Publications by authors named "Marc Bonneu"

51 Publications

Critical role of Aquaporin-1 and telocytes in infantile hemangioma response to propranolol beta blockade.

Proc Natl Acad Sci U S A 2021 Feb;118(7)

Inserm, Biothérapie des Maladies Génétiques Inflammatoires et Cancers (BMGIC), UMR 1035, University of Bordeaux, F-33076 Bordeaux, France.

Propranolol, a nonselective β-adrenergic receptor (ADRB) antagonist, is the first-line therapy for severe infantile hemangiomas (IH). Since the incidental discovery of propranolol efficacy in IH, preclinical and clinical investigations have shown evidence of adjuvant propranolol response in some malignant tumors. However, the mechanism for propranolol antitumor effect is still largely unknown, owing to the absence of a tumor model responsive to propranolol at nontoxic concentrations. Immunodeficient mice engrafted with different human tumor cell lines were treated with anti-VEGF bevacizumab to create a model sensitive to propranolol. Proteomics analysis was used to reveal propranolol-mediated protein alteration correlating with tumor growth inhibition, and Aquaporin-1 (AQP1), a water channel modulated in tumor cell migration and invasion, was identified. IH tissues and cells were then functionally investigated. Our functional protein association networks analysis and knockdown of ADRB2 and AQP1 indicated that propranolol treatment and AQP1 down-regulation trigger the same pathway, suggesting that AQP1 is a major driver of beta-blocker antitumor response. Examining AQP1 in human hemangioma samples, we found it exclusively in a perivascular layer, so far unrecognized in IH, made of telocytes (TCs). Functional in vitro studies showed that AQP1-positive TCs play a critical role in IH response to propranolol and that modulation of AQP1 in IH-TC by propranolol or shAQP1 decreases capillary-like tube formation in a Matrigel-based angiogenesis assay. We conclude that IH sensitivity to propranolol may rely, at least in part, on a cross talk between lesional vascular cells and stromal TCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2018690118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896303PMC
February 2021

A recurrent missense variant in EYA3 gene is associated with oculo-auriculo-vertebral spectrum.

Hum Genet 2021 Jun 21;140(6):933-944. Epub 2021 Jan 21.

Maladies Rares: Génétique et Métabolisme (MRGM), U 1211 INSERM, Univ. Bordeaux, 33000, Bordeaux, France.

Goldenhar syndrome or oculo-auriculo-vertebral spectrum (OAVS) is a complex developmental disorder characterized by asymmetric ear anomalies, hemifacial microsomia, ocular and vertebral defects. We aimed at identifying and characterizing a new gene associated with OAVS. Two affected brothers with OAVS were analyzed by exome sequencing that revealed a missense variant (p.(Asn358Ser)) in the EYA3 gene. EYA3 screening was then performed in 122 OAVS patients that identified the same variant in one individual from an unrelated family. Segregation assessment in both families showed incomplete penetrance and variable expressivity. We investigated this variant in cellular models to determine its pathogenicity and demonstrated an increased half-life of the mutated protein without impact on its ability to dephosphorylate H2AFX following DNA repair pathway induction. Proteomics performed on this cellular model revealed four significantly predicted upstream regulators which are PPARGC1B, YAP1, NFE2L2 and MYC. Moreover, eya3 knocked-down zebrafish embryos developed specific craniofacial abnormalities corroborating previous animal models and supporting its involvement in the OAVS. Additionally, EYA3 gene expression was deregulated in vitro by retinoic acid exposure. EYA3 is the second recurrent gene identified to be associated with OAVS. Moreover, based on protein interactions and related diseases, we suggest the DNA repair as a key molecular pathway involved in craniofacial development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-021-02255-6DOI Listing
June 2021

Proteomic and metabolomic profiling underlines the stage- and time-dependent effects of high temperature on grape berry metabolism.

J Integr Plant Biol 2020 Aug 31;62(8):1132-1158. Epub 2020 Jan 31.

UMR1287 EGFV, CNRS, INRAE, Bordeaux Sciences Agro, Bordeaux University, ISVV, 33140, Villenave d'Ornon, France.

