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1.
Nat Commun ; 11(1): 4664, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938919

RESUMO

Cardiorenal syndrome type 4 (CRS4) is a common complication of chronic kidney disease (CKD), but the pathogenic mechanisms remain elusive. Here we report that morphological and functional changes in myocardial mitochondria are observed in CKD mice, especially decreases in oxidative phosphorylation and fatty acid metabolism. High phosphate (HP), a hallmark of CKD, contributes to myocardial energy metabolism dysfunction by downregulating peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α). Furthermore, the transcriptional factor interferon regulatory factor 1 (IRF1) is revealed as the key molecule upregulated by HP through histone H3K9 acetylation, and responsible for the HP-mediated transcriptional inhibition of PGC1α by directly binding to its promoter region. Conversely, restoration of PGC1α expression or genetic knockdown of IRF1 significantly attenuates HP-induced alterations in vitro and in vivo. These findings demonstrate that IRF1-PGC1α axis-mediated myocardial energy metabolism remodeling plays a crucial role in the pathogenesis of CRS4.


Assuntos
Síndrome Cardiorrenal/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Síndrome Cardiorrenal/patologia , Modelos Animais de Doenças , Regulação para Baixo , Metabolismo Energético , Técnicas de Silenciamento de Genes , Taxa de Filtração Glomerular , Glucuronidase/genética , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Fator Regulador 1 de Interferon/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Ratos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismo , Adulto Jovem
2.
Nat Commun ; 11(1): 4243, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843625

RESUMO

Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1ß release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1ß release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.


Assuntos
Artrite Reumatoide/imunologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Calcinose , Cálcio/metabolismo , Células Cultivadas , Humanos , Inflamação , Interleucina-1beta/metabolismo , Camundongos , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Fosfatos/metabolismo , Pinocitose , Receptores de Detecção de Cálcio/deficiência , Transdução de Sinais , Células THP-1 , alfa-2-Glicoproteína-HS/metabolismo
3.
PLoS Genet ; 16(8): e1008783, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813693

RESUMO

Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here, we describe the role of ethanol in mixed-species co-cultures by dual-seq analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase, Adh1. Analysis of the RNA-Seq data using KEGG pathway enrichment and eADAGE methods revealed several P. aeruginosa responses to C. albicans-produced ethanol including the induction of a non-canonical low-phosphate response regulated by PhoB. C. albicans wild type, but not C. albicans adh1Δ/Δ, induces P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells and exhibits antifungal activity. Here, we show that C. albicans adh1Δ/Δ no longer activates P. aeruginosa PhoB and PhoB-regulated phosphatase activity, that exogenous ethanol complements this defect, and that ethanol is sufficient to activate PhoB in single-species P. aeruginosa cultures at permissive phosphate levels. The intersection of ethanol and phosphate in co-culture is inversely reflected in C. albicans; C. albicans adh1Δ/Δ had increased expression of genes regulated by Pho4, the C. albicans transcription factor that responds to low phosphate, and Pho4-dependent phosphatase activity. Together, these results show that C. albicans-produced ethanol stimulates P. aeruginosa PhoB activity and 5-MPCA-mediated antagonism, and that both responses are dependent on local phosphate concentrations. Further, our data suggest that phosphate scavenging by one species improves phosphate access for the other, thus highlighting the complex dynamics at play in microbial communities.


Assuntos
Antibiose , Candida albicans/fisiologia , Etanol/metabolismo , Fosfatos/metabolismo , Pseudomonas aeruginosa/fisiologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais , Transcriptoma
4.
PLoS One ; 15(7): e0235082, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634148

