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1.
Adv Exp Med Biol ; 1202: 35-65, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32034708

RESUMO

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y1, P2Y2, P2Y12, P2Y14 and the ionotropic P2X7 receptor in glioma C6 cells. P2Y1 and P2Y12 both respond to ADP, but while P2Y1 links to PLC and elevates cytosolic Ca2+ concentration, P2Y12 negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y1 receptor strongly decreases and P2Y12 becomes a major player responsible for ADP-evoked signal transduction. The P2Y12 receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y1 has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X7 receptor, often responsible for apoptotic fate, is not involved in Ca2+elevation in C6 cells. The shift in nucleotide receptor expression from P2Y1 to P2Y12 during serum withdrawal, the cross talk between both receptors and the lack of P2X7 activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.


Assuntos
Glioma/metabolismo , Nucleotídeos/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Difosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioma/patologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
2.
Adv Exp Med Biol ; 1202: 67-86, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32034709

RESUMO

Calcium signaling is probably one of the evolutionary oldest and the most common way by which the signal can be transmitted from the cell environment to the cytoplasmic calcium binding effectors. Calcium signal is fast and due to diversity of calcium binding proteins it may have a very broad effect on cell behavior. Being a crucial player in neuronal transmission it is also very important for glia physiology. It is responsible for the cross-talk between neurons and astrocytes, for microglia activation and motility. Changes in calcium signaling are also crucial for the behavior of transformed glioma cells. The present chapter summarizes molecular mechanisms of calcium signal formation present in glial cells with a strong emphasis on extracellular nucleotide-evoked signaling pathways. Some aspects of glioma C6 signaling such as the cross-talk between P2Y1 and P2Y12 nucleotide receptors in calcium signal generation will be discussed in-depth, to show complexity of machinery engaged in formation of this signal. Moreover, possible mechanisms of modulation of the calcium signal in diverse environments there will be presented herein. Finally, the possible role of calcium signal in glioma motility is also discussed. This is a very important issue, since glioma cells, contrary to the vast majority of neoplastic cells, cannot spread in the body with the bloodstream and, at least in early stages of tumor development, may expand only by means of sheer motility.


Assuntos
Sinalização do Cálcio , Glioma/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Glioma/patologia , Humanos , Nucleotídeos/metabolismo
3.
Adv Exp Med Biol ; 1202: 109-128, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32034711

RESUMO

This chapter describes signaling pathways, stimulated by the P2Y2 nucleotide receptor (P2Y2R), that regulate cellular processes dependent on actin cytoskeleton dynamics in glioma C6 cells. P2Y2R coupled with G-proteins, in response to ATP or UTP, regulates the level of iphosphatidylinositol-4,5-bisphosphate (PIP2) which modulates a variety of actin binding proteins and is involved in calcium response and activates Rac1 and RhoA proteins. The RhoA/ROCK signaling pathway plays an important role in contractile force generation needed for the assembly of stress fibers, focal adhesions and for tail retraction during cell migration. Blocking of this pathway by a specific Rho-kinase inhibitor induces changes in F-actin organization and cell shape and decreases the level of phosphorylated myosin II and cofilin. In glioma C6 cells these changes are reversed after UTP stimulation of P2Y2R. Signaling pathways responsible for this compensation are calcium signaling which regulates MLC kinase activation via calmodulin, and the Rac1/PAK/LIMK cascade. Stimulation of the Rac1 mediated pathway via Go proteins needs additional interaction between αvß5 integrins and P2Y2Rs. Calcium free medium, or growing of the cells in suspension, prevents Gαo activation by P2Y2 receptors. Rac1 activation is necessary for cofilin phosphorylation as well as integrin activation needed for focal complexes formation and stabilization of lamellipodium. Inhibition of positive Rac1 regulation prevents glioma C6 cells from recovery of control cell like morphology.


Assuntos
Citoesqueleto/metabolismo , Glioma/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Glioma/patologia , Humanos , Nucleotídeos/metabolismo , Fosforilação
4.
Chemistry ; 26(6): 1368-1379, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31682037

RESUMO

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.


