ANGUSTIFOLIA, a Plant Homolog of CtBP/BARS Localizes to Stress Granules and Regulates Their Formation.
ABSTRACT: The ANGUSTIFOLIA (AN) gene in Arabidopsis is important for a plethora of morphological phenotypes. Recently, AN was also reported to be involved in responses to biotic and abiotic stresses. It encodes a homolog of the animal C-terminal binding proteins (CtBPs). In contrast to animal CtBPs, AN does not appear to function as a transcriptional co-repressor and instead functions outside nucleus where it might be involved in Golgi-associated membrane trafficking. In this study, we report a novel and unexplored role of AN as a component of stress granules (SGs). Interaction studies identified several RNA binding proteins that are associated with AN. AN co-localizes with several messenger ribonucleoprotein granule markers to SGs in a stress dependent manner. an mutants exhibit an altered SG formation. We provide evidence that the NAD(H) binding domain of AN is relevant in this context as proteins carrying mutations in this domain localize to a much higher degree to SGs and strongly reduce AN dimerization and its interaction with one interactor but not the others. Finally, we show that AN is a negative regulator of salt and osmotic stress responses in Arabidopsis suggesting a functional relevance in SGs.
Project description:BACKGROUND: ANGUSTIFOLIA (AN), one of the CtBP family proteins, plays a major role in microtubule-dependent cell morphogenesis. Microarray analysis of mammalian AN homologs suggests that AN might function as a transcriptional activator and regulator of a wide range of genes. Genetic characterization of AN mutants suggests that AN might be involved in multiple biological processes beyond cell morphology regulation. RESULTS: Using a reverse genetic approach, we provide in this paper the genetic, biochemical, and physiological evidence for ANGUSTIFOLIA's role in other new biological functions such as abiotic and biotic stress response in higher plants. The T-DNA knockout an-t1 mutant exhibits not only all the phenotypes of previously described angustifolia null mutants, but also copes better than wild type under dehydration and pathogen attack. The stress tolerance is accompanied by a steady-state modulation of cellular H(2)O(2) content, malondialdehyde (MDA) derived from cellular lipid peroxidation, and over-expression of stress responsive genes. Our results indicate that ANGUSTIFOLIA functions beyond cell morphology control through direct or indirect functional protein interaction networks mediating other biological processes such as drought and pathogen attacks. CONCLUSIONS: Our results indicate that the ANGUSTIFOLIA gene participates in several biochemical pathways controlling cell morphogenesis, abiotic, and biotic stress responses in higher plants. Our results suggest that the in vivo function of plant ANGUSTIFOLIA has been overlooked and it needs to be further studied beyond microtubule-dependent cell morphogenesis.
Project description:The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.
Project description:We previously showed that the ANGUSTIFOLIA (AN) gene regulates the width of leaves of Arabidopsis thaliana, by controlling the polar elongation of leaf cells. In the present study, we found that the abnormal arrangement of cortical microtubules (MTs) in an leaf cells appeared to account entirely for the abnormal shape of the cells. It suggested that the AN gene might regulate the polarity of cell growth by controlling the arrangement of cortical MTs. We cloned the AN gene using a map-based strategy and identified it as the first member of the CtBP family to be found in plants. Wild-type AN cDNA reversed the narrow-leaved phenotype and the abnormal arrangement of cortical MTs of the an-1 mutation. In the animal kingdom, CtBPs self-associate and act as co-repressors of transcription. The AN protein can also self-associate in the yeast two-hybrid system. Furthermore, microarray analysis suggested that the AN gene might regulate the expression of certain genes, e.g. the gene involved in formation of cell walls, MERI5. A discussion of the molecular mechanisms involved in the leaf shape regulation is presented based on our observations.