RESUMO

Kidney donation results in reductions in kidney function and lasting perturbations in phosphate homeostasis, which may lead to adverse cardiovascular sequelae. However, the acute effects of kidney donation on bone mineral parameters including regulators of calcium and phosphate metabolism are unknown. We conducted a prospective observational controlled study to determine the acute effects of kidney donation on mineral metabolism and skeletal health. Biochemical endpoints were determined before and after donation on days 1, 2 and 3, 6 weeks and 12 months in donors and at baseline, 6 weeks and 12 months in controls. Baseline characteristic of donors (n = 34) and controls (n = 34) were similar: age (53±10 vs 50±14 years, p = 0.33), BMI (26.3±2.89 vs 25.9±3.65, p = 0.59), systolic BP (128±13 vs 130±6 mmHg, p = 0.59), diastolic BP (80±9 vs 81±9 mmHg, p = 0.68) and baseline GFR (84.4±20.2 vs 83.6±25.2 ml/min/1.73m2, p = 0.89). eGFR reduced from 84.4±20.2 to 52.3±17.5 ml/min/1.73m2 (p<0.001) by day 1 with incomplete recovery by 12 months (67.7±22.6; p = 0.002). Phosphate increased by day 1 (1.1(0.9-1.2) to 1.3(1.1-1.4) mmol/L, p <0.001) but declined to 0.8(0.8-1.0) mmol/L (p<0.001) before normalizing by 6 weeks. Calcium declined on day 1 (p = 0.003) but recovered at 6 weeks or 12 months. PTH and FGF-23 remained unchanged, but α-Klotho reduced by day 1 (p = 0.001) and remained low at 6 weeks (p = 0.02) and 1 year (p = 0.04). In this study, we conclude that kidney donation results in acute disturbances in mineral metabolism characterised by a reduced phosphate and circulating α-Klotho concentration without acute changes in the phosphaturic hormones FGF23 and PTH.


Assuntos
Densidade Óssea , Transplante de Rim , Minerais/metabolismo , Doadores de Tecidos , Adulto , Estudos de Casos e Controles , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , Estudos Prospectivos , Fatores de Tempo
5.
PLoS One ; 15(7): e0236520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730286

RESUMO

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.


Assuntos
Complexo de Golgi/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
6.
PLoS One ; 15(7): e0236361, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706793

RESUMO

MEdiator of cell MOtility1 (MEMO1) is a ubiquitously expressed redox protein involved in extracellular ligand-induced cell signaling. We previously reported that inducible whole-body Memo1 KO (cKO) mice displayed a syndrome of premature aging and disturbed mineral metabolism partially recapitulating the phenotype observed in Klotho or Fgf23-deficient mouse models. Here, we aimed at delineating the contribution of systemic mineral load on the Memo1 cKO mouse phenotype. We attempted to rescue the Memo1 cKO phenotype by depleting phosphate or vitamin D from the diet, but did not observe any effect on survival. However, we noticed that, by contrast to Klotho or Fgf23-deficient mouse models, Memo1 cKO mice did not present any soft-tissue calcifications and displayed even a decreased serum calcification propensity. We identified higher serum magnesium levels as the main cause of protection against calcifications. Expression of genes encoding intestinal and renal magnesium channels and the regulator epidermal growth factor were increased in Memo1 cKO. In order to check whether magnesium reabsorption in the kidney alone was driving the higher magnesemia, we generated a kidney-specific Memo1 KO (kKO) mouse model. Memo1 kKO mice also displayed higher magnesemia and increased renal magnesium channel gene expression. Collectively, these data identify MEMO1 as a novel regulator of magnesium homeostasis and systemic calcification propensity, by regulating expression of the main magnesium channels.


Assuntos
Calcinose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Rim/metabolismo , Magnésio/sangue , Animais , Calcinose/genética , Feminino , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatos/metabolismo , Vitamina D/metabolismo
7.
Nanotoxicology ; 14(6): 827-846, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32552239

RESUMO

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1ß, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences.


