina I/ultraestrutura , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Magnésio/metabolismo , Fusão de Membrana , Neurotransmissores/metabolismo , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Transmissão Sináptica , Sinaptotagmina I/metabolismo
4.
Biosci Biotechnol Biochem ; 83(9): 1766-1773, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31056002

RESUMO

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO3, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO3 and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO3. It was concluded that MBCa is a better calcium source than CaCO3 in terms of both calcium retention and absorption. Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.


Assuntos
Cálcio/metabolismo , Dissacarídeos/farmacologia , Absorção Intestinal/efeitos dos fármacos , Magnésio/metabolismo , Animais , Carbonato de Cálcio/administração & dosagem , Ceco/metabolismo , Dieta , Ácidos Graxos Voláteis/metabolismo , Concentração de Íons de Hidrogênio , Íleo/efeitos dos fármacos , Íleo/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Ratos , Ratos Wistar , Solubilidade
5.
Cell Mol Life Sci ; 76(17): 3301-3310, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31073743

RESUMO

The channel kinase (chanzyme) transient receptor potential melastatin-like 7 (TRPM7) has a unique dual protein structure composed of an ion channel with an α-kinase domain on its C-terminus. In the nervous system, under physiological conditions, TRPM7 contributes to critical neurobiological processes ranging from synaptic transmission to cognitive functions. Following certain pathological triggers, TRPM7 mediates neurotoxicity, neuro-injuries, and neuronal death. Here, we summarize the current knowledge of TRPM7 functions in neuronal systems in health and disease. The molecular mechanisms by which this chanzyme might regulate synaptic and cognitive functions are discussed. We also discuss the lack of knowledge regarding the molecular mechanisms responsible for turning TRPM7 into "a vicious tool" that mediates neuronal death following certain pathological triggers. Some synthetic and natural pharmacological modulators of the TRPM7 channel and its α-kinase are reviewed. We suggest that based on current knowledge, we should reshape our thinking regarding the implications of TRPM7 in neurological and neurodegenerative disorders. Moreover, we propose a paradigm shift concerning the targeting of TRPM7 as a therapeutic approach for treating certain neurological diseases. We agree that TRPM7 overexpression or overactivation may mediate neurodegenerative processes following certain triggers. However, TRPM7 dysfunction and/or downregulation might also be among the pathological changes leading to neurodegeneration. Consequently, further investigations are required before we decide whether blocking or activating the chanzyme is the correct therapeutic approach to treat certain neurological and/or neurodegenerative diseases.


Assuntos
Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/patologia , Canais de Cátion TRPM/metabolismo , Humanos , Magnésio/metabolismo , Doenças Neurodegenerativas/metabolismo , Plasticidade Neuronal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Zinco/metabolismo
6.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035385

RESUMO

Several drugs including diuretics and proton-pump inhibitors can cause magnesium loss and hypomagnesemia. Magnesium and drugs use the same transport and metabolism pathways in the body for their intestinal absorption, metabolism, and elimination. This means that when one or more drug is taken, there is always a potential risk of interaction with the magnesium status. Consequently the action of a drug may be adversely affected by magnesium (e.g., magnesium, calcium, and zinc can interfere with the gastrointestinal absorption of tetracycline antibiotics) and simultaneously the physiological function of minerals such as magnesium may be impaired by a drug (e.g., diuretics induce renal magnesium loss). Given the ever-increasing number of drugs on the market and the frequency with which they are used, greater attention must be paid in daily medical and pharmaceutical practice focused in particular on the adverse effects of drug therapy on magnesium status in order to minimize the potential risk to the health of patients.