Climate change scenarios predict an increase in mean air temperatures and in the frequency, intensity, and length of extreme temperature events in many wine-growing regions worldwide. Because elevated temperature has detrimental effects on berry growth and composition, it threatens the economic and environmental sustainability of wine production. Using Cabernet Sauvignon fruit-bearing cuttings, we investigated the effects of high temperature (HT) on grapevine berries through a label-free shotgun proteomic analysis coupled to a complementary metabolomic study. Among the 2,279 proteins identified, 592 differentially abundant proteins were found in berries exposed to HT. The gene ontology categories "stress," "protein," "secondary metabolism," and "cell wall" were predominantly altered under HT. High temperatures strongly impaired carbohydrate and energy metabolism, and the effects depended on the stage of development and duration of treatment. Transcript amounts correlated poorly with protein expression levels in HT berries, highlighting the value of proteomic studies in the context of heat stress. Furthermore, this work reveals that HT alters key proteins driving berry development and ripening. Finally, we provide a list of differentially abundant proteins that can be considered as potential markers for developing or selecting grape varieties that are better adapted to warmer climates or extreme heat waves.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jipb.12894DOI Listing
August 2020

Normalization of hippocampal retinoic acid level corrects age-related memory deficits in rats.

Neurobiol Aging 2020 01 26;85:1-10. Epub 2019 Sep 26.

University of Bordeaux, INRA, Bordeaux INP, NutriNeuro, Bordeaux, France. Electronic address:

Dietary micronutrients constitute a major environmental factor influencing aging processes. Vitamin A (vit. A) is the precursor of retinoic acid, a bioactive molecule that controls the expression of several genes involved in brain function. Evidence suggests a reduction of vit. A bioavailability with aging, but its impact on neuronal network is poorly understood. We investigated the mechanisms linking memory impairments with specific alterations of retinoic acid metabolism in the hippocampus. We compared young (10 weeks) and aged (16 months) rats, supplemented or not with dietary vit. A (20 IU retinol/g) for 4 weeks. Our study reveals that aging induced dysregulation of gene expression involved in vit. A and retinoic acid metabolism in the liver. Furthermore, vit. A supplementation restored the integrity of the hippocampal neuronal morphology altered by aging. Importantly, we found a high correlation between hippocampal levels of retinoic acid and memory performance. The present work establishes the link between collapse of retinoid metabolism and age-related cognitive decline, highlighting the role of vit. A in maintaining memory through aging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neurobiolaging.2019.09.016DOI Listing
January 2020

Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F.

Oncogene 2020 01 16;39(3):617-636. Epub 2019 Sep 16.

Bordeaux University, 146 rue Léo Saignat, 33000, Bordeaux, France.

The basic understanding of the biological effects of eukaryotic translation initiation factors (EIFs) remains incomplete, notably for their roles independent of protein translation. Different EIFs exhibit nuclear localization and DNA-related functions have been proposed, but the understanding of EIFs novel functions beyond protein translation lacks of integrative analyses between the genomic and the proteomic levels. Here, the noncanonical function of EIF3F was studied in human lung adenocarcinoma by combining methods that revealed both the protein-protein and the protein-DNA interactions of this factor. We discovered that EIF3F promotes cell metastasis in vivo. The underpinning molecular mechanisms involved the regulation of a cluster of 34 metastasis-promoting genes including Snail2, as revealed by proteomics combined with immuno-affinity purification of EIF3F and ChIP-seq/Q-PCR analyses. The interaction between EIF3F and signal transducer and activator of transcription 3 (STAT3) controlled the EIF3F-mediated increase in Snail2 expression and cellular invasion, which were specifically abrogated using the STAT3 inhibitor Nifuroxazide or knockdown approaches. Furthermore, EIF3F overexpression reprogrammed energy metabolism through the activation of AMP-activated protein kinase and the stimulation of oxidative phosphorylation. Our findings demonstrate the role of EIF3F in the molecular control of cell migration, invasion, bioenergetics, and metastasis. The discovery of a role for EIF3F-STAT3 interaction in the genetic control of cell migration and metastasis in human lung adenocarcinoma could lead to the development of diagnosis and therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-019-1009-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962096PMC
January 2020

Prenatal retinoic acid exposure reveals candidate genes for craniofacial disorders.

Sci Rep 2018 11 30;8(1):17492. Epub 2018 Nov 30.

