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1.
BMC Plant Biol ; 19(1): 368, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429706

RESUMO

BACKGROUND: We previously reported the involvement of nitric oxide (NO) and cyclic nucleotide-gated ion channel 6 (CNGC6) in the responses of plants to heat shock (HS) exposure. To elucidate their relationship with heat tolerance in Arabidopsis thaliana, we examined the effects of HS on several groups of seedlings: wild type, cngc6, and cngc6 complementation and overexpression lines. RESULTS: After HS exposure, the level of NO was lower in cngc6 seedlings than in wild-type seedlings but significantly elevated in the transgenic lines depending on CNGC6 expression level. The treatment of seeds with calcium ions (Ca2+) enhanced the NO level in Arabidopsis seedlings under HS conditions, whereas treatment with EGTA (a Ca2+ chelator) reduced it, implicating that CNGC6 stimulates the accumulation of NO depending on an increase in cytosolic Ca2+ ([Ca2+]cyt). This idea was proved by phenotypic observations and thermotolerance testing of transgenic plants overexpressing NIA2 and NOA1, respectively, in a cngc6 background. Western blotting indicated that CNGC6 stimulated the accumulation of HS proteins via NO. CONCLUSION: These data indicate that CNGC6 acts upstream of NO in the HS pathway, which improves our insufficient knowledge of the initiation of plant responses to high temerature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Óxido Nítrico/metabolismo , Termotolerância , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Canais de Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Citosol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Mutação , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Plântula/genética , Plântula/metabolismo
2.
Results Probl Cell Differ ; 67: 233-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435798

RESUMO

Morphology of Golgi apparatus changes frequently and diversely depending on various cellular conditions and these changes correlate with the balance between membrane inflow and outflow at the Golgi via vesicular transports. In a previous study, we introduced a semi-intact cell system suitable for the reconstitution of morphological changes that organelles undergo as well as the vesicular transport between them. Semi-intact cells are cells that have undergone plasma membrane permeabilization by the cholesterol-dependent pore-forming cytolysin, streptolysin O (SLO). Permeabilization enables the introduction of various molecules into the cells, as well as the substitution of the original cytosol with an exogenously made cytosol prepared from cells in various stages of cell cycle, differentiation, and disease progression. Coupled with a green fluorescent protein(GFP)-visualization technique, this cell-based system enables the analysis of the molecular mechanisms underlying biological processes that are highly dependent on the integrity of the intracellular architecture. In this chapter, we present a variety of reconstitution assays concerning biological reactions pertaining to the Golgi apparatus.


Assuntos
Permeabilidade da Membrana Celular , Complexo de Golgi/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citosol/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo de Golgi/efeitos dos fármacos
3.
Biochemistry (Mosc) ; 84(4): 358-369, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228927

RESUMO

Cytoplasmic actin structures are essential components of the eukaryotic cytoskeleton. According to the classic concepts, actin structures perform contractile and motor functions, ensuring the possibility of cell shape changes during cell spreading, polarization, and movement both in vitro and in vivo, from the early embryogenesis stages and throughout the life of a multicellular organism. Intracellular organization of actin structures, their biochemical composition, and dynamic properties play a key role in the realization of specific cellular and tissue functions and vary in different cell types. This paper is a review of recent studies on the organization and properties of actin structures in endotheliocytes, interaction of these structures with other cytoskeletal components and elements involved in cell adhesion, as well as their role in the functional activity of endothelial cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Caderinas/química , Caderinas/metabolismo , Citosol/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo
4.
Biochemistry (Mosc) ; 84(6): 583-592, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238858

RESUMO

Actin plays an important role in cellular adhesion, muscle and non-muscle contractility, migration, polarization, mitosis, and meiosis. Investigation of specific mechanisms underlying these processes is essential not only for fundamental research but also for clinical applications, since modulations of actin isoforms are directly or indirectly correlate with severe pathologies. In this review we summarize the isoform-specific functions of actin associated with adhesion structures, motility and division of normal and tumor cells; alterations of the expression and structural organization of actin isoforms in normal and tumor cells. Selective regulation of cytoplasmic ß- or γ-actin expression determines functional diversity between isoforms: ß-actin plays the predominant role in contraction and intercellular adhesion, and γ-actin is responsible for the cellular plasticity and motility. Similar data were obtained in different epithelial and mesenchymal neoplastic cell cultures, as well as in immunomorphological comparison of normal human tissues with tumor analogues. Reorganization of the actin cytoskeleton and cell-cell contacts is essential for proliferation control and acquisition of invasiveness in epithelial tumors.