Project description:Osteoarthritis (OA) is a common type of arthritis. Chronic inflammation is an important contributor to the pathogenesis of OA. The maturation and secretion of proinflammatory cytokines are controlled by inflammasomes, especially NLRP1 (NLR Family Pyrin Domain Containing 1) and NLRP3. In this study, we identified a transactivation mechanism of NLRP3 mediated by CtBPs (C-terminal-binding proteins). We found that both the mRNA and protein levels of CtBPs were significantly increased in OA biopsies. Analyzing the profiles of differentially expressed genes in CtBP-knockdown and overexpression cells, we found that the expression of NLRP3 was dependent on CtBP levels. By the knockdown or overexpression of transcription factors that potentially bind to the promoter of NLRP3, we found that only AP1 could specifically regulate the expression of NLRP3. Using immunoprecipitation (IP) and Co-IP assays, we found that AP1 formed a transcriptional complex with a histone acetyltransferase p300 and CtBPs. The knockdown of any member of this transcriptional complex resulted in a decrease in the expression of NLRP3. To explore the underlying mechanism of CtBP overexpression, we analyzed their promoters and found that they were abundant in CpG islands. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (AZA) or knockdown of DNMTs (DNA methyltransferases) resulted in the overexpression of CtBPs, while overexpression of DNMTs caused the reverse effects on CtBP expression. Collectively, our results suggest that the decreased DNA methylation levels in the promoters of CtBPs upregulate their expression. Increased CtBPs associated with p300 and AP1 to form a transcriptional complex and activate the expression of NLRP3 and its downstream signaling, eventually aggravating the inflammatory response and leading to the pathogenesis of OA.
Project description:C-terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate-binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide-binding domain, involved in NAD(H)-binding and dimerization. On the contrary, little is known about the structure of CtBP C-terminal region ( approximately 90 residues), hosting sites for post-translational modifications. In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small-angle X-ray scattering, and we show that the CtBP C-terminal region is intrinsically unstructured in the full-length CtBP and in constructs lacking the substrate- and/or the nucleotide-binding domains. The flexible nature of this protein region, and its structural transitions, may be instrumental for CtBP recognition and binding to diverse molecular partners.
Project description:C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons.
Project description:GATA transcription factors have been implicated in controlling adipogenesis in Drosophila and in mammals. In mammals, both GATA2 and GATA3 have been shown to be present in preadipocytes, and their silencing allows the onset of adipogenesis. Overexpression of GATA proteins blocks adipogenesis in cellular assays. GATA factors have been found to operate through recruiting cofactors of the Friend of GATA (FOG) family. FOG proteins, in turn, recruit co-regulators, including C-terminal binding proteins (CTBPs). We have investigated whether FOGs and CTBPs influence adipogenesis. We found that both FOG1 and FOG2 are expressed in cells prior to adipogenesis but are down-regulated as adipogenesis proceeds. Overexpression of FOG1 or FOG2 interferes with adipogenesis. Mutant versions of FOG2 unable to bind CTBP or GATA proteins are impaired in their inability to inhibit adipogenesis. Finally, a mutant version of GATA2, unable to associate with FOGs, also displays abnormal activity and causes enhanced cell proliferation. These results implicate FOGs and CTBPs as partners of GATA proteins in the control of adipocyte proliferation and differentiation.
Project description:We used high-throughput sequencing to identify conserved and nonconserved miRNAs and other short RNAs in Typha angustifolia under control and cadmium stressed condition. A total of 114 conserved miRNAs and 41 novel candidate miRNAs encoded by 66 hairpin precursors were identified in both small RNA libraries. 4 conserved and 6 novel miRNAs showed specific expression, which, combined with function of target genes, suggests that miRNAs may play a role in plant Cd stress response.These results provide a framework for further analysis of miRNAs and their role in regulating Typha angustifolia response to cadmium stress. Overall design: Two small RNA libraries, with (Cd) and without (CK) cadmium-treatment, were sequenced by Illumina sequencing technology
Project description:MicroRNAs (miRNAs) play important roles in plant responses to environmental stress. In this work, we used high-throughput sequencing to analyze transcriptome and small RNAs (sRNAs) in Typha angustifolia under cadmium (Cd) stress. 57,608,230 raw reads were obtained from deep sequencing of a pooled cDNA library. Sequence assembly and analysis yielded 102,473 unigenes. We subsequently sequenced two sRNA libraries from T. angustifolia with or without Cd exposure respectively. Based on transcriptome data of T. angustifolia, we catalogued and analyzed the sRNAs, resulting in the identification of 114 conserved miRNAs and 41 novel candidate miRNAs in both small RNA libraries. In silico analysis revealed 764 targets for 89 conserved miRNAs and 21 novel miRNAs. Statistical analysis on sequencing reads abundance and experimental validation revealed that 4 conserved and 6 novel miRNAs showed specific expression. Combined with function of target genes, these results suggested that miRNAs might play a role in plant Cd stress response. This study provided the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in T. angustifolia, which provide a framework for further analysis of miRNAs and their role in regulating plant responses to Cd stress.
