Publications (38)

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    [Show abstract] [Hide abstract] ABSTRACT: Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.
    Full-text Article · Jul 2016 · Scientific Reports
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    Full-text Dataset · Jul 2016
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    Full-text Dataset · Jul 2016
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    Full-text Dataset · Jul 2016
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    [Show abstract] [Hide abstract] ABSTRACT: SILAC quantitation of Ki-67 peptides from WT and Mki67-mutant NIH-3T3 cells.Table showing identity and H/L ratio of Ki-67 peptides purified from chromatin of NIH-3T3 Wt, Mki67 mutant clones 14 and 21, recovered in excised bands 1 (>250 kDa) or 2 (130-250 kDa). Data have been uploaded into the massIVE repository with accession number MSV000079492.DOI: http://dx.doi.org/10.7554/eLife.13722.018
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: ELife digest Living cells divide in two to produce new cells. In mammals, cell division is strictly controlled so that only certain groups of cells in the body are actively dividing at any time. However, some cells may escape these controls so that they divide rapidly and form tumors. A protein called Ki-67 is only produced in actively dividing cells, where it is located in the nucleus – the structure that contains most of the cell’s DNA. Researchers often use Ki-67 as a marker to identify which cells are actively dividing in tissue samples from cancer patients, and previous studies indicated that Ki-67 is needed for cells to divide. However, the exact role of this protein was not clear. Before cells can divide they need to make large amounts of new proteins using molecular machines called ribosomes and it has been suggested that Ki-67 helps to produce ribosomes. Now, Sobecki et al. used genetic techniques to study the role of Ki-67 in mice. The experiments show that Ki-67 is not required for cells to divide in the laboratory or to make ribosomes. Instead, Ki-67 alters the way that DNA is packaged in the nucleus. Loss of Ki-67 from mice cells resulted in DNA becoming less compact, which in turn altered the activity of genes in those cells. Sobecki et al. also identified many other proteins that interact with Ki-67, so the next step following on from this research is to understand how Ki-67 alters DNA packaging at the molecular level. Another future challenge will be to find out if inhibiting the activity of Ki-67 can hinder the growth of cancer cells. DOI: http://dx.doi.org/10.7554/eLife.13722.002
    Full-text Article · Mar 2016 · eLife Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: Ki-67 interacting proteome.Table showing proteins identified by mass spectrometry eluted from immunoprecipitates of cells transfected with FLAG-tagged Ki-67, an unrelated protein (FLAG-tagged TRIM39) or empty vector. Proteins specifically interacting only with Ki-67 are presented on the first worksheet. Data are available via ProteomeXchange with identifier PXD003551.DOI: http://dx.doi.org/10.7554/eLife.13722.025
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: Ki-67-dependent transcriptome in U2OS cells.Table showing statistically significant (corrected p value < 0.02, Fold-change >1.5) changes of transcript abundance from Agilent Gene chip analysis of cDNA from control U2OS (pGIPZ-shRNA non silencing control) and U2OS stably silenced (pGIPZ-Ki-67 shRNA) for Ki-67.DOI: http://dx.doi.org/10.7554/eLife.13722.033
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: Ki-67-dependent transcriptome in HeLa cells.Table showing statistically significant (corrected p value <0.02, Fold-change >1.5) changes of transcript abundance from Agilent Gene chip analysis of cDNA from control HeLa (pGIPZ-shRNA non silencing control) and HeLa stably silenced (pGIPZ-Ki-67 shRNA) for Ki-67.DOI: http://dx.doi.org/10.7554/eLife.13722.034
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.
    Full-text Article · Mar 2016 · Transgenic Research
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    [Show abstract] [Hide abstract] ABSTRACT: Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.
    Full-text Article · Oct 2015 · Plant physiology
  • [Show abstract] [Hide abstract] ABSTRACT: Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In IRLC legumes such as Medicago, the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated and polyploid state. Here we show that in Aeschynomene legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene species and that these bacteroids are terminally differentiated and polyploid, similarly to bacteroids in IRLC legumes. Transcriptome, in situ hybridization and proteome analysis demonstrated that the symbiotic cells in the Aeschynomene nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNAi-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes. Copyright © 2015, Plant Physiology.
