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1.
FASEB J ; 35(9): e21827, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383980

RESUMO

Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.


Assuntos
Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Sumoilação/genética , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinase do Ponto de Checagem 2/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Hipocampo/metabolismo , Homeostase/genética , Humanos , Camundongos , Neuroblastoma/genética , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/genética , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
2.
Analyst ; 143(18): 4387-4393, 2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-30132006

RESUMO

Bicarbonate salts are used in various industrial processes and could even serve as an alternative source of carbon in bioprocesses involving photosynthetic organisms. Industrial productions require efficient monitoring and control to ensure that their output will meet target specifications. To this end, a simple and rapid in situ quantification method was developed for bicarbonate, carbonate and phosphate ions using Attenuated Total Reflectance-Fourier Transform Infrared (ATR FT-IR) spectroscopy combined with partial least squares (PLS). The resulting multivariate approach allows the simultaneous determination of inorganic carbon and orthophosphate ions concentrations in aqueous solutions (R2 > 0.98, root-mean-square-errors of the cross validation RMSECV < 3.3%). Validation of the method was achieved through replicability and repeatability tests. Univariate calibration graphs are linear over a concentration range of 150 mM (R2 > 0.9990). Quantification limits for those ions were in the 6.9-17.2 mM range, as determined from univariate models. The multivariate model was successfully applied to a microalgal culture of Scenedesmus obliquus using bicarbonate as the carbon source and a phosphate buffer to maintain the pH. This analytical technique did not require extraction or chemical treatment and no sample preparation is needed. The results demonstrate the potential of ATR FT-IR method to study inorganic carbon and phosphate species during a bioprocess.

3.
J Mol Cell Biol ; 9(3): 195-208, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28201649

RESUMO

Farnesoid X receptor (FXR) is a ligand-activated nuclear receptor that plays a central role in regulating genes involved in bile acid homeostasis, and fat and glucose metabolism. Here, we demonstrate a post-translational interplay between FXR phosphorylation, SUMOylation, and ubiquitination that directs the receptor into an activation-degradation pathway in hepatocytes. We identify a non-canonical SUMOylation motif termed pSuM that conjugates SUMO2 at Lys-325 of FXR under the direct control of casein kinase 2 (CK2), which provides the required negative charge for Ubc9 and PIAS1 to perform SUMOylation, by phosphorylating Ser-327. Lys-325 SUMOylation is indispensable to the promotion of efficient ligand activation and transcriptional coactivation of FXR. Constitutive pSuM activation using a phospho-mimic Ser-327 mutant or catalytic CK2 expression strongly induces SUMO2 conjugation, which directs FXR ubiquitination and proteasome-dependent degradation. We also determine that such SUMOylation-dependent ubiquitination of FXR is mediated by the E3 ubiquitin ligase RNF4, which is required to achieve maximal induction of FXR and optimal up- or downregulation of responsive genes involved in bile acid homeostasis and liver regeneration. Our findings identify a highly regulated atypical SUMO conjugation motif that serves to coordinate FXR transcriptional competence, thereby expanding the intricate dynamics of the SUMOylation process used by incoming signals to govern metabolic gene regulation.


Assuntos
Caseína Quinase II/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Células HEK293 , Hepatócitos/metabolismo , Humanos , Lisina/metabolismo , Mutação , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas de Ligação a RNA/genética , Serina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitinação
4.
Chem Cent J ; 10(1): 54, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27570541

RESUMO

BACKGROUND: Nanomaterials are widely used in industry for their specific properties. Silver nanoparticles (Ag NPs) are largely used in several consumer products notably for their antibacterial properties and will likely be found in wastewater, then in the receiving environment. The development of a product capable to sequestrate those released contaminants is needed. Under environmental conditions, the biopolymer chitosan can generally coordinate the cationic metals. Ag NPs present unique properties due to their high surface/mass ratio which are promising for their sequestration. RESULTS: The immobilization of chitosan on functionalized silica assisted by microwaves gives a sequestering agent of silver while being easily recoverable. The IR spectrum confirmed the immobilization of N,N-dimethylchitosan (DMC) on silica core. The immobilized DMC gave a percentage of sequestration of Ag NPs (120 µg L(-1)) in nanopure water of 84.2 % in 4 h. The sequestration efficiency was largely dependent of temperature. By addition of hydrosulfide ions, the percentage of sequestration increased up to 100 %. The immobilized DMC recovered a large portion of silver regardless the speciation (Ag NP or Ag(+)). In wastewater, the immobilized DMC sequestered less Ag NPs (51.7 % in 97 % wastewater). The presence of anionic (sodium dodecyl sulfate and sodium N-lauroylsarcosinate) and non-ionic surfactants (cetyl alcohol) increased the hydrophobicity of Ag NPs and decreased the percentage of sequestration. CONCLUSIONS: The immobilized DMC is a promising tool for sequestrating such emerging pollutant at low concentrations in a large volume of sample that would allow the characterization of concentrated Ag NPs by transmission electron microscopy. The efficiency of the support to sequestrate would likely be influenced by the chemical environment of the Ag NP solution.