Assuntos
Actinas/fisiologia , Isoformas de Proteínas/fisiologia , Actinas/química , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Transformação Celular Neoplásica , Citoplasma/metabolismo , Citosol/metabolismo , Humanos , Mamíferos , Isoformas de Proteínas/química , Relação Estrutura-Atividade
5.
Chem Commun (Camb) ; 55(52): 7474-7477, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31184664

RESUMO

Inspired by clinical studies on alcohol abuse induced endoplasmic reticulum disruption, we designed a N-hydroxylethyl peptide assembly to regulate the ER stress response in cancer cells. Upon coupling with a coumarin derivative via an ester linkage, a prodrug was synthesized to promote esterase-facilitated self-delivery of N-hydroxylethyl peptide assemblies around the ER, inducing ER dilation. Following this, ER-specific apoptosis was effectively and efficiently activated in various types of cancer cells including drug resistant and metastatic ones.


Assuntos
Citosol/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peptídeos/farmacologia , Apoptose/efeitos dos fármacos , Carboxilesterase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/química , Humanos , Microscopia de Fluorescência , Peptídeos/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia
6.
Plant Physiol Biochem ; 141: 353-369, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207496

RESUMO

Reactive oxygen species (ROS) - the byproducts of aerobic metabolism - influence numerous aspects of the plant life cycle and environmental response mechanisms. In plants, ROS act like a double-edged sword; they play multiple beneficial roles at low concentrations, whereas at high concentrations ROS and related redox-active compounds cause cellular damage through oxidative stress. To examine the dual role of ROS as harmful oxidants and/or crucial cellular signals, this review elaborates that (i) how plants sense and respond to ROS in various subcellular organelles and (ii) the dynamics of subsequent ROS-induced signaling processes. The recent understanding of crosstalk between various cellular compartments in mediating their redox state spatially and temporally is discussed. Emphasis on the beneficial effects of ROS in maintaining cellular energy homeostasis, regulating diverse cellular functions, and activating acclimation responses in plants exposed to abiotic and biotic stresses are described. The comprehensive view of cellular ROS dynamics covering the breadth and versatility of ROS will contribute to understanding the complexity of apparently contradictory ROS roles in plant physiological responses in less than optimum environments.


Assuntos
Oxirredução , Estresse Oxidativo , Fenômenos Fisiológicos Vegetais , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estresse Fisiológico , Aclimatação , Antioxidantes/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica , Genes de Plantas , Mitocôndrias/metabolismo , Oryza/metabolismo , Oxigênio/metabolismo , Peroxissomos/metabolismo , Fotossíntese , Populus/metabolismo
7.
Genes Dev ; 33(15-16): 1031-1047, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196865

RESUMO

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.


Assuntos
Aneuploidia , Citosol/metabolismo , Compensação de Dosagem (Genética)/fisiologia , Agregados Proteicos/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Agregação Patológica de Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Nat Commun ; 10(1): 2299, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127110

RESUMO

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.


Assuntos
Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Animais , Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Células Cultivadas , Masculino , Células Musculares/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Membrana Nuclear/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
9.
Nat Plants ; 5(5): 525-538, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31061535

RESUMO

Communication between organelles and the nucleus is essential for fitness and survival. Retrograde signals are cues emitted from the organelles to regulate nuclear gene expression. GENOMES UNCOUPLED1 (GUN1), a protein of unknown function, has emerged as a central integrator, participating in multiple retrograde signalling pathways that collectively regulate the nuclear transcriptome. Here, we show that GUN1 regulates chloroplast protein import through interaction with the import-related chaperone cpHSC70-1. We demonstrated that overaccumulation of unimported precursor proteins (preproteins) in the cytosol causes a GUN phenotype in the wild-type background and enhances the GUN phenotype of the gun1 mutant. Furthermore, we identified the cytosolic HSP90 chaperone complex, induced by overaccumulated preproteins, as a central regulator of photosynthetic gene expression that determines the expression of the GUN phenotype. Taken together, our results suggest a model in which protein import capacity, folding stress and the cytosolic HSP90 complex control retrograde communication.