Assuntos
Inativação Gênica , Nucleotídeos/química , Oligonucleotídeos Antissenso/química , Ribonuclease H/metabolismo , Sequência de Bases , Estabilidade Enzimática , Células HeLa , Humanos , Concentração Inibidora 50 , Desnaturação de Ácido Nucleico , Nucleotídeos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA/química , RNA/metabolismo , Temperatura Ambiente , Transfecção
5.
PLoS One ; 14(12): e0221745, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877133

RESUMO

To comprehensively characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), metabolites in two CHH target tissues of the crayfish Procambarus clarkii, whose levels were significantly different between CHH knockdown and control (saline-treated) animals, were analyzed using bioinformatics tools provided by an on-line analysis suite (MetaboAnalyst). Analysis with Metabolic Pathway Analysis (MetPA) indicated that in the muscle Glyoxylate and dicarboxylate metabolism, Nicotinate and nicotinamide metabolism, Alanine, aspartate and glutamate metabolism, Pyruvate metabolism, and Nitrogen metabolism were significantly affected by silencing of CHH gene expression at 24 hours post injection (hpi), while only Nicotinate and nicotinamide metabolism remained significantly affected at 48 hpi. In the hepatopancreas, silencing of CHH gene expression significantly impacted, at 24 hpi, Pyruvate metabolism and Glycolysis or gluconeogenesis, and at 48 hpi, Glycine, serine and threonine metabolism. Moreover, analysis using Metabolite Set Enrichment Analysis (MSEA) showed that many metabolite sets were significantly affected in the muscle at 24hpi, including Ammonia recycling, Nicotinate and nicotinamide metabolism, Pyruvate metabolism, Purine metabolism, Warburg effect, Citric acid cycle, and metabolism of several amino acids, and at 48 hpi only Nicotinate and nicotinamide metabolism, Glycine and serine metabolism, and Ammonia recycling remained significantly affected. In the hepatopancreas, MSEA analysis showed that Fatty acid biosynthesis was significantly impacted at 24 hpi. Finally, in the muscle, levels of several amino acids decreased significantly, while those of 5 other amino acids or related compounds significantly increased in response to CHH gene silencing. Levels of metabolites related to nucleotide metabolism significantly decreased across the board at both time points. In the hepatopancreas, the effects were comparatively minor with only levels of thymine and urea being significantly decreased at 24 hpi. The combined results showed that the metabolic effects of silencing CHH gene expression were far more diverse than suggested by previous studies that emphasized on carbohydrate and energy metabolism. Based on the results, metabolic roles of CHH on the muscle and hepatopancreas are suggested: CHH promotes carbohydrate utilization in the hepatopancreas via stimulating glycolysis and lipolysis, while its stimulatory effect on nicotinate and nicotinamide metabolism plays a central role in coordinating metabolic activity in the muscle with diverse and wide-ranging consequences, including enhancing the fluxes of glycolysis, TCA cycle, and pentose phosphate pathway, leading to increased ATP supply and elevated protein and nucleic acid turnovers.


Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/genética , Astacoidea/metabolismo , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Metabolismo Energético , Inativação Gênica , Glicólise , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Redes e Vias Metabólicas , Músculo Esquelético/metabolismo , Músculos/metabolismo , Nucleotídeos/metabolismo , Taiwan
6.
Biochemistry (Mosc) ; 84(9): 1008-1020, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693460

RESUMO

Nucleotide excision repair (NER) is one of the major DNA repair pathways aimed at maintaining genome stability. Correction of DNA damage by the NER system is a multistage process that proceeds with the formation of multiple DNA-protein and protein-protein intermediate complexes and requires precise coordination and regulation. NER proteins undergo post-translational modifications, such as ubiquitination, sumoylation, phosphorylation, acetylation, and poly(ADP-ribosyl)ation. These modifications affect the interaction of NER factors with DNA and other proteins and thus regulate either their recruitment into the complexes or dissociation from these complexes at certain stages of DNA repair, as well as modulate the functional activity of NER proteins and control the process of DNA repair in general. Here, we review the data on the post-translational modifications of NER factors and their effects on DNA repair. Protein poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerase 1 and its impact on NER are discussed in detail, since such analysis has not been done before.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Nucleotídeos/metabolismo , Processamento de Proteína Pós-Traducional , Humanos
7.
Nat Commun ; 10(1): 5126, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719534

RESUMO

N1-methyladenosine (m1A) was proposed to be a highly prevalent modification in mRNA 5'UTRs based on mapping studies using an m1A-binding antibody. We developed a bioinformatic approach to discover m1A and other modifications in mRNA throughout the transcriptome by analyzing preexisting ultra-deep RNA-Seq data for modification-induced misincorporations. Using this approach, we detected appreciable levels of m1A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m1A-mapping approach to detect m1A at single-nucleotide resolution, and confirmed that the commonly used m1A antibody maps sites to the transcription-start site in mRNA 5'UTRs. However, further analysis revealed that these were false-positives caused by binding of the antibody to the m7G-cap. A different m1A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5'UTRs. These results demonstrate that high-stoichiometry m1A sites are exceedingly rare in mRNAs and that previous mappings of m1A to 5'UTRs were the result of antibody cross-reactivity to the 5' cap.


Assuntos
Regiões 5' não Traduzidas/genética , Adenosina/análogos & derivados , Anticorpos/imunologia , Reações Cruzadas/imunologia , Adenosina/metabolismo , Animais , Sequência de Bases , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Nucleotídeos/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
9.
PLoS Genet ; 15(8): e1008136, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381575

RESUMO

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/genética , Hidroxiureia/toxicidade , Nucleotídeos/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética
10.
Elife ; 82019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31290743

RESUMO

The bacterial Sec translocon is a multi-protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Nucleotídeos/metabolismo , Canais de Translocação SEC/metabolismo , /metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/química , Ativação do Canal Iônico , Conformação Proteica , Canais de Translocação SEC/química , /química
11.
Poult Sci ; 98(10): 4375-4383, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31329966

RESUMO

Nucleotide-rich yeast extract (YN) was investigated for its effects on growth performance, jejunal histomorphology and mucosal immunoglobulin A (IgA), immune organs weight and apparent retention (AR) of components in broiler chickens challenged with Eimeria. A total of 336 day-old male chicks (Ross x Ross 708) were placed in floor pens and provided a corn-soybean meal-based diet without or with YN (500 g/mt) (n = 14). On day 10, 7 replicates per diet were orally administered with 1 mL of sporulated E. acervulina and E. maxima oocysts and the rest (non-challenged control) administered equivalent distilled water creating a 2 × 2 factorial arrangement for the post-challenge period (day 11 to 35). Feed intake (FI), BWG, and FCR responses were measured in pre- and post-challenge periods. Excreta samples were collected on day 14 to 17 and 31 to 34 for oocyst count and AR of components, respectively. On day 15 and 35, 5 birds/pen were necropsied for intestinal samples. Spleen, bursa, and thymus weights were also recorded at both time points and breast yield on day 35. Diet had no effect (P > 0.05) on pre-challenge growth performance. Interaction (P = 0.046) between Eimeria and YN on FI was such that Eimeria challenge increased FI (day 11 to 35) in non-YN birds. There was no interaction (P > 0.05) between Eimeria and YN on other post-challenge responses. Eimeria reduced (P < 0.05) BWG, FCR, caloric efficiency, day 15 jejunal villi height and IgA concentration, and increased (P < 0.01) day 15 spleen weight. On day 35, YN increased bursa weight (1.57 vs. 1.78 mg/g BW, P = 0.04). There was a tendency for an interaction effect (P = 0.09) on day 35 thymus weight, such that in challenged birds, YN fed birds tended to have a lighter thymus relative to non-YN fed birds. In conclusions, independent of Eimeria challenge, supplemental YN had no effect on growth performance, caloric efficiency, and intestinal function but increased immune organ weights.


Assuntos
Galinhas , Coccidiose/veterinária , Nucleotídeos/metabolismo , Doenças das Aves Domésticas/imunologia , Fermento Seco/metabolismo , Ração Animal/análise , Animais , Galinhas/anatomia & histologia , Galinhas/crescimento & desenvolvimento , Coccidiose/imunologia , Coccidiose/metabolismo , Coccidiose/parasitologia , Dieta/veterinária , Suplementos Nutricionais/análise , Eimeria/fisiologia , Jejuno/anatomia & histologia , Jejuno/parasitologia , Masculino , Nucleotídeos/administração & dosagem , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/parasitologia , Fermento Seco/administração & dosagem
12.
EMBO J ; 38(17): e100772, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31355487

RESUMO

Bacterial usage of the cyclic dinucleotide c-di-GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c-di-GMP metabolism, particularly on regulatory mechanisms governing control of EAL c-di-GMP phosphodiesterases. Herein, we provide high-resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full-length cAMP-bound form reveals the sensory N-terminus to be a domain-swapped variant of the cNMP/CRP family, which in the cAMP-activated state holds the C-terminal EAL enzyme in a phosphodiesterase-active conformation. Using a truncation mutant, we trap both a half-occupied and inactive apo-form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c-di-GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c-di-GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing "action potentials" to be generated by each GGDEF protein to effect their specific functions.


Assuntos
Bdellovibrio bacteriovorus/metabolismo , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bdellovibrio bacteriovorus/química , Bdellovibrio bacteriovorus/genética , Sítios de Ligação , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Nucleotídeos/metabolismo , Diester Fosfórico Hidrolases/genética , Ligação Proteica , Conformação Proteica , Transdução de Sinais
13.
Biochem Soc Trans ; 47(4): 1013-1027, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31296733

RESUMO

Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2'-deoxynucleoside-5'-triphosphate) pool by catalysing the hydrolysis of dNTP into 2'-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi-Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer-monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.


Assuntos
Antivirais , Proteína 1 com Domínio SAM e Domínio HD/fisiologia , Replicação Viral/fisiologia , Regulação Alostérica , Catálise , Proliferação de Células/fisiologia , DNA Viral/biossíntese , Homeostase , Humanos , Mutação , Nucleotídeos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
14.
J Sci Food Agric ; 99(13): 6108-6113, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31177538

RESUMO

BACKGROUND: Nucleotides are key constituents of milk, where they are utilized in cell replication, although there are limited studies for weaned piglets. This study evaluated the effects of uridine monophosphate (UMP) with uridine (UR) feed supplementation on the intestinal development and nucleotide transport in weaned piglets. RESULTS: Supplementation with UMP significantly increased (P < 0.05) plasma glucose, and UR supplementation significantly reduced (0.05 < P < 0.10) the plasma total cholesterol (TC) of piglets when compared with that of the control group, although non-significant difference (P > 0.05) in growth performance was observed among three groups. Piglets fed supplementary UR exhibited greater (P < 0.05) crypt depth in the duodenum and ileum when compared with those in the supplementary UMP and control groups. Real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that UR supplementation increased (P < 0.05) the relative mRNA levels of genes encoding the transmembrane proteins ZO-1 and occludin in the duodenum mucosa, and ZO-1 in the jejunum mucosa (P < 0.05). Similarly, UR supplementation increased (P < 0.05) expression of solute carriers SLC28A1 and SLC29A1 in the duodenum mucosa. Conversely, claudin-1 expression in the duodenum mucosa was inhibited (P < 0.05) by dietary supplementation with UMP or UR. CONCLUSION: Collectively, our data indicated that dietary supplementation with UMP or UR was conducive to stimulating intestinal development and promoting nucleotide transport in weaned piglets. © 2019 Society of Chemical Industry.


Assuntos
Intestino Delgado/crescimento & desenvolvimento , Nucleotídeos/metabolismo , Suínos/crescimento & desenvolvimento , Uridina Monofosfato/metabolismo , Uridina/metabolismo , Animais , Transporte Biológico , Claudina-1/genética , Claudina-1/metabolismo , Suplementos Nutricionais/análise , Feminino , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Suínos/genética , Suínos/metabolismo , Desmame
15.
Int J Biol Macromol ; 136: 253-265, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170491

RESUMO

Ribokinase (RK) is an ATP dependent sugar kinase that enables the entry of ribose in the metabolism. Leishmania accumulates ribose into the cytosol through hydrolysis of nucleosides and by transport from the extracellular environment. Activation by RK is critical to mobilize the ribose into the metabolism of Leishmania. To understand the catalytic role, the crystal structure of RK (LdRK) from L. donovani was determined in the apo and complex forms with several nucleotides (ATP, AMPPCP and ADP) in the presence of Na+ ion. The dual insertion of five amino acid stretches makes LdRK structurally unique from other reported structures of RKs. The structure of LdRK-ATP provided the basis for positioning of γ-phosphate of ATP by conserved -GAGD- motif. Liganded and unliganded structures of LdRK exists in similar conformation, which suggests binding of nucleotides does not make any significant conformational changes in nucleotide-bound structures. Substitution of a conserved asparagine with phenylalanine in ribose binding pocket differentiates the LdRK from other RKs. Glycerol molecule bound in the substrate binding pocket mimics the enzyme-substrate interactions but in turn, hampers the binding of ribose to LdRK. Comparative structural analysis revealed the flexibility of γ-phosphate, which adopts multiple conformations in the absence of divalent metal ion and ribose. Similar to other RKs, LdRK is also dependent on monovalent as well as divalent cations for its catalytic activity.


Assuntos
Leishmania donovani/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Nucleotídeos/metabolismo , Fosfatos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Conformação Proteica
16.
Biochem Mol Biol Educ ; 47(5): 532-537, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31125165

RESUMO

Students always encounter difficulties in studying biochemical pathways. They are especially weak in understanding the relationships between metabolic pathways and their integration because these pathways are always taught one-by-one in class. In the past, various online resources have been developed to facilitate students' understanding toward energy metabolism. Although these learning materials enable students to understand individual metabolic pathway in a clearer manner, many of them fail to demonstrate the linkages between these pathways. The "AG City" is a self-learning tool which aims to arouse students' interest in exploring nucleotide metabolism. We have designed a metro map as a concept map to allow students to have an overview of different pathways as well as their integration. Major pathways are presented as railway lines in an easy-to-understand and interactive manner using navigation, animations, and narrations. Key molecules involved in the pathways are presented as "railway stations". Students can easily identify common "railway stations" presented in different pathways and link the concepts that they have learnt together. This interactive self-learning tool has been packaged as a courseware using the Articulate Storyline eLearning authoring software. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):532-537, 2019.


Assuntos
Bioquímica/educação , Instrução por Computador/métodos , Aprendizagem , Mapas como Assunto , Redes e Vias Metabólicas , Nucleotídeos/metabolismo , Ferrovias , Software , Compreensão , Currículo , Avaliação Educacional , Humanos , Estudantes
17.
Biochem J ; 476(11): 1637-1651, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31085558

RESUMO

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.


Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Apirase/química , Apirase/metabolismo , Animais , Antígenos CD/genética , Apirase/genética , Linhagem Celular , Elementos de DNA Transponíveis/genética , Nucleotídeos/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Solubilidade , Transfecção , Regulação para Cima
18.
Nat Commun ; 10(1): 2136, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086188

RESUMO

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Simulação por Computador , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA de Transferência de Metionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , eIF-2 Quinase/metabolismo
19.
Nucleic Acids Res ; 47(13): 6685-6698, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31127308

RESUMO

The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.


Assuntos
Proteínas de Bactérias/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Iniciação da Transcrição Genética , Proteínas de Escherichia coli/metabolismo , Heparina/farmacologia , Cinética , Modelos Químicos , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade da Espécie , Iniciação da Transcrição Genética/efeitos dos fármacos
20.
Nucleic Acids Res ; 47(10): 5405-5419, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30949702

RESUMO

Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5' end of nascent DNA. A subsequent 5'-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5'-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA-DNA junctions to remove the 5'-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA-DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA-DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5'-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Endonucleases/metabolismo , Exonucleases/metabolismo , Ribonuclease H/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Endonucleases/genética , Exonucleases/genética , Humanos , Mitocôndrias/genética , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA/genética , Ribonucleotídeos/metabolismo
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