    Article · Aug 2015 · Plant physiology
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    Guowei Li · Marie Boudsocq · Sonia Hem · [...] · VÉRONIQUE SANTONI
    Full-text Article · Jul 2015
  • [Show abstract] [Hide abstract] ABSTRACT: The unicellular pathogenic protozoan Trypanosoma brucei gambiense is responsible for the chronic form of sleeping sickness. This vector-borne disease is transmitted to humans by the tsetse fly of the group Glossina palpalis, including the subspecies G. p. gambiensis, in which the parasite completes its developmental cycle. Sleeping sickness control strategies can therefore target either the human host or the fly vector. Indeed, suppression of one step in the parasite developmental cycle could abolish parasite transmission to humans, with consequences on the spreading of the disease. In order to develop this type of approach, we have identified, at the proteome level, events resulting from the tripartite interaction between the tsetse fly G. p. gambiensis, its microbiome, and the trypanosome. Proteomes were analyzed from four biological replicates of midguts from flies sampled three days post-feeding on either a trypanosome-infected (stimulated flies) or a non-infected (non-stimulated flies) bloodmeal. Over 500 proteins were identified in the midguts of flies from both feeding groups, thirteen of which were shown to be differentially expressed in trypanosome-stimulated versus non-stimulated flies. Functional annotation revealed that several of these proteins have important functions that could be involved in modulating the fly infection process by trypanosomes (and thus fly vector competence), including anti-oxidant and anti-apoptotic, cellular detoxifying, trypanosome agglutination, and immune stimulating or depressive effects. The results show a strong potential for diminishing or even disrupting fly vector competence, and their application holds great promise for improving the control of sleeping sickness.
    Article · May 2015 · Frontiers in Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher up-stream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
    Full-text Article · Mar 2015 · Journal of Experimental Botany
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    [Show abstract] [Hide abstract] ABSTRACT: We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Full-text Article · Feb 2015 · Nucleic Acids Research
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    Guowei Li · Marie Boudsocq · Sonia Hem · [...] · Véronique Santoni
    [Show abstract] [Hide abstract] ABSTRACT: The hydraulic conductivity of plant roots (Lpr ) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the Plasma membrane Intrinsic Protein 2 (PIP2) sub-family, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lpr of knock-out Arabidopsis plants for four Ca(2+) -dependent protein kinases. cpk7 plants showed a 30% increase in Lpr due to a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lpr of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins.
    Full-text Article · Nov 2014 · Plant Cell and Environment
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  • Article · Aug 2014
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    [Show abstract] [Hide abstract] ABSTRACT: An excess of NaCl in the soil is detrimental for plant growth. It interferes with mineral nutrition and water uptake and leads to accumulation of toxic ions in the plant. Understanding the response of roots to NaCl stress may facilitate the development of crops with increased tolerance to this and other stresses. Since controls achieved at the post-translational level are of critical importance for regulating protein function, the present work used a robust label-free quantitative proteomic methodology to quantify phosphorylation events that affect root membrane proteins in Arabidopsis, in response to short term (up to 2h) NaCl treatments. This work identified 302 proteotypic phosphopeptides including 77 novel phosphorylated sites. NaCl treatment significantly altered the abundance of 74 phosphopeptides, giving novel insights into the regulation of major classes of membrane proteins including ATPases, sodium transporters and aquaporins. The data provide a unique access to phosphorylation reprogramming of ionic equilibrium in plant cells under NaCl stress. The use of predictive bioinformatic tools for kinase motifs suggested that root membrane proteins are substrates of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and protein kinase C (PKC) families, also called AGC kinases, arguing for an important role of lipid signaling in abiotic stress responses. It also pointed to cross-talks between protein kinase families during NaCl stress. This article is protected by copyright. All rights reserved.
    Full-text Article · May 2014 · Proteomics