Assuntos
Proteínas de Arabidopsis/fisiologia , Proteínas de Ligação a DNA/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transdução de Sinais/fisiologia , Transcriptoma
10.
Parasitol Res ; 118(6): 1785-1797, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31062084

RESUMO

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.


Assuntos
Citosol/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Mitocôndrias/metabolismo , Taenia solium/genética , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cysticercus/genética , Cysticercus/isolamento & purificação , Cysticercus/metabolismo , Citosol/química , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Fases de Leitura Aberta , Coelhos , Taenia solium/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo
11.
MBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064825

RESUMO

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Adaptação Fisiológica , Transporte Biológico , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions , Citosol/química , Citosol/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/patogenicidade
12.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075903

RESUMO

Boron (B) is a micronutrient for plant development, and its deficiency alters many physiological processes. However, the current knowledge on how plants are able to sense the B-starvation signal is still very limited. Recently, it has been reported that B deprivation induces an increase in cytosolic calcium concentration ([Ca2+]cyt) in Arabidopsis thaliana roots. The aim of this work was to research in Arabidopsis whether [Ca2+]cyt is restored to initial levels when B is resupplied and elucidate whether apoplastic Ca2+ is the major source for B-deficiency-induced rise in [Ca2+]cyt. The use of chemical compounds affecting Ca2+ homeostasis showed that the rise in root [Ca2+]cyt induced by B deficiency was predominantly owed to Ca2+ influx from the apoplast through plasma membrane Ca2+ channels in an IP3-independent manner. Furthermore, B resupply restored the root [Ca2+]cyt. Interestingly, expression levels of genes encoding Ca2+ transporters (ACA10, plasma membrane PIIB-type Ca2+-ATPase; and CAX3, vacuolar cation/proton exchanger) were upregulated by ethylene glycol tetraacetic acid (EGTA) and abscisic acid (ABA). The results pointed out that ACA10, and especially CAX3, would play a major role in the restoration of Ca2+ homeostasis after 24 h of B deficiency.


Assuntos
Arabidopsis/metabolismo , Boro/deficiência , Sinalização do Cálcio , Cálcio/metabolismo , Citosol/metabolismo , Arabidopsis/genética , Boro/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Citosol/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo
13.
Nat Commun ; 10(1): 1629, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967547

RESUMO

Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.


Assuntos
Retículo Endoplasmático/virologia , Vírus da Influenza A/fisiologia , Influenza Humana/patologia , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Células A549 , Animais , Citosol/metabolismo , Citosol/virologia , Cães , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Ligação Proteica , RNA Viral/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
14.
Nat Commun ; 10(1): 1703, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979871

RESUMO

Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.


Assuntos
Polaridade Celular , Citoesqueleto/fisiologia , Mandíbula/embriologia , Mandíbula/fisiologia , Proteína Wnt-5a/fisiologia , Citoesqueleto de Actina , Actomiosina/metabolismo , Animais , Cálcio/metabolismo , Ciclo Celular , Citosol/metabolismo , Elasticidade , Células Epiteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Mutação , Oscilometria , Transdução de Sinais , Estresse Mecânico , Vinculina/metabolismo , Viscosidade
15.
Nat Immunol ; 20(5): 527-533, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962589

RESUMO

Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1ß and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.


Assuntos
Caspases/imunologia , Citosol/imunologia , Imunidade Inata/imunologia , Lipopolissacarídeos/imunologia , Animais , Caspases/metabolismo , Citosol/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Modelos Imunológicos , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Piroptose/imunologia
16.
Int J Mol Sci ; 20(7)2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30987055

RESUMO

BACKGROUND: Previous studies demonstrated that calcium/calmodulin (Ca2+/CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway. METHODS: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays. RESULTS: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+-free or 1 mM of extracellular Ca2+ concentration modified Tyrode's solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5. CONCLUSIONS: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Bradicinina/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Suínos
17.
J Zhejiang Univ Sci B ; 20(4): 310-321, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30932376

RESUMO

OBJECTIVE: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli. METHODS: Modified microfluidics and imaging techniques were used to determine O2 •- levels and construct an O2 •- reaction network. To elucidate the consequences of increased O2 •- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca2+ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. RESULTS AND CONCLUSIONS: Results from a modified microchip demonstrated that 1 mmol/L H2O2 induced a rapid increase in cellular O2 •- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca2+ and mitochondrial Ca2+ by confocal laser scanning microscopy and confirmed that Ca2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca2+ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.


