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1.
PLoS Comput Biol ; 16(9): e1008202, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925922

RESUMO

Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well established, the quantitative and mechanistic aspects of H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H2O2 reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 will be in the low nM range (2-4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 generation rates ~50 µM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H2O2 generation rates. At longer times, substantial efflux of H2O2 from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.


Assuntos
Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Biologia Computacional , Citosol/química , Citosol/metabolismo , Células HeLa , Humanos , Cinética , Mitocôndrias/química , Neoplasias/química , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
2.
Chem Biol Interact ; 330: 109247, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866466

RESUMO

This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.


Assuntos
Fígado/metabolismo , Oxazinas/metabolismo , Frações Subcelulares/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Feminino , Herbicidas/química , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Ratos , Fatores Sexuais , Estereoisomerismo
3.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
4.
Nat Commun ; 11(1): 3439, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651385

RESUMO

Various stress conditions induce the nuclear translocation of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), but its nuclear function in plant stress responses remains elusive. Here we show that GAPC interacts with a transcription factor to promote the expression of heat-inducible genes and heat tolerance in Arabidopsis. GAPC accumulates in the nucleus under heat stress. Overexpression of GAPC enhances heat tolerance of seedlings and the expression of heat-inducible genes whereas knockout of GAPCs has opposite effects. Screening of Arabidopsis transcription factors identifies nuclear factor Y subunit C10 (NF-YC10) as a GAPC-binding protein. The effects of GAPC overexpression are abolished when NF-YC10 is deficient, the heat-induced nuclear accumulation of GAPC is suppressed, or the GAPC-NF-YC10 interaction is disrupted. GAPC overexpression also enhances the binding ability of NF-YC10 to its target promoter. The results reveal a cellular and molecular mechanism for the nuclear moonlighting of a glycolytic enzyme in plant response to environmental changes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Citosol/metabolismo , Citosol/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plântula/genética , Plântula/metabolismo , Plântula/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Mol Cell ; 79(2): 320-331.e9, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645369

RESUMO

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.


Assuntos
Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Catálise , Citosol/metabolismo , Células HEK293 , Humanos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteólise , Solubilidade , Ubiquitinação
6.
Nat Commun ; 11(1): 3393, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636388

RESUMO

Meiotic divisions in oocytes are extremely asymmetric and require pre- and post-anaphase-onset phases of spindle migration. The latter induces membrane protrusion that is moulded around the spindle thereby reducing cytoplasmic loss. Here, we find that depleting the NAD biosynthetic enzyme, nicotinamide phosphoribosyl-transferase (Nampt), in mouse oocytes results in markedly longer spindles and compromises asymmetry. By analysing spindle speed in live oocytes, we identify a striking and transient acceleration after anaphase-onset that is severely blunted following Nampt-depletion. Slow-moving midzones of elongated spindles induce cortical furrowing deep within the oocyte before protrusions can form, altogether resulting in larger oocyte fragments being cleaved off. Additionally, we find that Nampt-depletion lowers NAD and ATP levels and that reducing NAD using small molecule Nampt inhibitors also compromises asymmetry. These data show that rapid midzone displacement is critical for extreme asymmetry by delaying furrowing to enable protrusions to form and link metabolic status to asymmetric division.


Assuntos
Anáfase , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Oócitos/citologia , Fuso Acromático , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ciclo Celular , Segregação de Cromossomos , Citoplasma/metabolismo , Citosol/metabolismo , Feminino , Meiose , Camundongos , Microscopia Confocal , NAD/química
7.
Nat Commun ; 11(1): 3527, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669538

RESUMO

Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.


Assuntos
Ciclina D1/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hipertensão Pulmonar/metabolismo , NF-kappa B/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Sinalização do Cálcio , Proliferação de Células , Citosol/metabolismo , Humanos , Hipóxia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxigênio/metabolismo , Artéria Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Transtornos Respiratórios/metabolismo , Transdução de Sinais
8.
Nature ; 585(7824): 288-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32641834

RESUMO

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciona intestinalis/enzimologia , Ciclo do Ácido Cítrico , Citosol/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Lactobacillus brevis/enzimologia , Masculino , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neoplasias/enzimologia , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquinona/metabolismo
9.
Nucleic Acids Res ; 48(14): 7623-7639, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32644123

RESUMO

RNA therapeutics are a promising strategy to treat genetic diseases caused by the overexpression or aberrant splicing of a specific protein. The field has seen major strides in the clinical efficacy of this class of molecules, largely due to chemical modifications and delivery strategies that improve nuclease resistance and enhance cell penetration. However, a major obstacle in the development of RNA therapeutics continues to be the imprecise, difficult, and often problematic nature of most methods used to measure cell penetration. Here, we review these methods and clearly distinguish between those that measure total cellular uptake of RNA therapeutics, which includes both productive and non-productive uptake, and those that measure cytosolic/nuclear penetration, which represents only productive uptake. We critically analyze the benefits and drawbacks of each method. Finally, we use key examples to illustrate how, despite rigorous experimentation and proper controls, our understanding of the mechanism of gymnotic uptake of RNA therapeutics remains limited by the methods commonly used to analyze RNA delivery.