Project description:BACKGROUND:Sphaeralcea angustifolia (Malvaceae) is extensively used in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. OBJECTIVE:The current study was to validate the traditional use of S. angustifolia for the treatment of diarrhea and dysentery on biological grounds using in vitro antiprotozoal activity and computational experiments. MATERIALS AND METHODS:The ethanol extract, subsequent fractions, flavonoids, phenolic acids, and a sterol were evaluated on Entamoeba histolytica and Giardia lamblia trophozoites. Moreover, molecular docking studies on tiliroside were performed; it was tested for its affinity against pyruvate:ferredoxin oxidoreductase (PFOR) and fructose-1,6-bisphosphate aldolase (G/FBPA), two glycolytic enzymes of anaerobic protozoa. RESULTS:Bioassay-guided fractionation of extract of the aerial parts of S. angustifolia gives tiliroside and apigenin, caffeic acid, protocatechuic acid, and ?-sitosterol. The in vitro antiprotozoal assay showed that tiliroside was the most potent antiprotozoal compound on both protozoa with 50% inhibitory concentration values of 17.5 ?g/mL for E. histolytica and 17.4 ?g/mL for G. lamblia. Molecular docking studies using tiliroside showed its probable antiprotozoal mechanism with PFOR and G/FBPA. In both cases, tiliroside showed high affinity and inhibition constant theoretic for PFOR (lowest free binding energy from -9.92 kcal/mol and 53.57 ?M, respectively) and G/FBPA (free binding energy from -7.17 kcal/mol and 55.5 ?M, respectively), like to metronidazole, revealing its potential binding mode at molecular level. CONCLUSION:The results suggest that tiliroside seems to be a potential antiprotozoal compound responsible for antiamoebic and antigiardial activities of S. angustifolia. Its in vitro antiprotozoal activities are in good agreement with the traditional medicinal use of S. angustifolia in gastrointestinal disorders such as diarrhea and dysentery. SUMMARY:Bioassay-guided fractionation of extract of the aerial parts of S. angustifolia gives: tiliroside and apigenin, caffeic acid, protocatechuic acid) and ?-sitosterol. The in vitro antiprotozoal assay showed that tiliroside was the most potent antiprotozoal compound on both protozoa with IC50 values of 17.5 mg/mL for E. histolytica and 17.4 ?g/mL for G. lamblia. Molecular docking studies using tiliroside showed its probable antiprotozoal mechanism with PFOR and G/FBPA. In both cases tiliroside showed high affinity and inhibition constant theoretic for PFOR (lowest free binding energy from -9.92 kcal/mol and 53.57 mM, respectively) and G/FBPA (free binding energy from -7.17 kcal/mol, respectively and 55.5 ?M), like to metronidazole, revealing its potential binding mode at molecular level. The results suggest that tiliroside seems to be a potential antiprotozoal compound responsible for antiamoebic and antigiardial activities of Sphaeralcea angustifolia. Abbreviations Used: PFOR: Pyruvate:ferredoxin oxidoreductase; G/FBPA: Fructose 1,6 bisphosphate aldolase.