University Bordeaux, Maladies Rares: Génétique et Métabolisme (MRGM), U 1211 INSERM, F-33000, Bordeaux, France.

Syndromes that display craniofacial anomalies comprise a major class of birth defects. Both genetic and environmental factors, including prenatal retinoic acid (RA) exposure, have been associated with these syndromes. While next generation sequencing has allowed the discovery of new genes implicated in these syndromes, some are still poorly characterized such as Oculo-Auriculo-Vertebral Spectrum (OAVS). Due to the lack of clear diagnosis for patients, developing new strategies to identify novel genes involved in these syndromes is warranted. Thus, our study aimed to explore the link between genetic and environmental factors. Owing to a similar phenotype of OAVS reported after gestational RA exposures in humans and animals, we explored RA targets in a craniofacial developmental context to reveal new candidate genes for these related disorders. Using a proteomics approach, we detected 553 dysregulated proteins in the head region of mouse embryos following their exposure to prenatal RA treatment. This novel proteomic approach implicates changes in proteins that are critical for cell survival/apoptosis and cellular metabolism which could ultimately lead to the observed phenotype. We also identified potential molecular links between three major environmental factors known to contribute to craniofacial defects including maternal diabetes, prenatal hypoxia and RA exposure. Understanding these links could help reveal common key pathogenic mechanisms leading to craniofacial disorders. Using both in vitro and in vivo approaches, this work identified two new RA targets, Gnai3 and Eftud2, proteins known to be involved in craniofacial disorders, highlighting the power of this proteomic approach to uncover new genes whose dysregulation leads to craniofacial defects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-35681-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269437PMC
November 2018

Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR.

J Biol Chem 2019 01 26;294(3):805-815. Epub 2018 Nov 26.

From the Université de Bordeaux, IBGC UMR 5095, F-33077 Bordeaux, France,

5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-β Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.004964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341394PMC
January 2019

Characterization of a Cell-Assembled extracellular Matrix and the effect of the devitalization process.

Acta Biomater 2018 12 6;82:56-67. Epub 2018 Oct 6.

Univ. Bordeaux, Inserm, Laboratory for the Bioengineering of Tissues, U1026, F-33076, Bordeaux, France. Electronic address:

We have previously shown that the Cell-Assembled extracellular Matrix (CAM) synthesized by normal, human, skin fibroblasts in vitro can be assembled in a completely biological vascular graft that was successfully tested in the clinic. The goal of this study was to perform a detailed analysis of the composition and the organization of this truly bio-material. In addition, we investigated whether the devitalization process (dehydration) used to store the CAM, and thus, make the material available "off-the-shelf," could negatively affect its organization and mechanical properties. We demonstrated that neither the thickness nor the mechanical strength of CAM sheets were significantly changed by the dehydration/freezing/rehydration cycle. The identification of over 50 extracellular matrix proteins highlighted the complex composition of the CAM. Histology showed intense collagen and glycosaminoglycan staining throughout the CAM sheet. The distribution of collagen I, collagen VI, thrombospondin-1, fibronectin-1, fibrillin-1, biglycan, decorin, lumican and versican showed various patterns that were not affected by the devitalization process. Transmission electron microscopy analysis revealed that the remarkably dense collagen network was oriented in the plane of the sheet and that neither fibril density nor diameter was changed by devitalization. Second harmonic generation microscopy revealed an intricate, multi-scale, native-like collagen fiber orientation. In conclusion, this bio-material displayed many tissue-like properties that could support normal cell-ECM interactions and allow implantation without triggering degradative responses from the host's innate immune system. This is consistent with its success in vivo. In addition, the CAM can be devitalized without affecting its mechanical or unique biological architecture. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) defines biological function and mechanical properties of tissues and organs. A number of promising tissue engineering approaches have used processed ECM from cadaver/animal tissues or cell-assembled ECM in vitro combined with scaffolds. We have shown the clinical potential of a scaffold-free approach based on an entirely biological material produced by human cells in culture without chemical processing. Here, we perform a comprehensive analysis of the properties of what can truly be called a bio-material. We also demonstrate that this material can be stored dried without losing its remarkable biological architecture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2018.10.006DOI Listing
December 2018

Energy Metabolism Rewiring Precedes UVB-Induced Primary Skin Tumor Formation.

Cell Rep 2018 06;23(12):3621-3634

Inserm U 1035, BMGIC, 33076 Bordeaux, France; Université de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France; Centre de Référence pour les Maladies Rares de la Peau, CHU de Bordeaux, France. Electronic address:

Although growing evidence indicates that bioenergetic metabolism plays an important role in the progression of tumorigenesis, little information is available on the contribution of reprogramming of energy metabolism in cancer initiation. By applying a quantitative proteomic approach and targeted metabolomics, we find that specific metabolic modifications precede primary skin tumor formation. Using a multistage model of ultraviolet B (UVB) radiation-induced skin cancer, we show that glycolysis, tricarboxylic acid (TCA) cycle, and fatty acid β-oxidation are decreased at a very early stage of photocarcinogenesis, while the distal part of the electron transport chain (ETC) is upregulated. Reductive glutamine metabolism and the activity of dihydroorotate dehydrogenase (DHODH) are both necessary for maintaining high ETC. Mice with decreased DHODH activity or impaired ETC failed to develop pre-malignant and malignant lesions. DHODH activity represents a major link between DNA repair efficiency and bioenergetic patterning during skin carcinogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.05.060DOI Listing
June 2018

A tyrosine kinase-STAT5-miR21-PDCD4 regulatory axis in chronic and acute myeloid leukemia cells.

Oncotarget 2017 Sep 12;8(44):76174-76188. Epub 2017 Jul 12.

University of Bordeaux, INSERM U1035, Bordeaux, France.

MicroRNAs (miRNAs) are regulators of several key patho-physiological processes, including cell cycle and apoptosis. Using microarray-based miRNA profiling in K562 cells, a model of chronic myeloid leukemia (CML), we found that the oncoprotein BCR-ABL1 regulates the expression of miR-21, an "onco-microRNA", found to be overexpressed in several cancers. This effect relies on the presence of two STAT binding sites on the promoter of miR-21, and on the phosphorylation status of STAT5, a transcription factor activated by the kinase activity of BCR-ABL1. Mir-21 regulates the expression of PDCD4 (programmed cell death protein 4), a tumor suppressor identified through a proteomics approach. The phosphoSTAT5 - miR-21 - PDCD4 pathway was active in CML primary CD34 cells, but also in acute myeloid leukemia (AML) models like MV4.11 and MOLM13, where the constitutively active tyrosine kinase FLT3-ITD plays a similar role to BCR-ABL1 in the K562 cell line.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.19192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652696PMC
September 2017

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

PLoS One 2017 5;12(7):e0180341. Epub 2017 Jul 5.

Spectrométrie de Masse des Macromolécules Biologiques, Chimie Biologie des Membranes et Nanoobjets, UMR CNRS 5248, Institut National Polytechnique de Bordeaux, Université de Bordeaux, Bordeaux, France.

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180341PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498041PMC
October 2017

A second Wpl1 anti-cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4.

EMBO J 2017 05 24;36(10):1364-1378. Epub 2017 Apr 24.

Institut de Biochimie et Génétique Cellulaires, UMR 5095 CNRS - Université de Bordeaux, Bordeaux, France.

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201696050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430217PMC
May 2017

High glucose repatterns human podocyte energy metabolism during differentiation and diabetic nephropathy.

FASEB J 2017 01 17;31(1):294-307. Epub 2016 Oct 17.

INSERM Unité 1211, Laboratory of Rare Diseases, Metabolism, and Genetics, Bordeaux University, Bordeaux, France.

Podocytes play a key role in diabetic nephropathy pathogenesis, but alteration of their metabolism remains unknown in human kidney. By using a conditionally differentiating human podocyte cell line, we addressed the functional and molecular changes in podocyte energetics during in vitro development or under high glucose conditions. In 5 mM glucose medium, we observed a stepwise activation of oxidative metabolism during cell differentiation that was characterized by peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)-dependent stimulation of mitochondrial biogenesis and function, with concomitant reduction of the glycolytic enzyme content. Conversely, when podocytes were cultured in high glucose (20 mM), stepwise oxidative phosphorylation biogenesis was aborted, and a glycolytic switch occurred, with consecutive lactic acidosis. Expression of the master regulators of oxidative metabolism transcription factor A mitochondrial, PGC-1α, AMPK, and serine-threonine liver kinase B1 was altered by high glucose, as well as their downstream signaling networks. Focused transcriptomics revealed that myocyte-specific enhancer factor 2C (MEF2C) and myogenic factor 5 (MYF5) expression was inhibited by high glucose levels, and endoribonuclease-prepared small interfering RNA-mediated combined inhibition of those transcription factors phenocopied the glycolytic shift that was observed in high glucose conditions. Accordingly, a reduced expression of MEF2C, MYF5, and PGC-1α was found in kidney tissue sections that were obtained from patients with diabetic nephropathy. These findings obtained in human samples demonstrate that MEF2C-MYF5-dependent bioenergetic dedifferentiation occurs in podocytes that are confronted with a high-glucose milieu.-Imasawa, T., Obre, E., Bellance, N., Lavie, J., Imasawa, T., Rigothier, C., Delmas, Y., Combe, C., Lacombe, D., Benard, G., Claverol, S., Bonneu, M., Rossignol, R. High glucose repatterns human podocyte energy metabolism during differentiation and diabetic nephropathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201600293RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161522PMC
January 2017

Copper stress-induced changes in leaf soluble proteome of Cu-sensitive and tolerant Agrostis capillaris L. populations.

Proteomics 2016 05;16(9):1386-97

UMR1202 BIOGECO, University of Bordeaux, Pessac Cedex, France.

Changes in leaf soluble proteome were explored in 3-month-old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC-MS/MS. In both populations, Cu excess impacted both light-dependent (OEE, cytochrome b6-f complex, and chlorophyll a-b binding protein), and -independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin-NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S-containing amino-acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 (http//proteomecentral.proteomexchange.org/dataset/PXD001930).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201500083DOI Listing
May 2016

Beyond plant defense: insights on the potential of salicylic and methylsalicylic acid to contain growth of the phytopathogen Botrytis cinerea.

Front Plant Sci 2015 16;6:859. Epub 2015 Oct 16.

Laboratoire Mixte UMR 5240, Plateforme de Protéomique, Centre National de la Recherche Scientifique Lyon, France.

Using Botrytis cinerea we confirmed in the present work several previous studies showing that salicylic acid, a main plant hormone, inhibits fungal growth in vitro. Such an inhibitory effect was also observed for the two salicylic acid derivatives, methylsalicylic and acetylsalicylic acid. In marked contrast, 5-sulfosalicylic acid was totally inactive. Comparative proteomics from treated vs. control mycelia showed that both the intracellular and extracellular proteomes were affected in the presence of salicylic acid or methylsalicylic acid. These data suggest several mechanisms that could potentially account for the observed fungal growth inhibition, notably pH regulation, metal homeostasis, mitochondrial respiration, ROS accumulation and cell wall remodeling. The present observations support a role played by the phytohormone SA and derivatives in directly containing the pathogen. Data are available via ProteomeXchange with identifier PXD002873.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2015.00859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607878PMC
November 2015

Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

J Proteome Res 2015 Aug 8;14(8):3188-203. Epub 2015 Jul 8.

†INRA, UMR 1202 BIOGECO, F-33610 Cestas, France.

Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.5b00140DOI Listing
August 2015

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

EMBO Rep 2015 Mar 4;16(3):332-40. Epub 2015 Feb 4.

Team "Endoplasmic Reticulum stress and cancer", INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Centre Régional de Lutte Contre le Cancer Eugène Marquis, Rennes, France

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201439123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364872PMC
March 2015

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

PLoS One 2014 10;9(12):e114628. Epub 2014 Dec 10.

Fakultät für Biologie, Genetik, Ludwig-Maximilians-Universität München, Biozentrum, Martinsried, Germany.

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114628PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262433PMC
October 2017

Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

J Invest Dermatol 2015 Apr 1;135(4):1108-1118. Epub 2014 Dec 1.

Inserm U1035, Bordeaux, France; Université de Bordeaux, Bordeaux, France; Centre de Référence pour les Maladies Rares de la Peau, CHU de Bordeaux, France. Electronic address:

Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2014.511DOI Listing
April 2015

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies.

BMC Microbiol 2014 Sep 30;14:253. Epub 2014 Sep 30.

Background: Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth.

Results: Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated.

Conclusions: Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12866-014-0253-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189659PMC
September 2014

Role of Campylobacter jejuni gamma-glutamyl transpeptidase on epithelial cell apoptosis and lymphocyte proliferation.

Gut Pathog 2014 12;6:20. Epub 2014 Jun 12.

Bacteriology Laboratory, University of Bordeaux, F-33000 Bordeaux, France ; INSERM U853, F-33000 Bordeaux, France ; INSERM U853, Bacteriology Laboratory, Université de Bordeaux (site Carreire), F-33076 Bordeaux, France.

Background: A gamma-glutamyl transpeptidase (GGT) is produced by up to 31% of strains of Campylobacter jejuni isolates. C. jejuni GGT is close to Helicobacter pylori GGT suggesting a conserved activity but unlike the latter, C. jejuni GGT has not been studied extensively. In line with the data available for H. pylori, our objectives were to purify C. jejuni GGT from the bacteria, and to evaluate its inhibitory and proapoptotic activities on epithelial cells and human lymphocytes.

Methods: C. jejuni GGT was purified from culture supernatants by chromatography. After verification of the purity by using mass spectrometry of the purified enzyme, its action on two epithelial cell lines and human lymphocytes was investigated. Cell culture as well as flow cytometry experiments were developed for these purposes.

Results: This study demonstrated that C. jejuni GGT is related to Helicobacter GGTs and inhibits the proliferation of epithelial cells with no proapoptotic activity. C. jejuni GGT also inhibits lymphocyte proliferation by causing a cell cycle arrest in the G0/G1 phase. These effects are abolished in the presence of a specific pharmacological inhibitor of GGT.

Conclusion: C. jejuni GGT activity is comparable to that of other Epsilonproteobacteria GGTs and more generally to Helicobacter bilis (inhibition of epithelial cell and lymphocyte proliferation, however with no proapoptotic activity). It could therefore be considered as a pathogenicity factor and promote, via the inhibition of lymphocyte proliferation, the persistence of the bacteria in the host. These observations are consistent with a role of this enzyme in the pathophysiology of chronic infections associated with C. jejuni.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1757-4749-6-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080688PMC
July 2014

Differential accumulation of soluble proteins in roots of metallicolous and nonmetallicolous populations of Agrostis capillaris L. exposed to Cu.

Proteomics 2014 Aug 16;14(15):1746-58. Epub 2014 Jun 16.

UMR1202 BIOGECO, University of Bordeaux, Talence, France; INRA, UMR1202 BIOGECO, Cestas, France.

Differential expression of soluble proteins was explored in roots of metallicolous (M) and non-M (NM) plants of Agrostis capillaris L. exposed to increasing Cu to partially identify molecular mechanisms underlying higher Cu tolerance in M plants. Plants were cultivated for 2 months on perlite with a CuSO4 (1-30 μM) spiked-nutrient solution. Soluble proteins extracted by the trichloroacetic acid/acetone procedure were separated with 2DE (linear 4-7 pH gradient). After Coomassie Blue staining and image analysis, 19 proteins differentially expressed were identified using LC-MS/MS and Expressed Sequence Tag (ESTs) databases. At supra-optimal Cu exposure (15-30 μM), glycolysis was likely altered in NM roots with increased production of glycerone-P and methylglyoxal based on overexpression of triosephosphate isomerase and fructose bisphosphate aldolase. Changes in tubulins and higher expressions of 5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase and S-adenosylmethionine synthase underpinned impacts on the cytoskeleton and stimulation of ethylene metabolism. Increased l-methionine and S-adenosylmethionine amounts may also facilitate production of nicotianamine, which complexes Cu, and of l-cysteine, needed for metallothioneins and GSH. In M roots, the increase of [Cu/Zn] superoxide dismutase suggested a better detoxification of superoxide, when Cu exposure rose. Higher Cu-tolerance of M plants would rather result from simultaneous cooperation of various processes than from a specific mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201300168DOI Listing
August 2014

Proteomic remodeling of proteasome in right heart failure.

J Mol Cell Cardiol 2014 Jan 30;66:41-52. Epub 2013 Oct 30.

Univ. Bordeaux, Centre de recherche Cardio-Thoracique de Bordeaux, U1045, F-33000 Bordeaux, France; INSERM, Centre de recherche Cardio-Thoracique de Bordeaux, U1045, F-33000 Bordeaux, France. Electronic address:

The development of right heart failure (RHF) is characterized by alterations of right ventricle (RV) structure and function, but the mechanisms of RHF remain still unknown. Thus, understanding the RHF is essential for improved therapies. Therefore, identification by quantitative proteomics of targets specific to RHF may have therapeutic benefits to identify novel potential therapeutic targets. The objective of this study was to analyze the molecular mechanisms changing RV function in the diseased RHF and thus, to identify novel potential therapeutic targets. For this, we have performed differential proteomic analysis of whole RV proteins using two experimental rat models of RHF. Differential protein expression was observed for hundred twenty six RV proteins including proteins involved in structural constituent of cytoskeleton, motor activity, structural molecule activity, cytoskeleton protein binding and microtubule binding. Interestingly, further analysis of down-regulated proteins, reveals that both protein and gene expressions of proteasome subunits were drastically decreased in RHF, which was accompanied by an increase of ubiquitinated proteins. Interestingly, the proteasomal activities chymotrypsin and caspase-like were decreased whereas trypsin-like activity was maintained. In conclusion, this study revealed the involvement of ubiquitin-proteasome system (UPS) in RHF. Three deregulated mechanisms were discovered: (1) decreased gene and protein expressions of proteasome subunits, (2) decreased specific activity of proteasome; and (3) a specific accumulation of ubiquitinated proteins. This modulation of UPS of RV may provide a novel therapeutic avenue for restoration of cardiac function in the diseased RHF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yjmcc.2013.10.015DOI Listing
January 2014

The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1.

Biochem Biophys Res Commun 2013 Mar 27;433(1):1-5. Epub 2013 Feb 27.

Univ. Bordeaux, Institut de Biochimie et Génétique Cellulaires, UMR CNRS 5095, F-33000 Bordeaux, France.

Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2013.02.081DOI Listing
March 2013

Functional characterization of a chimeric soluble Fas ligand polymer with in vivo anti-tumor activity.

PLoS One 2013 9;8(1):e54000. Epub 2013 Jan 9.

Unité Mixte de Recherche Centre National de la Recherche Scientifique 5164, Université de Bordeaux 2, Bordeaux, France.

Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054000PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541234PMC
July 2013

Pseudomonas putida KT2440 response to nickel or cobalt induced stress by quantitative proteomics.

Metallomics 2013 Jan;5(1):68-79

Université de Lyon, Lyon, France.

Nickel and cobalt are obligate nutrients for the gammaproteobacteria but when present at high concentrations they display toxic effects. These two metals are present in the environment, their origin being either from natural sources or from industrial use. In this study, the effect of inhibitory concentrations of Ni or Co was assessed on the soil bacterium Pseudomonas putida KT2440 using a proteomic approach. The identification of more than 400 spots resulted in the quantification of 160 proteins that underwent significant variations in cells exposed to Co and Ni. This analysis allowed us to depict the cellular response of P. putida cells toward metallic stress. More precisely, the parallel comparison of the two proteomes showed distinct responses of P. putida to Ni or Co toxicity. The most striking effect of Co was revealed by the accumulation of several proteins involved in the defense against oxidative damage, which include proteins involved in the detoxification of the reactive oxygen species, superoxides and peroxides. The up-regulation of the genes encoding these enzymes was confirmed using qRT-PCR. Interestingly, in the Ni-treated samples, sodB, encoding superoxide dismutase, was up-regulated, indicating the apparition of superoxide radicals due to the presence of Ni. However, the most striking effect of Ni was the accumulation of several proteins involved in the synthesis of amino acids. The measurement of the amount of amino acids in Ni-treated cells revealed a strong accumulation of glutamate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c2mt20147jDOI Listing
January 2013

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation.

Microbiologyopen 2012 Sep 1;1(3):326-39. Epub 2012 Sep 1.

Biotechnologie des Protéines Recombinantes à Visée Santé, University Bordeaux EA4135, F-33000, Bordeaux, France.

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mbo3.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496976PMC
September 2012

Impact of foliar symptoms of "Esca proper" on proteins related to defense and oxidative stress of grape skins during ripening.

Proteomics 2013 Jan;13(1):108-18

Unité de recherche OENOLOGIE, Univ. Bordeaux, ISVV, EA 4577, Villenave d'Ornon, France.

Esca is one of the major diseases affecting vineyards with direct impact on product yield; nevertheless, scientific studies concerning its impact on grape quality are scarce. As an attempt to better understand the mechanisms behind "Esca proper" development in grapes, this work focused on the identification of proteins whose expression is altered by the disease. 2-DEs were performed on protein extracts from grape skins at different stages of maturity for two consecutive vintages. Grapes were collected in 2009 and in 2010 from plants that did not present signs of infection by Esca proper since the 2004 vintage and from plants that presented cast leaf symptoms at least once since 2004. For the first time, 13 proteins were shown to be influenced by Esca proper during the ripening process. Extensive bioinformatics analysis allowed the grouping of proteins involved in (i) stress tolerance and defense response, (ii) oxidative phosphorylation, (iii) oxidation-reduction processes in mitochondria, and (iv) oxidation-reduction processes in chloroplasts. Of these 13 proteins, cysteine synthase is the only one implicated in a metabolic pathway of oenological interest. This study shows how foliar symptoms of Esca proper may impact stress-related pathways in grapes, which are characterized by modifications in the chain of oxidative phosphorylation and redox scavenging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201200194DOI Listing
January 2013

Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria.

Biochimie 2013 Apr 14;95(4):759-71. Epub 2012 Nov 14.

MCMP, UMR 5234, CNRS, Université Bordeaux Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

DNA replication occurs in various compartments of eukaryotic cells such as the nuclei, mitochondria and chloroplasts, the latter of which is used in plants and algae. Replication appears to be simpler in the mitochondria than in the nucleus where multiple DNA polymerases, which are key enzymes for DNA synthesis, have been characterized. In mammals, only one mitochondrial DNA polymerase (pol γ) has been described to date. However, in the mitochondria of the yeast Saccharomyces cerevisiae, we have found and characterized a second DNA polymerase. To identify this enzyme, several biochemical approaches such as proteinase K treatment of sucrose gradient purified mitochondria, analysis of mitoplasts, electron microscopy and the use of mitochondrial and cytoplasmic markers for immunoblotting demonstrated that this second DNA polymerase is neither a nuclear or cytoplasmic contaminant nor a proteolytic product of pol γ. An improved purification procedure and the use of mass spectrometry allowed us to identify this enzyme as DNA polymerase α. Moreover, tagging DNA polymerase α with a fluorescent probe demonstrated that this enzyme is localized both in the nucleus and in the organelles of intact yeast cells. The presence of two replicative DNA polymerases may shed new light on the mtDNA replication process in S. cerevisiae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2012.11.003DOI Listing
April 2013

Proteomic analysis of proteins secreted by Botrytis cinerea in response to heavy metal toxicity.

Metallomics 2012 Aug 18;4(8):835-46. Epub 2012 Jun 18.

CNRS/Université de Lyon/Bayer CropScience Joint Laboratory (UMR CNRS 5240), 14-20 rue Pierre Baizet, F-69263, Lyon cedex 09, France.

Although essential in many cellular processes, metals become toxic when they are present in excess and constitute a global environmental hazard. To overcome this stress, fungi have evolved several mechanisms at both intracellular and extracellular levels. In particular, fungi are well known for their ability to secrete a large panel of proteins. However, their role in the adaptation of fungi to metal toxicity has not yet been investigated. To address this question, here, the fungus Botrytis cinerea was challenged to copper, zinc, nickel or cadmium stress and secreted proteins were collected and separated by 2D-PAGE. One hundred and sixteen spots whose volume varied under at least one tested condition were observed on 2D gels. Densitometric analyses revealed that the secretome signature in response to cadmium was significantly different from those obtained with the other metals. Fifty-five of these 116 spots were associated with unique proteins and functional classification revealed that the production of oxidoreductases and cell-wall degrading enzymes was modified in response to metals. Promoter analysis disclosed that PacC/Rim101 sites were statistically over-represented in the upstream sequences of the 31 genes corresponding to the varying unique spots suggesting a possible link between pH regulation and metal response in B. cinerea.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c2mt20041dDOI Listing
August 2012