Assuntos
RNA/metabolismo , RNA/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Núcleo Celular/metabolismo , Citosol/metabolismo , Doenças Genéticas Inatas/tratamento farmacológico , Técnicas Genéticas , Humanos , MicroRNAs/uso terapêutico , Microscopia Eletrônica , Oligonucleotídeos Antissenso/uso terapêutico , RNA/química , RNA/farmacocinética , RNA Interferente Pequeno/uso terapêutico , Espectrometria de Fluorescência
10.
Proc Natl Acad Sci U S A ; 117(32): 19399-19407, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719124

RESUMO

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/genética
11.
Nat Commun ; 11(1): 2729, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483166

RESUMO

Aggregation and spreading of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, thus monitoring human αSyn (hαSyn) in animal models or cell cultures is vital for the field. However, the detection of native hαSyn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind hαSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hαSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hαSyn. Here, we exploit this feature to design a new Fluorescent Reporter for hαSyn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hαSyn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable αSyn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics.


Assuntos
Citosol/metabolismo , Proteínas Luminescentes/metabolismo , Anticorpos de Domínio Único/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Citosol/química , Feminino , Fluorescência , Células HEK293 , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Masculino , Microscopia de Fluorescência por Excitação Multifotônica , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/metabolismo , Ratos Wistar , Anticorpos de Domínio Único/genética , alfa-Sinucleína/líquido cefalorraquidiano , alfa-Sinucleína/genética
12.
Proc Natl Acad Sci U S A ; 117(26): 15343-15353, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32546525

RESUMO

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thaliana Chloride Channel a) is a vacuolar NO3 -/H+ exchanger regulating stomata aperture in A thaliana Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo in Arabidopsis guard cells. We first found that ClopHensor is not only a Cl- but also, an NO3 - sensor. We were then able to quantify the variations of NO3 - and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3 - In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3 - and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Citosol/química , Homeostase/fisiologia , Nitratos/química , Proteínas de Arabidopsis/genética , Canais de Cloreto/genética , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Concentração de Íons de Hidrogênio , Nitratos/metabolismo
13.
PLoS Comput Biol ; 16(6): e1007752, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479491

RESUMO

We study the pathogenesis of Francisella tularensis infection with an experimental mouse model, agent-based computation and mathematical analysis. Following inhalational exposure to Francisella tularensis SCHU S4, a small initial number of bacteria enter lung host cells and proliferate inside them, eventually destroying the host cell and releasing numerous copies that infect other cells. Our analysis of disease progression is based on a stochastic model of a population of infectious agents inside one host cell, extending the birth-and-death process by the occurrence of catastrophes: cell rupture events that affect all bacteria in a cell simultaneously. Closed expressions are obtained for the survival function of an infected cell, the number of bacteria released as a function of time after infection, and the total bacterial load. We compare our mathematical analysis with the results of agent-based computation and, making use of approximate Bayesian statistical inference, with experimental measurements carried out after murine aerosol infection with the virulent SCHU S4 strain of the bacterium Francisella tularensis, that infects alveolar macrophages. The posterior distribution of the rate of replication of intracellular bacteria is consistent with the estimate that the time between rounds of bacterial division is less than 6 hours in vivo.


Assuntos
Francisella tularensis/citologia , Pulmão/microbiologia , Tularemia/microbiologia , Animais , Teorema de Bayes , Biologia Computacional , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Teóricos , Fagossomos/metabolismo , Probabilidade , Processos Estocásticos , Virulência
14.
Nat Commun ; 11(1): 3238, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591540

RESUMO

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/NAD+ ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/NAD+ ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle.


Assuntos
Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Luz , Proteínas Luminescentes/metabolismo , NADP/metabolismo , NAD/metabolismo , Fotossíntese/efeitos da radiação , Respiração Celular/efeitos da radiação , Cloroplastos/metabolismo , Citosol/metabolismo , Transporte de Elétrons/efeitos da radiação , Malatos/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Oxirredução , Peroxissomos/metabolismo , Plântula/metabolismo , Plântula/efeitos da radiação
15.
Nat Commun ; 11(1): 3024, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541684

RESUMO

The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potássio/metabolismo , Citosol/química , Citosol/metabolismo , Condutividade Elétrica , Impedância Elétrica , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Transporte de Íons , Íons/química , Íons/metabolismo , Simulação de Dinâmica Molecular , Potássio/química , Conformação Proteica , Água/metabolismo
16.
Ecotoxicol Environ Saf ; 202: 110876, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32563953