Assuntos
Cério/toxicidade , Fígado/efeitos dos fármacos , Nanopartículas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Baço/efeitos dos fármacos , Animais , Cério/química , Cério/metabolismo , Feminino , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Nanopartículas/química , Nanopartículas/metabolismo , Fosfatos/metabolismo , Especificidade da Espécie , Baço/metabolismo , Propriedades de Superfície , Distribuição Tecidual
8.
Life Sci ; 257: 117999, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585244

RESUMO

AIM: This paper was mainly aimed at synthesis of Ce-containing nano-Mg-phosphate ceramic as a multifunctional material. MATERIALS AND METHODS: Two ceramics based on Mg3(PO4)2 and Ce0.2Mg2.8(PO4)2 formulas (MP and MP-C, respectively) were synthesized. The synthesized powders were characterized by XRD, TEM, Zeta potential, and FTIR. Also, their dissolution behavior was tested in Tris-HCl buffer solution. Moreover, the antimicrobial efficacy was evaluated against gram-positive bacteria (Bacillus sphaericus MTCC 511 &Staphylococcus aureus MTCC 87) and gram-negative bacteria (Enterobacter aerogenes MTCC 111 &Pseudomonas aeruginosa MTCC 1034) using dick diffusion assay and microdilution method. Furthermore, the cell viability test was performed for the ceramics on Vero cells (African green monkey kidney cells), and their antitumor activity was determined by PC3 cell line (prostatic cancer). Also, the cellular uptake was determined by the flow cytometry. KEY FINDINGS: The results showed that the substitution of Mg by Ce decreased the particle size from 40 to 90 nm for MP sample to 2-10 nm for MP-C sample and increased the degradation rate. Both samples showed excellent antimicrobial activities. Moreover, MP demonstrated more cell viability than MP-C on Vero cells at high concentrations, whereas, MP-C showed more antitumor activity on PC3 cells than MP sample. Moreover, MP-C showed a higher cell uptake than MP due to its smaller size and more negative charge. SIGNIFICANCE: Mg-phosphate ceramic can be used in this study successfully as a delivery system for cerium ions and showed a high antitumor activity, which makes it highly recommended as safe and effective cancer treatment materials.


Assuntos
Cerâmica/farmacologia , Cério/farmacologia , Compostos de Magnésio/farmacologia , Fosfatos/farmacologia , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bacillaceae/efeitos dos fármacos , Osso e Ossos/microbiologia , Osso e Ossos/cirurgia , Sobrevivência Celular , Cério/metabolismo , Chlorocebus aethiops , Enterobacter aerogenes/efeitos dos fármacos , Humanos , Compostos de Magnésio/metabolismo , Testes de Sensibilidade Microbiana/métodos , Células PC-3 , Fosfatos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Células Vero
9.
Proc Natl Acad Sci U S A ; 117(25): 14322-14330, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32518115

RESUMO

Phosphorothioate (PT) DNA modifications-in which a nonbonding phosphate oxygen is replaced with sulfur-represent a widespread, horizontally transferred epigenetic system in prokaryotes and have a highly unusual property of occupying only a small fraction of available consensus sequences in a genome. Using Salmonella enterica as a model, we asked a question of fundamental importance: How do the PT-modifying DndA-E proteins select their GPSAAC/GPSTTC targets? Here, we applied innovative analytical, sequencing, and computational tools to discover a novel behavior for DNA-binding proteins: The Dnd proteins are "parked" at the G6mATC Dam methyltransferase consensus sequence instead of the expected GAAC/GTTC motif, with removal of the 6mA permitting extensive PT modification of GATC sites. This shift in modification sites further revealed a surprising constancy in the density of PT modifications across the genome. Computational analysis showed that GAAC, GTTC, and GATC share common features of DNA shape, which suggests that PT epigenetics are regulated in a density-dependent manner partly by DNA shape-driven target selection in the genome.