5.
Chem Cent J ; 9: 34, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26075020

RESUMO

BACKGROUND: Engineered nanoparticles have unique properties compared to bulk materials and their commercial uses growing rapidly. They represent a potential risk for environment and health and could be eventually released in water. Silver nanoparticles (Ag NP) are applied in various products and are well-known for their antibacterial properties. Nowadays, pre-concentration and separation methods for Ag NP possess some limitations. Here, we present a simple, green method to sequestrate and concentrate Ag NP from different aqueous media. RESULTS: Supported polysaccharides on glass beads synthesized in water by a single step reaction show high sequestration capacity of citrate-coated Ag NP in aqueous media. Supported polysaccharides were characterized by infrared spectroscopy, scanning electron microscopy (SEM) and elemental analysis. Sequestration of 83.0 % of Ag NP was attained from a 20 µg.L(-1) aqueous solution with supported chitosan in water whereas supported 2-hydroxyethylcellulose (HEC) reached 64.0 % in synthetic seawater in 2 h. The influence of polymer/glass beads ratio and molecular weight of polysaccharides was also studied. The effect of the salinity and humic acids on sequestration of Ag NP was investigated. Supported polymers have shown high performance for sequestration of ionic silver. Sequestration of 82.5 % and 80.8 % were obtained from a 60 µg.L(-1) silver ion (as nitrate salt) with supported HEC and chitosan, respectively. Sequestrated Ag NP was characterized with transmission electron microscopy (TEM) where images showed Ag NP with unchanged size and shape. CONCLUSIONS: This sequestration method, involving green synthesis, allows efficient concentration and characterization of Ag NP from different aqueous media. This simple and fast method is a potential sustainable technique for elimination of Ag NP and ionic silver from waste waters and waters at different salinities.

6.
eNeuro ; 2(5)2015.
Artigo em Inglês | MEDLINE | ID: mdl-26730399

RESUMO

Guidance molecules regulate the navigation of retinal ganglion cell (RGC) projections toward targets in the visual thalamus. In this study, we demonstrate that the G-protein-coupled receptor 55 (GPR55) is expressed in the retina during development, and regulates growth cone (GC) morphology and axon growth. In vitro, neurons obtained from gpr55 knock-out (gpr55(-/-) ) mouse embryos have smaller GCs, less GC filopodia, and have a decreased outgrowth compared with gpr55(+/+) neurons. When gpr55(+/+) neurons were treated with GPR55 agonists, lysophosphatidylinositol (LPI) and O-1602, we observed a chemo-attractive effect and an increase in GC size and filopodia number. In contrast, cannabidiol (CBD) decreased the GC size and filopodia number inducing chemo-repulsion. In absence of the receptor (gpr55(-/-) ), no pharmacologic effects of the GPR55 ligands were observed. In vivo, compared to their wild-type (WT) littermates, gpr55(-/-) mice revealed a decreased branching in the dorsal terminal nucleus (DTN) and a lower level of eye-specific segregation of retinal projections in the superior colliculus (SC) and in the dorsal lateral geniculate nucleus (dLGN). Moreover, a single intraocular injection of LPI increased branching in the DTN, whereas treatment with CBD, an antagonist of GPR55, decreased it. These results indicate that GPR55 modulates the growth rate and the targets innervation of retinal projections and highlight, for the first time, an important role of GPR55 in axon refinement during development.


Assuntos
Axônios/fisiologia , Cones de Crescimento/fisiologia , Neurônios/fisiologia , Receptores de Canabinoides/metabolismo , Animais , Axônios/efeitos dos fármacos , Canabidiol/análogos & derivados , Canabidiol/farmacologia , Crescimento Celular , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Feminino , Cones de Crescimento/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Masculino , Mesocricetus , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Receptores de Canabinoides/genética , Retina/citologia , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Retina/fisiologia , Vias Visuais/citologia , Vias Visuais/efeitos dos fármacos , Vias Visuais/crescimento & desenvolvimento , Vias Visuais/fisiologia
7.
Bioresour Technol ; 144: 664-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23876970

RESUMO

The objective of this work is to develop a quick and simple method for the in situ monitoring of sugars in biological cultures. A new technology based on Attenuated Total Reflectance-Fourier Transform Infrared (FT-IR/ATR) spectroscopy in combination with an external light guiding fiber probe was tested, first to build predictive models from solutions of pure sugars, and secondly to use those models to monitor the sugars in the complex culture medium of mixotrophic microalgae. Quantification results from the univariate model were correlated with the total dissolved solids content (R(2)=0.74). A vector normalized multivariate model was used to proportionally quantify the different sugars present in the complex culture medium and showed a predictive accuracy of >90% for sugars representing >20% of the total. This method offers an alternative to conventional sugar monitoring assays and could be used at-line or on-line in commercial scale production systems.


Assuntos
Carboidratos/análise , Meios de Cultura/química , Microalgas/química , Modelos Teóricos , Scenedesmus/química , Calibragem , Galactose/análise , Cromatografia Gasosa-Espectrometria de Massas , Glucose/análise , Lactose/análise , Análise dos Mínimos Quadrados , Análise Multivariada , Espectroscopia de Infravermelho com Transformada de Fourier
8.
Carbohydr Res ; 351: 87-92, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22330754

RESUMO

A simple and efficient pivaloylation of primary alcohols was realized on N-phthaloylchitosan that was regioselectively and entirely protected. The selectivity of this mild esterification was demonstrated by comparison with (1)H NMR chemical shifts of H-1 and H-3 of complete 3,6-O-dipivaloylated derivatives. The selective hydrazinolysis of N-phthaloyl groups in the presence of pivaloyl ester was achieved in ethanol/water. High molecular weight 6-O-pivaloylchitosan, purified by ultrafiltration, with solubility in organic solvents was obtained. The selective introduction of a phosphorus moiety in O-3 using chlorodiphenylphosphine has led to 3-O-diphenylphosphinito-N-phthaloyl-6-O-pivaloylchitosan with a ds of 0.97 from energy dispersive X-ray spectroscopy, which demonstrates the potential applications of O-6 pivaloyl protection. The pivaloylation of O-6 alcohols can enhance the solubility of some chitosan derivatives and therefore contributes to the development of applications involving chitosan through regioselective modifications of alcohol and amino groups.


Assuntos
Quitina/análogos & derivados , Quitina/química , Hidrazinas/química , Ácidos Pentanoicos/química , Solubilidade , Estereoisomerismo , Especificidade por Substrato
9.
Biomacromolecules ; 8(6): 1812-5, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17487971

RESUMO

N-Phthaloylchitosan represents a key intermediate for the regioselective modification of chitosan in organic media. Chemoselective protection of primary alcohols on N-phthaloylchitosan was achieved with tert-butyldimethylsilyl (TBDMS) and tert-butyldiphenylsilyl (TBDPS) groups in imidazole/DMF and DMAP/pyridine. Influence of experimental conditions such as solvent, choice of base, stoichiometry, temperature, and time of reaction was studied regarding the degree of substitution (ds) of silyl groups. TBDMS groups allow higher ds than TBDPS groups. Higher reaction temperatures in different conditions led to lower ds and incomplete substitution. However, regioselective silylation of N-phthaloylchitosan was realized with ds up to 0.92 at room temperature. Silylated derivatives were characterized by elemental analysis, IR, and CP/MAS 13C NMR spectroscopies.


Assuntos
Quitina/análogos & derivados , Quitosana/química , Silanos/química , Siloxanas/química , Estirenos/química , Álcoois/química , Quitina/química , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Químicos , Conformação Molecular , Solubilidade , Solventes/química , Espectrofotometria Infravermelho , Temperatura
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