RESUMO

This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium (45Ca2+) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca2+ was involved in the effects of BPA on testicular toxicity. In vitro studies on 45Ca2+ influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of 45Ca2+ and BPA to testes to study the mechanism of action of BPA. The involvement of intracellular calcium from stores on lactate dehydrogenase (LDH) release and on triacylglycerol (TAG) content were carried out after in vitro incubation of testes with BPA for 1 h. Furthermore, gamma-glutamyl transpeptidase (GGT) and aspartate aminotransferase (AST) activities were analyzed in the liver at 1 h after in vitro BPA incubation of D. rerio. Our data show that the acute in vitro treatment of D. rerio testes with BPA at very low concentration activates plasma membrane ionic channels, such as voltage-dependent calcium channels and calcium-dependent chloride channels, and protein kinase C (PKC), which stimulates Ca2+ influx. In addition, BPA increased cytosolic Ca2+ by activating inositol triphosphate receptor (IP3R) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic reticulum, contributing to intracellular Ca2+ overload. The protein kinases, PKC, MEK 1/2 and PI3K, are involved in the mechanism of action of BPA, which may indicate a crosstalk between the non-genomic initiation effects mediated by PLC/PKC/IP3R signaling and genomic responses of BPA mediated by the estrogen receptor (ESR). In vitro exposure to a higher concentration of BPA caused cell damage and plasma membrane injury with increased LDH release and TAG content; both effects were dependent on intracellular Ca2+ and mediated by IP3R. Furthermore, BPA potentially induced liver damage, as demonstrated by increased GGT activity. In conclusion, in vitro effect of BPA in a low concentration triggers cytosolic Ca2+ overload and activates downstream protein kinases pointing to a crosstalk between its non-genomic and genomic effects of BPA mediated by ESR. Moreover, in vitro exposure to a higher concentration of BPA caused intracellular Ca2+-dependent testicular cell damage and plasma membrane injury. This acute toxicity was reinforced by increased testicular LDH release and GGT activity in the liver.


Assuntos
Compostos Benzidrílicos/toxicidade , Cálcio/metabolismo , Fenóis/toxicidade , Testículo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/fisiologia , Animais , Membrana Celular/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canais Iônicos , Masculino , Proteína Quinase C/metabolismo , Proteína Quinase C/farmacologia , Transdução de Sinais/efeitos dos fármacos , Testículo/metabolismo , Peixe-Zebra/metabolismo
17.
PLoS Comput Biol ; 16(6): e1007572, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502205

RESUMO

Ventricular contraction is roughly proportional to the amount of calcium released from the Sarcoplasmic Reticulum (SR) during systole. While it is rather straightforward to measure calcium levels and contractibility under different physiological conditions, the complexity of calcium handling during systole and diastole has made the prediction of its release at steady state impossible. Here we approach the problem analyzing the evolution of intracellular and extracellular calcium fluxes during a single beat which is away from homeostatic balance. Using an in-silico subcellular model of rabbit ventricular myocyte, we show that the high dimensional nonlinear problem of finding the steady state can be reduced to a two-variable general equilibrium condition where pre-systolic calcium level in the cytosol and in the SR must fulfill simultaneously two different equalities. This renders calcium homeostasis as a problem that can be studied in terms of its equilibrium structure, leading to precise predictions of steady state from single-beat measurements. We show how changes in ion channels modify the general equilibrium, as shocks would do in general equilibrium macroeconomic models. This allows us to predict when an enhanced entrance of calcium in the cell reduces its contractibility and explain why SERCA gene therapy, a change in calcium handling to treat heart failure, might fail to improve contraction even when it successfully increases SERCA expression.


Assuntos
Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Íons , Células Musculares/metabolismo , Animais , Simulação por Computador , Citosol/metabolismo , Homeostase , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sístole
18.
Mol Cell ; 79(1): 68-83.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533918

RESUMO

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.


Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos
19.
Nat Commun ; 11(1): 3068, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555155

RESUMO

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.


Assuntos
Cálcio/química , Epitélio/metabolismo , Aderências Teciduais/metabolismo , Animais , Sinalização do Cálcio , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional , Citoesqueleto/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complicações Pós-Operatórias , Análise de Componente Principal , RNA Interferente Pequeno/metabolismo , Análise de Célula Única
20.
Nat Commun ; 11(1): 2381, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404906

RESUMO

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.


Assuntos
Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Bactérias Gram-Negativas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/microbiologia , Células Eucarióticas/microbiologia , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/fisiologia , Humanos , Luz , Microscopia de Fluorescência , Optogenética/métodos , Transporte Proteico/efeitos da radiação , Análise Espacial , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/fisiologia
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