Assuntos
Bactérias/genética , Bactérias/metabolismo , DNA Bacteriano/metabolismo , Epigênese Genética/fisiologia , Epigenômica , Fosfatos/metabolismo , 2-Aminopurina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Consenso , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Genoma Bacteriano , Salmonella enterica/genética
10.
PLoS One ; 15(6): e0233896, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492057

RESUMO

Food insecurity is a looming threat for the burgeoning world population. Phosphorus (P), which is absorbed from soil as inorganic phosphate (Pi), is an essential macronutrient for the growth of all agricultural crops. This study reports phenotype analysis for P responses in natural field and greenhouse conditions, using 54 genotypes of foxtail millet (Setaria italica) representing wide geographic origins. The genotype responses were assessed in natural field conditions in two different seasons (monsoon and summer) under Pi-fertilized (P+) and unfertilized (P-) soil for eight above-ground traits. Enormous variations were seen among the genotypes in phenotypic responses for all the measured parameters under low P stress conditions. Variations were significant for plant height, leaf number and length, tillering ability and seed yield traits. Genotypes ISe 1234 and ISe 1541 were P+ responders, and the genotypes ISe 1181, ISe 1655, ISe 783 and ISe 1892 showed tolerance to low P for total seed yield. Genotypes that performed well under P- conditions were almost as productive as genotypes that performed well under P+ conditions suggesting some genotypes are well adapted to nutrient-poor soils. In the greenhouse, most of the genotypes produced changes in root architecture that are characteristic of P- stress, but to differing degrees. Significant variation was seen in root hair density and root hair number and in fresh and dry weight of shoot and root under P- stress. However, there was not much difference in the shoot and root total P and Pi levels of five selected high and low responding genotypes. We noticed contrasting responses in the greenhouse and natural field experiments for most of these genotypes. The leads from the study form the basis for breeding and improvement of foxtail millet for better Pi-use efficiency.


Assuntos
Produtos Agrícolas/genética , Regulação da Expressão Gênica de Plantas , Fosfatos/metabolismo , Melhoramento Vegetal , Setaria (Planta)/genética , Produtos Agrícolas/metabolismo , Genoma de Planta , Genótipo , Redes e Vias Metabólicas/genética , Filogenia , Setaria (Planta)/metabolismo , Solo/química
11.
PLoS One ; 15(6): e0232633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555651

RESUMO

Maize, a main crop worldwide, establishes a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi providing nutrients to the roots from soil volumes which are normally not in reach of the non-colonized root. The mycorrhizal phosphate uptake pathway (MPU) spans from extraradical hyphae to root cortex cells housing fungal arbuscules and promotes the supply of phosphate to the mycorrhizal host in exchange for photosynthetic carbon. This symbiotic association with the mycobiont has been shown to affect plant host nutritional status and growth performance. However, whether and how the MPU affects the root microbial community associated with mycorrhizal hosts in association with neighboring plants, remains to be demonstrated. Here the maize germinal Mu transposon insertion mutant pht1;6, defective in mycorrhiza-specific Pi transporter PHT1;6 gene, and wild type B73 (wt) plants were grown in mono- and mixed culture and examined under greenhouse and field conditions. Disruption of the MPU in pht1;6 resulted in strongly diminished growth performance, in reduced P allocation to photosynthetic source leaves, and in imbalances in leaf elemental composition beyond P. At the microbial community level a loss of MPU activity had a minor effect on the root-associated fungal microbiome which was almost fully restricted to AM fungi of the Glomeromycotina. Moreover, while wt grew better in presence of pht1;6, pht1;6 accumulated little biomass irrespective of whether it was grown in mono- or mixed culture and despite of an enhanced fungal colonization of its roots in co-culture with wt. This suggested that a functional MPU is prerequisite to maintain maize growth and that neighboring plants competed for AM fungal Pi in low P soil. Thus future strategies towards improving yield in maize populations on soils with low inputs of P fertilizer could be realized by enhancing MPU at the individual plant level while leaving the root-associated fungal community largely unaffected.