Assuntos
Magnésio/metabolismo , Animais , Humanos , Absorção Intestinal/efeitos dos fármacos , Deficiência de Magnésio/metabolismo , Inibidores da Bomba de Prótons/uso terapêutico , Canais de Cátion TRPM/metabolismo , Tetraciclina/metabolismo
7.
Chemosphere ; 229: 409-417, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31082708

RESUMO

Antibiotics represent a novel type of environment pollutants which modify chlorophyll content in plants. Spectroscopic methods were employed to investigate the effect of tetracycline on chlorophyll degradation. Changes in absorbance and fluorescence demonstrated that tetracycline reaction with chlorophyll results in the formation of pheophytin, which was confirmed by new bands typical of pheophytin which appeared in the absorbance spectrum. The rate of pheophytin formation depended on ratio tetracycline to chlorophyll concentration in solution. In solutions with chlorophyll concentration of C = 1 × 10-5 M and tetracycline concentrations of C = 1 × 10-3 M and C = 1 × 10-2 M, pheophytin was formed after 28 h and 25 min, respectively. The obtained lifetime for pheophytin formed during chlorophyll reaction - with tetracycline hydrochloride was τ = 5.71 ±â€¯0.02 ns and its value coincides, within the error limits, with the value obtained for pure pheophytin purchased from ChromaDex. The experiment demonstrated two mechanisms of chlorophyll degradation to pheophytin by tetracycline hydrochloride, i.e. 1) loss of Mg2+ ions from the chlorophyll molecule as a result of the presence of H+ ions in solution (i.e. as a result of medium acidification), and 2) removal of Mg2+ ions directly from chlorophyll by tetracycline which binds Mg2+ ions from the chlorophyll. We demonstrated that magnesium occurring in low concentrations attached to a tetracycline molecule in the BCD ring, and that the second ion of Mg2+ may attach to the A ring of tetracycline at higher Mg2+ concentrations. Two fluorescence bands appeared which indicated such magnesium attachments indeed occurred.


Assuntos
Clorofila/química , Feofitinas/química , Tetraciclina/química , Antibacterianos , Cinética , Magnésio/metabolismo , Plantas/química , Plantas/efeitos dos fármacos , Espectrometria de Fluorescência , Tetraciclina/metabolismo
8.
Microb Pathog ; 132: 293-301, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31082531

RESUMO

Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg2+ and Ca2+ precipitation and turbidity. A catheter blockage study was performed in a synthetic bladder model mimicking natural UTI in the presence of allicin at sub-MIC concentrations (MIC = 64 µg/ml). The results of crystallization study showed that allicin inhibited pH rise and consequently turbidity and precipitation of ions in a dose-dependent manner. The results of catheter blockage study showed that allicin at sub-MIC concentrations (2, 4, 8 µg/ml) significantly increased the time for catheter blockage to occur to 61, 74 and 92 h respectively compared to allicin-free control (48 h). In a similar way, the results showed that allicin delayed the increase of SU pH level in bladder model in a dose-dependent manner compared to allicin-free control. The results also showed that following the increase of allicin concentration, Mg2+ and Ca2+ deposition in catheters were much lower compared to allicin-free control, further confirmed by direct observation of the catheters' eyehole and cross sections. We conclude that allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheters by delaying pH increase and lowering Mg2+ and Ca2+ deposition in a dose-dependent manner.


Assuntos
Infecções por Proteus/prevenção & controle , Proteus/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Bexiga Urinária/microbiologia , Cálcio/metabolismo , Cristalização , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Testes de Sensibilidade Microbiana , Proteus/crescimento & desenvolvimento , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/crescimento & desenvolvimento , Urease , Infecções Urinárias/microbiologia , Infecções Urinárias/prevenção & controle , Urina
9.
Biosci Biotechnol Biochem ; 83(9): 1616-1623, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31032741

RESUMO

To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5'-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p1, p4-di(adenosine) 5'-tetraphosphate, and adenosine-5'-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 µM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively). Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5'-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap4A, P1, P4-di(adenosine) 5'-tetraphosphate; AMP: adenosine-5'-monophosphate; PAR: pyrophosphate amplification rate.


Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Difosfatos/metabolismo , Escherichia coli/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Especificidade por Substrato , Temperatura Ambiente
10.
Toxicol Ind Health ; 35(4): 277-293, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30983556

RESUMO

The aim of the study was to investigate the effect of physical training on bone parameters of rats exposed to alcohol (Al) and/or cadmium (Cd). Young female rats were divided into one control group and six groups exposed to Cd and/or Al. Al (36% calories of diet) and Cd (20 mg Cd/kg feed) were administered with liquid diet. Half of the rats from the treated groups were subjected to treadmill training (20 m/min for 0.5 h, 4 days a week). The experiment was carried out for 5 months. Al decreased the concentration of calcium (Ca) and iron (Fe) in the femur, whereas Cd and Cd + Al intake reduced the contents of Ca, Fe and zinc. Al and/or Cd caused an increase in both C-terminal telopeptide of type I collagen (CTX1; bone resorption marker) and osteocalcin (OC; formation indicator) and enhanced the degree of porosity and flexural strength of the femur. Al partially prevented the loss of Fe from the bone caused by Cd, but intensified the inhibition of growth of body weight in comparison with separate exposure to Cd. In rats co-exposed to Cd + Al, the levels of CTX1 were greater compared with those treated with Al or Cd separately, and the density was less than that in rats exposed to Al separately. The training caused increases of magnesium and Ca contents, decreases in CTX1, as well as increases in OC and bone density, decreasing their porosity. The effect of training on the bone status, however, was limited (especially in rats co-exposed to Cd and Al) because of the increase in their mineralization, stimulated by exercises, was insufficient in relation to collagen production intensity. In conclusion, training had favourable effects on some bone parameters, but did not compensate for the negative effects of Al and/or Cd exposure on the poor mineralization and histopathological and morphological changes in the femur.


Assuntos
Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Cádmio/farmacologia , Etanol/farmacologia , Condicionamento Físico Animal/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Reabsorção Óssea , Cálcio/metabolismo , Colágeno Tipo I/metabolismo , Feminino , Fêmur/efeitos dos fármacos , Ferro/metabolismo , Magnésio/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Zinco/metabolismo
11.
Mol Biotechnol ; 61(6): 432-441, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963480

RESUMO

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.


Assuntos
Trifosfato de Adenosina/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biotransformação , Carboidratos Epimerases/genética , Cátions Bivalentes , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Magnésio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo
12.
Int J Mol Sci ; 20(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995736

RESUMO

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed chanzyme that possesses an ion channel permeable to the divalent cations Mg2+, Ca2+, and Zn2+, and an α-kinase that phosphorylates downstream substrates. TRPM7 and its homologue TRPM6 have been implicated in a variety of cellular functions and is critically associated with intracellular signaling, including receptor tyrosine kinase (RTK)-mediated pathways. Emerging evidence indicates that growth factors, such as EGF and VEGF, signal through their RTKs, which regulate activity of TRPM6 and TRPM7. TRPM6 is primarily an epithelial-associated channel, while TRPM7 is more ubiquitous. In this review we focus on TRPM7 and its association with growth factors, RTKs, and downstream kinase signaling. We also highlight how interplay between TRPM7, Mg2+ and signaling kinases influences cell function in physiological and pathological conditions, such as cancer and preeclampsia.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Magnésio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/metabolismo , Animais , Feminino , Humanos , Neoplasias/metabolismo , Pré-Eclâmpsia/metabolismo , Gravidez
13.
Methods Mol Biol ; 1981: 55-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016647

RESUMO

Transporters play a crucial role in the uptake of endo- and exogenous molecules in hepatocytes and efflux into the bile. The bile salt export pump (BSEP; ABCB11) is of major importance for efflux of bile salts to the bile and BSEP inhibition frequently provokes drug-induced cholestasis. This chapter describes two assays to determine inhibition of BSEP-mediated bile salt excretion. The first assay uses inside-out membrane vesicles, prepared from BSEP-transfected cell lines. The cholestasis potential of compounds can be determined by specifically investigating the ability to inhibit BSEP-mediated uptake of tauro-nor-THCA-24-DBD, a fluorescent bile salt derivative. For the second assay, relative accumulation of tauro-nor-THCA-24-DBD in sandwich-cultured hepatocytes, which represents a more biorelevant in vitro system, is investigated. Through incubation with standard or Ca2+/Mg2+-free buffer, the substrate signal can be determined in the cells and bile or the cells alone, respectively. Performing this assay in the presence and absence of potentially interfering compounds of interest enables exploration of the relative effect of these compounds on biliary excretion of the probe substrate.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Hepatócitos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Cálcio/metabolismo , Colestase/metabolismo , Humanos , Magnésio/metabolismo
14.
J Dairy Sci ; 102(6): 5566-5576, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30981486

