Search Results (11)

Celery (Apium graveolens) can be considered as a model plant for studying pectin-enriched primary cell walls. In addition to parenchyma cells with xyloglucan-deficient walls, celery petioles contain collenchyma, a mechanical tissue with thickened cell walls of similar composition. This study presents a comprehensive analysis of these tissues at both early and late developmental stages, integrating data on polysaccharide yield, composition, localization, and transcriptome analysis. Our results reveal that young collenchyma walls possess distinct polysaccharide compositions, including higher levels of rhamnogalacturonan I (RG-I), branched galactans, esterified homogalacturonan, and xyloglucan, compared to parenchyma cells. A significant number of genes encoding proteins involved in pectin methylesterification and acetylation were upregulated in young collenchyma. Different gene isoforms encoding glycosyltransferases involved in RG-I biosynthesis were activated in both collenchyma and parenchyma, suggesting potential variations in RG-I structure and function across different primary cell walls. We identified a set of potential glycosyltransferases involved in RG-I biosynthesis in collenchyma and proposed synthase complexes for heteromannan and heteroxylan. The transcriptome data not only confirmed known biochemical traits of celery cell walls but also provided deeper insights into the peculiarities of cell wall polysaccharide metabolism, thereby helping to narrow down candidate genes for further molecular genetic studies. Full article

Background: The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3′untranslated regions (3′UTRs) are known to regulate the levels of their mRNA splice variants. Methods: We constructed recombinant pmir-GLO vectors with a truncated 3′ regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs. Results: The dual-luciferase reporter assay found that regions +401–+789, +789–+1152, and +1562–+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789–+1152 region in the 3′UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853–+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells. Conclusions: Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853–+859. Full article

The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction—idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells. Full article

p190RhoGAP, which exists in two paralogs, p190RhoGAP-A (p190A) and p190RhoGAP-B (p190B), is a GTPase activating protein (GAP) contributing to the regulation of the cellular activity of RhoGTPases. Recent data showed that M₂ muscarinic acetylcholine receptor (M₂R) stimulation in neonatal rat cardiac myocytes (NRCM) induces the binding of p190RhoGAP to the long isoform of the regulator of G protein signaling 3 (RGS3L). This complex formation alters the substrate preference of p190RhoGAP from RhoA to Rac1. By analyzing carbachol-stimulated GAP activity, we show herein that p190A, but not p190B, alters its substrate preference in NRCM. Based on data that the RhoGAP activity of p190A in endothelial cells is diminished upon nitration by endothelial nitric oxide synthase (eNOS)-derived peroxynitrite, we studied whether carbachol-induced NO/peroxynitrite formation contributes to the carbachol-induced RhoA activation in NRCM. Interestingly, the carbachol-induced RhoA activation in NRCM was suppressed by the eNOS-preferring inhibitor L-NIO as well as the non-selective NOS inhibitor L-NAME. Using L-NIO, we firstly verified the carbachol-induced NO production concurrent with eNOS activation and, secondly, the carbachol-induced nitration of p190A in NRCM. By co-immunoprecipitation, the carbachol-induced complex formation of eNOS, p190A, RGS3L and caveolin-3 was detected. We thus conclude that the NO production by M₂R-induced eNOS activation in caveolae in NRCM is required for the nitration of p190A, leading to the binding to RGS3L and the change in substrate preference from RhoA to Rac1. In line with this interpretation, the disruption of caveolae in NRCM by methyl-β-cyclodextrin suppressed carbachol-induced RhoA activation in NRCM to a similar extent as the inhibition of NO production. Full article

Several molecular subtypes of cancer are highly dependent on splicing for cell survival. There is a general interest in the therapeutic targeting of splicing by small molecules. E7107, a first-in-class spliceosome inhibitor, showed strong growth inhibitory activities against a large variety of human cancer xenografts. Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous hematologic malignancy, with approximately 90% of cases being TP53 wild-type at diagnosis. An increasing number of studies are evaluating alternative targeted agents in CLL, including MDM2–p53 binding antagonists. In this study, we report the effect of splicing modulation on key proteins in the p53 signalling pathway, an important cell death pathway in B cells. Splicing modulation by E7107 treatment reduced full-length MDM2 production due to exon skipping, generating a consequent reciprocal p53 increase in TP53^WT cells. It was especially noteworthy that a novel p21^WAF1 isoform with compromised cyclin-dependent kinase inhibitory activity was produced due to intron retention. E7107 synergized with the MDM2 inhibitor RG7388, via dual MDM2 inhibition; by E7107 at the transcript level and by RG7388 at the protein level, producing greater p53 stabilisation and apoptosis. This study provides evidence for a synergistic MDM2 and spliceosome inhibitor combination as a novel approach to treat CLL and potentially other haematological malignancies. Full article

Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10⁻⁸ M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function. Full article

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography–mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation. Full article

The ZNF518B gene, which is up-regulated in colorectal cancer, plays a role in cell dissemination and metastasis. It encodes a zinc-finger protein, which interacts with histone methyltransferases G9A and EZH2. The expression of the two major mRNA isoforms 1 (coding for the full protein) and 2 was quantified by RT-qPCR in a cohort of 66 patients. The effects of silencing ZNF518B on the transcriptome of DLD1 and HCT116 cells were analysed by Clariom-S assays and validated by RT-qPCR. The recruitment of methyltransferases and the presence of H3K27me3 were studied by chromatin immunoprecipitation (ChIP). The ratio (isoform 2)/(isoform 1) negatively correlated with the relapsing of disease. The study of the transcriptome of DLD1 and HCT116 cells revealed that many genes affected by silencing ZNF518B are related to cancer. After crossing these results with the list of genes affected by silencing the histone methyltransferases (retrieved in silico), five genes were selected. ChIP analysis revealed that the recruitment of EZH2 is ZNF518B-dependent in KAT2B, RGS4 and EFNA5; the level of H3K27me3 changes in accordance. G9A also binds RGS4 and PADI3 in a ZNF518B-dependent manner. The results highlight the importance of epigenetics in cancer and open a novel therapeutic possibility, as inhibition of histone methyltransferases may reverse the disease-linked histone marks. Full article

The enzymatic complex Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (NOx) may be the principal source of reactive oxygen species (ROS). The NOX2 and NOX4 isoforms are tissue-dependent and are differentially expressed in slow-twitch fibers (type I fibers) and fast-twitch fibers (type II fibers) of skeletal muscle, making them different markers of ROS metabolism induced by physical exercise. The aim of this study was to investigate NOx signaling, as a non-adaptive and non-cumulative response, in the predominant fiber types of rat skeletal muscles 24 h after one strenuous treadmill exercise session. The levels of mRNA, reduced glycogen, thiol content, NOx, superoxide dismutase, catalase, glutathione peroxidase activity, and PPARGC1α and SLC2A4 gene expression were measured in the white gastrocnemius (WG) portion, the red gastrocnemius (RG) portion, and the soleus muscle (SOL). NOx activity showed higher values in the SOL muscle compared to the RG and WG portions. The same was true of the NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, glycogen content. Twenty-four hours after the strenuous exercise session, NOx expression increased in slow-twitch oxidative fibers. The acute strenuous exercise condition showed an attenuation of oxidative stress and an upregulation of antioxidant activity through PPARGC1α gene activity, antioxidant defense adaptations, and differential gene expression according to the predominant fiber type. The most prominent location of detoxification (indicated by NOX4 activation) in the slow-twitch oxidative SOL muscle was the mitochondria, while the fast-twitch oxidative RG portion showed a more cytosolic location. Glycolytic metabolism in the WG portion suggested possible NOX2/NOX4 non-regulation, indicating other possible ROS regulation pathways. Full article

The HepaRG cell line is a highly differentiated human hepatoma cell line, displaying the expression of various drug transporters. However, functional expression of nucleoside transporters remains poorly characterized in HepaRG cells, although these transporters play a key role in hepatic uptake of antiviral and anticancer drugs. The present study was, therefore, designed to characterize the expression, activity and regulation of equilibrative (ENT) and concentrative (CNT) nucleoside transporter isoforms in differentiated HepaRG cells. These cells were found to exhibit a profile of nucleoside transporter mRNAs similar to that found in human hepatocytes, i.e., notable expression of ENT1, ENT2 and CNT1, with very low or no expression of CNT2 and CNT3. ENT1 activity was, next, demonstrated to be the main uridine transport activity present in HepaRG cells, like in cultured human hepatocytes. Various physiological factors, such as protein kinase C (PKC) activation or treatment by inflammatory cytokines or hepatocyte growth factor (HGF), were additionally found to regulate expression of ENT1, ENT2 and CNT1; PKC activation and HGF notably concomitantly induced mRNA expression and activity of ENT1 in HepaRG cells. Overall, these data suggest that HepaRG cells may be useful for analyzing cellular pharmacokinetics of nucleoside-like drugs in human hepatic cells, especially of those handled by ENT1. Full article

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein–protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein (click beetle luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins. Full article