Assuntos
Micorrizas/metabolismo , Fosfatos/metabolismo , Zea mays/microbiologia , Zea mays/fisiologia , Biomassa , Mutação com Perda de Função , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Solo/química , Simbiose/fisiologia , Zea mays/genética , Zea mays/crescimento & desenvolvimento
12.
Nat Commun ; 11(1): 2642, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457313

RESUMO

Bacteria acquire phosphate (Pi) by maintaining a periplasmic concentration below environmental levels. We recently described an extracellular Pi buffer which appears to counteract the gradient required for Pi diffusion. Here, we demonstrate that various treatments to outer membrane (OM) constituents do not affect the buffered Pi because bacteria accumulate Pi in the periplasm, from which it can be removed hypo-osmotically. The periplasmic Pi can be gradually imported into the cytoplasm by ATP-powered transport, however, the proton motive force (PMF) is not required to keep Pi in the periplasm. In contrast, the accumulation of Pi into the periplasm across the OM is PMF-dependent and can be enhanced by light energy. Because the conventional mechanism of Pi-specific transport cannot explain Pi accumulation in the periplasm we propose that periplasmic Pi anions pair with chemiosmotic cations of the PMF and millions of accumulated Pi pairs could influence the periplasmic osmolarity of marine bacteria.


Assuntos
Bactérias/metabolismo , Fosfatos/metabolismo , Alphaproteobacteria/metabolismo , Alphaproteobacteria/efeitos da radiação , Oceano Atlântico , Bactérias/efeitos da radiação , Membrana Celular/metabolismo , Transporte de Íons/efeitos da radiação , Luz , Modelos Biológicos , Concentração Osmolar , Osmose , Periplasma/metabolismo , Fitoplâncton/metabolismo , Fitoplâncton/efeitos da radiação , Prochlorococcus/metabolismo , Prochlorococcus/efeitos da radiação , Força Próton-Motriz , Água do Mar/microbiologia , Synechococcus/metabolismo , Synechococcus/efeitos da radiação
13.
Proc Natl Acad Sci U S A ; 117(24): 13519-13528, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461373

RESUMO

Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to <1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name "young/strong" and "old/weak." The "young/strong" state 1 has adenosine 5'-diphosphate (ADP)-P i bound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the "old/weak" state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP-Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP-P i -Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling "old" actin filament branches in cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Fosfatos/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Fator de Maturação da Glia/metabolismo , Microfluídica , Microscopia de Fluorescência , Modelos Biológicos , Ligação Proteica , Coelhos , Schizosaccharomyces/metabolismo , Estresse Mecânico
14.
Arterioscler Thromb Vasc Biol ; 40(7): 1664-1679, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32434409

RESUMO

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.


Assuntos
Transdiferenciação Celular , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteogênese , Fatores de Transcrição/metabolismo , Calcificação Vascular/metabolismo , Idoso , Animais , Células Cultivadas , Colecalciferol , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Hiperfosfatemia/induzido quimicamente , Hiperfosfatemia/metabolismo , Hiperfosfatemia/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosfatos/metabolismo , Transdução de Sinais , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Calcificação Vascular/genética , Calcificação Vascular/patologia , Calcificação Vascular/prevenção & controle
15.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140441, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32371149

RESUMO

Dengue represents a substantial public health burden, particularly in low-resource countries. Non-structural protein 3 (NS3) is a multifunctional protein critical in the virus life cycle and has been identified as a promising anti-viral drug target. Despite recent crystallographic studies of the NS3 helicase domain, only subtle structural nucleotide-dependent differences have been identified, such that its coupled ATPase and helicase activities remain mechanistically unclear. Here we use molecular dynamics simulations to explore the nucleotide-dependent conformational landscape of the Dengue virus NS3 helicase and identify substantial changes in the protein flexibility during the ATP hydrolysis cycle. We relate these changes to the RNA-protein interactions and proposed translocation models for other monomeric helicases. Furthermore, we report a novel open-loop conformation with a likely escape route for Pi after hydrolysis, providing new insight into the conformational changes that underlie the ATPase activity of NS3.


Assuntos
Trifosfato de Adenosina/química , Vírus da Dengue/química , Fosfatos/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Vírus da Dengue/enzimologia , Hidrólise , Simulação de Dinâmica Molecular , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/metabolismo
16.
Chemosphere ; 252: 126470, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32443258

RESUMO

Concerns have been raised over the neurotoxicity of triphenyl phosphate (TPP), but there have been few studies of the neurotoxic effects of TPP on mammals and the underlying mechanisms. In this study, weaned male mice (C57/BL6) were used and exposed to 0, 50, or 150 mg/kg TPP daily by oral gavage for 30 days. The blood brain barrier (BBB) permeability of TPP and its metabolite diphenyl phosphate (DPP) in the brain, and TPP induced metabolomic and transcriptomic changes of the brain were investigated. The results showed that TPP and DPP can cross the BBB of mice. Histopathological examination of the brain revealed abnormalities in the hippocampus, cortex and thalamus, and mice treated with high doses showed a potential inflammation in the thalamus and hippocampus. Untargeted metabolomic results revealed that the changed level of glutamic acid, N-acetyl CoA metabolites, and organic acid in the brain of treated mice, suggest that amino acid and lipid metabolism was interfered. RNA-seq data indicated that neuronal transcription processes and cell apoptosis pathway (forkhead box (FOXO), and mitogen-activated protein kinase (MAPK) signaling pathways) were significantly affected by TPP exposure. RT-PCR showed proinflammation cytokine tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6)) levels were increased, while antioxidant genes including nuclear factor-E2-related factor 2 (Nrf2), heme oxygenase1 (HO-1) and superoxide dismutase (SOD1) decreased. These results suggest that TPP could cause a degree of neurotoxicity by inducing neuroinflammation and neuronal apoptosis, which are related to oxidative stress. The potential implications for neurophysiology and behavioral regulation cannot be ignored.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Sistema Nervoso/efeitos dos fármacos , Organofosfatos/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose , Encéfalo/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Inflamação/metabolismo , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Síndromes Neurotóxicas , Estresse Oxidativo/efeitos dos fármacos , Fosfatos/metabolismo , Superóxido Dismutase/metabolismo , Testes de Toxicidade
18.
Nucleic Acids Res ; 48(9): 4811-4826, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32282918

RESUMO

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis-trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3' processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3' processing/termination that acts via Ssu72.


Assuntos
Regulação Fúngica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/genética , Fosfoproteínas Fosfatases/genética , Processamento de Terminações 3' de RNA , Proteínas de Schizosaccharomyces pombe/genética , Terminação da Transcrição Genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Deleção de Genes , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteínas Nucleares/genética , Fosfatos/metabolismo , Fosforilação , Domínios Proteicos/genética , Pirofosfatases/genética , RNA Polimerase II/genética , RNA-Seq , Regulon , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Serina/metabolismo , Treonina/metabolismo
19.
Nat Microbiol ; 5(7): 917-928, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32251370

RESUMO

Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD-SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behaviour. SspABCD confers single-stranded and high-frequency PTs with SspB acting as a nickase and possibly introducing nicks to facilitate sulfur incorporation. Strikingly, SspABCD coupled with SspE provides protection against phages in unusual ways: (1) SspE senses sequence-specific PTs by virtue of its PT-stimulated NTPase activity to exert its anti-phage activity, and (2) SspE inhibits phage propagation by introducing nicking damage to impair phage DNA replication. These results not only expand our knowledge about the diversity and functions of DNA PT modification but also enhance our understanding of the known arsenal of defence systems.


Assuntos
Bacteriófagos/fisiologia , Interações Hospedeiro-Patógeno , Fosfatos/metabolismo , Streptomyces/fisiologia , Streptomyces/virologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sítios de Ligação , Ativação Enzimática , Genoma Bacteriano , Interações Hospedeiro-Patógeno/genética , Modelos Moleculares , Fases de Leitura Aberta , Fosfatos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica
20.
Proc Natl Acad Sci U S A ; 117(19): 10234-10245, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341157

RESUMO

The nitrogen-related phosphotransferase system (PTSNtr) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTSNtr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTSNtr is widely conserved in proteobacteria, highlighting its global importance.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Fosfatos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Rhizobium leguminosarum/metabolismo , Proteínas de Bactérias/genética , Ciclo do Ácido Cítrico , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilação , Regiões Promotoras Genéticas , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/crescimento & desenvolvimento
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