RESUMO

Hydrogen is a key metabolite that connects microbial fermentation and methanogenesis in the rumen. This study was to investigate the effects of elevated H2 produced by elemental Mg on rumen fermentation and methanogenesis in dairy cows. Four nonlactating Chinese Holstein dairy cows were employed for this experiment in a replicated crossover design. The 2 dietary treatments included a basal diet supplemented with Mg(OH)2 (14.5 g/kg of feed dry matter) or elemental Mg (6.00 g/kg of feed dry matter). When compared with Mg(OH)2 treatment, cows fed diet with elemental Mg had similar rumen Mg2+ concentration, but higher rumen dissolved H2 and methane concentrations at 2.5 h after morning feeding. Also, elemental Mg supplementation decreased feed digestibility, rumen volatile fatty acid concentration, and relative abundance of group Ruminococcaceae_UCG-014, genus Bifidobacterium, and group Mollicutes_RF9, increased acetate to propionate ratio, succinate concentration, and abundance of family Christensenellaceae. Elemental Mg supplementation increased enteric CH4 emission, altered methanogen community with increased abundance of order Methanomassiliicoccales, 16S ribosomal RNA gene copies of methanogens, and order Methanobacteriales. In summary, the pulse of elevated dissolved H2 after feeding produced by elemental Mg inhibited rumen fermentation and feed digestibility by decreasing the abundance of carbohydrate-degrading bacteria, promoted H2 incorporation into succinate by increasing family Christensenellaceae and genus Bacteroidales_BS11, and increased H2 utilization for methanogenesis by favoring growth of methanogens.


Assuntos
Bovinos/metabolismo , Hidrogênio/metabolismo , Magnésio/metabolismo , Metano/metabolismo , Rúmen/metabolismo , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Ácidos Graxos Voláteis/metabolismo , Feminino , Fermentação , Lactação , Propionatos/metabolismo
15.
Nat Commun ; 10(1): 1142, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850593

RESUMO

FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg2+ and Ca2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Cálcio/metabolismo , Quimiocina CCL7/química , Proteínas de Choque Térmico/química , Processamento de Proteína Pós-Traducional , Monofosfato de Adenosina/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Cálcio/química , Cátions Bivalentes , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Magnésio/química , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
16.
RNA ; 25(6): 737-746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926754

RESUMO

Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.


Assuntos
Proteínas 14-3-3/química , Biomarcadores Tumorais/química , DNA de Cadeia Simples/química , Exorribonucleases/química , Magnésio/química , Proteínas Mitocondriais/química , Oligorribonucleotídeos/química , RNA/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sítios de Ligação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Magnésio/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA/genética , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
J Biochem ; 166(2): 149-156, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30825308

RESUMO

A cutinase from Saccharomonospora viridis AHK190, Cut190, can hydrolyze polyethylene terephthalate and has a unique feature that the activity and stability are regulated by Ca2+ binding. Our recent structural and functional analyses showed three Ca2+ binding sites and their respective roles. Here, we analysed the binding thermodynamics of Mn2+, Zn2+ and Mg2+ to Cut190 and their effects on the catalytic activity and thermal stability. The binding affinities of Mn2+ and Zn2+ were higher than that of Mg2+ and are all entropy driven with a binding stoichiometry of three, one and one for Zn2+, Mn2+ and Mg2+, respectively. The catalytic activity was measured in the presence of the respective metals, where the activity of 0.25 mM Mn2+ was comparable to that of 2.5 mM Ca2+. Our 3D Reference Interaction Site Model calculations suggested that all the ions exhibited a high occupancy rate for Site 2. Thus, Mn2+ and Mg2+ would most likely bind to Site 2 (contributes to stability) with high affinity, while to Sites 1 and 3 (contributes to activity) with low affinity. We elucidate the metal-dependent structural and functional properties of Cut190 and show the subtle balance on structure stability and flexibility is controlled by specific metal ions.