Assuntos
Peróxido de Hidrogênio/química , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/química , Apoptose , Cálcio/metabolismo , Sinalização do Cálcio , Caspases/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Citosol/metabolismo , Glutationa/metabolismo , Humanos , Oxirredução , Transdução de Sinais
18.
PLoS Genet ; 15(4): e1008094, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31034471

RESUMO

As a component of the Cytosolic Iron-sulfur cluster Assembly (CIA) pathway, DRE2 is essential in organisms from yeast to mammals. However, the roles of DRE2 remain incompletely understood largely due to the lack of viable dre2 mutants. In this study, we successfully created hypomorphic dre2 mutants using the CRISPR/Cas9 technology. Like other CIA pathway mutants, the dre2 mutants have accumulation of DNA lesions and show constitutive DNA damage response. In addition, the dre2 mutants exhibit DNA hypermethylation at hundreds of loci. The mutant forms of DRE2 in the dre2 mutants, which bear deletions in the linker region of DRE2, lost interaction with GRXS17 but have stronger interaction with NBP35, resulting in the CIA-related defects of dre2. Interestingly, we find that DRE2 is also involved in auxin response that may be independent of its CIA role. DRE2 localizes in both the cytoplasm and the nucleus and nuclear DRE2 associates with euchromatin. Furthermore, DRE2 directly associates with multiple auxin responsive genes and maintains their normal expression. Our study highlights the importance of the linker region of DRE2 in coordinating CIA-related protein interactions and identifies the canonical and non-canonical roles of DRE2 in maintaining genome stability, epigenomic patterns, and auxin response.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Alelos , Sistemas CRISPR-Cas , Citosol/metabolismo , Dano ao DNA , Metilação de DNA , Ácidos Indolacéticos/metabolismo , Família Multigênica , Mutação , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas
19.
BMC Plant Biol ; 19(1): 89, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819104

RESUMO

BACKGROUND: HMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation. RESULTS: We cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 µM CdCl2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation. CONCLUSION: BjHMA4R can specifically bind Cd2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations.


Assuntos
Cádmio/metabolismo , Citosol/metabolismo , Metais Pesados/metabolismo , Mostardeira/metabolismo , Zinco/metabolismo , Regulação da Expressão Gênica de Plantas
20.
Pharmacol Rep ; 71(2): 257-265, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30822619

RESUMO

BACKGROUND: Non-opioid and opioid analgesics, as over-the-counter or prescribed medications, are widely used for the management of a diverse array of pathophysiological conditions. Previous studies have demonstrated the involvement of human cytosolic sulfotransferase (SULT) SULT1A1 in the sulfation of acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol. The current study was designed to investigate the impact of single nucleotide polymorphisms (SNPs) of the human SULT1A1 gene on the sulfation of these analgesic compounds by SULT1A1 allozymes. METHODS: Human SULT1A1 genotypes were identified by database search. cDNAs corresponding to nine SULT1A1 nonsynonymous missense coding SNPs (cSNPs) were generated by site-directed mutagenesis. Recombinant wild-type and SULT1A1 allozymes were bacterially expressed and affinity-purified. Purified SULT1A1 allozymes were analyzed for sulfation activity using an established assay procedure. RESULTS: Compared with the wild-type enzyme, SULT1A1 allozymes were shown to display differential sulfating activities toward three analgesic compounds, acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol, as well as the prototype substrate 4NP. CONCLUSION: Results obtained indicated clearly the impact of genetic polymorphisms on the drug-sulfation activity of SULT1A1 allozymes. Such information may contribute to a better understanding about the differential metabolism of acetaminophen, O-DMN, and tapentadol in individuals with different SULT1A1 genotypes.


Assuntos
Acetaminofen/metabolismo , Arilsulfotransferase/genética , Naproxeno/análogos & derivados , Tapentadol/metabolismo , Analgésicos não Entorpecentes/metabolismo , Analgésicos Opioides/metabolismo , Citosol/metabolismo , Escherichia coli/citologia , Genótipo , Humanos , Isoenzimas , Mutagênese Sítio-Dirigida , Naproxeno/metabolismo , Polimorfismo de Nucleotídeo Único , Sulfatos/metabolismo
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