Atypic SUMOylation of Nor1/NR4A3 regulates neural cell viability and redox sensitivity.

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.

Development and validation of an in situ and real-time quantification method for bicarbonate, carbonate and orthophosphate ions by ATR FT-IR spectroscopy in aqueous solutions.

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.

SUMOylation of nuclear receptor Nor1/NR4A3 coordinates microtubule cytoskeletal dynamics and stability in neuronal cells.

BACKGROUND: Nor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive. METHODS: We used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy. RESULTS: Here, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals. CONCLUSIONS: Our study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.

Determination of the degree of quaternization of N,N,N-trimethylchitosan by CP-MAS 13C NMR.

Chitosan is used in several fields such as medicine, environment and advanced functional materials. The N-alkylation of chitosan into N,N,N-trimethylchitosan (TMC) allows to improve some properties. The current quantification methods of the degree of quaternization (DQ) like titration and 1H NMR spectroscopy require the solubilization of TMC. In this study, a solid-state 13C NMR quantification method was developed for insoluble TMCs. For this purpose, four TMC derivatives acting as reference were synthesized and their degrees of quaternization, N,N-dimethylation (DD) and acetylation (DA) were determined in solution by 1H NMR. CP-MAS 13C NMR spectra of those derivatives were deconvolved with Lorentz functions. Several ratios of the 13C NMR peak areas were correlated with the degrees of substitution obtained in 1H NMR. The best quantification method of DQ involved the correlation of the carbon signal of methyl groups. The method was also applied for the determination of the DD and DA of TMCs.

Proton induced DNA double strand breaks at the Bragg peak: Evidence of enhanced LET effect.

Purpose: To investigate DNA double strand breaks (DSBs) induced by therapeutic proton beams in plateau and Bragg peak to demonstrate DSB induction due to the higher LET in the Bragg peak. Materials and Methods: pUC19 plasmid DNA samples were irradiated to doses of 1000 and 3000 Gy on a Mevion S250i proton system with a monoenergetic, 110 MeV, proton beam at depths of 2 and 9.4 cm, corresponding to a position on the plateau and distal Bragg peak of the beam, respectively. The irradiated DNA samples were imaged by atomic force microscopy for visualization of individual DNA molecules, either broken or intact, and quantification of the DNA fragment length distributions for each of the irradiated samples. Percentage of the broken DNA and average number of DSBs per DNA molecule were obtained. Results: Compared to irradiation effects in the plateau region, DNA irradiated at the Bragg peak sustained more breakage at the same dose, yielding more short DNA fragments and higher numbers of DSB per DNA molecule. Conclusion: The higher LET of proton beams at the Bragg peak results in more densely distributed DNA DSBs, which supports an underlying mechanism for the increased cell killing by protons at the Bragg peak.

A CK2-RNF4 interplay coordinates non-canonical SUMOylation and degradation of nuclear receptor FXR.

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.

New method for sequestration of silver nanoparticles in aqueous media: in route toward municipal wastewater.

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.

Simple and green technique for sequestration and concentration of silver nanoparticles by polysaccharides immobilized on glass beads in aqueous media.

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.

Role of GPR55 during Axon Growth and Target Innervation.

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.

FT-IR/ATR univariate and multivariate calibration models for in situ monitoring of sugars in complex microalgal culture media.

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.

Regioselective pivaloylation of N-phthaloylchitosan: a promising soluble intermediate for chitosan chemistry.

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.

Regioselective silylation of N-phthaloylchitosan with TBDMS and TBDPS groups.

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.