Assuntos
Actinobacteria/enzimologia , Cálcio/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Zinco/metabolismo , Sítios de Ligação , Cálcio/química , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática , Magnésio/química , Manganês/química , Modelos Moleculares , Mutação , Termodinâmica , Zinco/química
18.
Talanta ; 198: 179-184, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876547

RESUMO

Nucleic acids-based biosensors are extremely important in modern life sciences and have been widely used for the detection of many biomarkers of disease and extensively applied in many fields, such as medical analysis, gene therapy, and pathogen determination. Therefore, it is necessary to develop some sensitive and selective methods for rapid detection of nucleic acids. In this work, an ultrasensitive and non-enzyme electrochemical biosensor has been developed for nucleic acids detection based on entropy-driven amplification (EDA) strategy and Mg2+-dependent DNAzyme cleavage method. In the presence of target DNA (T-DNA), the T-DNA could hybridize with the premade three-strand duplex (TD) through the toehold region to initiate the EDA process (Cycle I), leading to the generation of Mg2+-dependent DNAzyme served for Cycle II. The newly formed Mg2+-dependent DNAzyme could hybridize with the methylene blue (MB)-labeled hairpin DNA (MB-HP) on the gold electrode surface which induced the cleavage process of Mg2+, resulting in the recycle of Mg2+-dependent DNAzyme, accompanied by the release of MB-labeled DNA fragment from the gold electrode surface. Based on the proposed strategy, the developed electrochemical biosensor exhibited a wide linear relationship in the range from 5 fM to 1 nM with a limit of detection (LOD) of 2.7 fM (S/N = 3), which gave the developed electrochemical biosensor a great promising for the detection of nucleic acids in biomedical research and disease diagnosis.


Assuntos
DNA Catalítico/metabolismo , DNA/análise , Entropia , Magnésio/metabolismo , DNA/metabolismo , Humanos , Magnésio/química , Azul de Metileno/química , Técnicas de Amplificação de Ácido Nucleico
19.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845649

RESUMO

The cyclin and cystathionine ß-synthase (CBS) domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg2+/Co2+ efflux protein CorC and the Mg2+ channel MgtE.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Proteínas de Transporte de Cátions/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica
20.
Mol Biol Evol ; 36(5): 999-1007, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30861070

RESUMO

The ribosome is responsible for protein synthesis in all living organisms. It is best known to exist around 3.5-3.7 Ga whereat life on Earth inhabited anoxic environment with abundant soluble irons. The RNAs and proteins are the two biopolymers that constitute the ribosome. However, both proteins and RNAs require metal cations to fold and to function. There are four Mg-microcluster (Mg2+-µc) structures conserved in core of large subunit, and the 23S ribosomal RNA (rRNA) was shown to catalyze electron transfer in an anoxic environment in the presence of Fe2+. The Mg2+-µc features two idiosyncratic Mg2+ ions that are chelated and bridged by a common phosphate group and along with that, the adjacent residues of RNA backbone together forming ten-membered chelation ring(s). Here, we utilized four rRNA fragments of the large subunit 23S rRNA of Haloarcula marismortui, that includes the residues that form the four Mg2+-µc's. These four rRNA fragments are shown competent to assemble with Mg2+. Our results show that when these rRNA fragments fold or assembly in the presence of Fe2+ under anoxic conditions, each Fe2+-microcluster can catalyze electron transfer. We propose that Fe2+-microclusters of the ribosome, which use Fe2+ as a cofactor to regulate electron transfer, are pivotal and primordial and may be an origin in evolution of the ribosome.


Assuntos
Transporte de Elétrons , Ferro/metabolismo , Magnésio/metabolismo , RNA Ribossômico 23S/metabolismo , Ribossomos , Haloarcula marismortui
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA