Publications by authors named "Thibaut Léger"

50 Publications

Acetaminophen metabolism revisited using non-targeted analyses: Implications for human biomonitoring.

Environ Int 2021 04 29;149:106388. Epub 2021 Jan 29.

Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, F-35000 Rennes, France; Danish Headache Center, Department of Neurology, Rigshospitalet-Glostrup, Denmark.

The analgesic paracetamol/acetaminophen (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, these conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of a non-persistent chemical such as APAP.
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http://dx.doi.org/10.1016/j.envint.2021.106388DOI Listing
April 2021

Bariatric surgery induces a new gastric mucosa phenotype with increased functional glucagon-like peptide-1 expressing cells.

Nat Commun 2021 01 4;12(1):110. Epub 2021 Jan 4.

Université de Paris, Inserm U1149, Centre de Recherche sur l'inflammation, Paris, France.

Glucagon-Like Peptide-1 (GLP-1) undergoes rapid inactivation by dipeptidyl peptidase-4 (DPP4) suggesting that target receptors may be activated by locally produced GLP-1. Here we describe GLP-1 positive cells in the rat and human stomach and found these cells co-expressing ghrelin or somatostatin and able to secrete active GLP-1 in the rats. In lean rats, a gastric load of glucose induces a rapid and parallel rise in GLP-1 levels in both the gastric and the portal veins. This rise in portal GLP-1 levels was abrogated in HFD obese rats but restored after vertical sleeve gastrectomy (VSG) surgery. Finally, obese rats and individuals operated on Roux-en-Y gastric bypass and SG display a new gastric mucosa phenotype with hyperplasia of the mucus neck cells concomitant with increased density of GLP-1 positive cells. This report brings to light the contribution of gastric GLP-1 expressing cells that undergo plasticity changes after bariatric surgeries, to circulating GLP-1 levels.
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http://dx.doi.org/10.1038/s41467-020-20301-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782689PMC
January 2021

From Metabolomics to HRMS-Based Exposomics: Adapting Peak Picking and Developing Scoring for MS1 Suspect Screening.

Anal Chem 2021 01 22;93(3):1792-1800. Epub 2020 Dec 22.

Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail)-UMR_S 1085, F-35000 Rennes, France.

The technological advances of cutting-edge high-resolution mass spectrometry (HRMS) have set the stage for a new paradigm for exposure assessment. However, some adjustments of the metabolomics workflow are needed before HRMS-based methods can detect the low-abundant exogenous chemicals in human matrixes. It is also essential to provide tools to speed up marker identifications. Here, we first show that metabolomics software packages developed for automated optimization of XCMS parameters can lead to a false negative rate of up to 80% for chemicals spiked at low levels in blood. We then demonstrate that manual selection criteria in open-source (XCMS, MZmine2) and vendor software (MarkerView, Progenesis QI) allow to decrease the rate of false negative up to 4% (MZmine2). We next report an MS1 automatized suspect screening workflow that allows for a rapid preannotation of HRMS data sets. The novelty of this suspect screening workflow is to combine several predictors based on /, retention time () prediction models, and isotope ratio to generate intermediate and global scorings. Several prediction models were tested and hierarchized (PredRet, Retip, retention time indices, and a log model), and a nonlinear scoring was developed to account for variations observed within individual runs. We then tested the efficiency of this suspect screening tool to detect spiked and nonspiked chemicals in human blood. Compared to other existing annotation tools, its main advantages include the use of predictors using different models, its speed, and the use of efficient scoring algorithms to prioritize preannotated markers and reduce false positives.
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http://dx.doi.org/10.1021/acs.analchem.0c04660DOI Listing
January 2021

Singular manifolds of proteomic drivers to model the evolution of inflammatory bowel disease status.

Sci Rep 2020 11 4;10(1):19066. Epub 2020 Nov 4.

INSERM, Research Centre of Inflammation, Laboratoire d'excellence Inflamex, BP 416, Paris, France.

The conditions used to describe the presence of an immune disease are often represented by interaction graphs. These informative, but intricate structures are susceptible to perturbations at different levels. The mode in which that perturbation occurs is still of utmost importance in areas such as cell reprogramming and therapeutics models. In this sense, module identification can be useful to well characterise the global graph architecture. To help us with this identification, we perform topological overlap-related measures. Thanks to these measures, the location of highly disease-specific module regulators is possible. Such regulators can perturb other nodes, potentially causing the entire system to change behaviour or collapse. We provide a geometric framework explaining such situations in the context of inflammatory bowel diseases (IBD). IBD are severe chronic disorders of the gastrointestinal tract whose incidence is dramatically increasing worldwide. Our approach models different IBD status as Riemannian manifolds defined by the graph Laplacian of two high throughput proteome screenings. It also identifies module regulators as singularities within the manifolds (the so-called singular manifolds). Furthermore, it reinterprets the characteristic nonlinear dynamics of IBD as compensatory responses to perturbations on those singularities. Then, particular reconfigurations of the immune system could make the disease status move towards an innocuous target state.
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http://dx.doi.org/10.1038/s41598-020-76011-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643119PMC
November 2020

The Interactome analysis of the Respiratory Syncytial Virus protein M2-1 suggests a new role in viral mRNA metabolism post-transcription.

Sci Rep 2019 10 24;9(1):15258. Epub 2019 Oct 24.

UMR1173 INSERM, Université Paris-Saclay - UVSQ, Montigny-le-Bretonneux, France.

Human respiratory syncytial virus (RSV) is a globally prevalent negative-stranded RNA virus, which can cause life-threatening respiratory infections in young children, elderly people and immunocompromised patients. Its transcription termination factor M2-1 plays an essential role in viral transcription, but the mechanisms underpinning its function are still unclear. We investigated the cellular interactome of M2-1 using green fluorescent protein (GFP)-trap immunoprecipitation on RSV infected cells coupled with mass spectrometry analysis. We identified 137 potential cellular partners of M2-1, among which many proteins associated with mRNA metabolism, and particularly mRNA maturation, translation and stabilization. Among these, the cytoplasmic polyA-binding protein 1 (PABPC1), a candidate with a major role in both translation and mRNA stabilization, was confirmed to interact with M2-1 using protein complementation assay and specific immunoprecipitation. PABPC1 was also shown to colocalize with M2-1 from its accumulation in inclusion bodies associated granules (IBAGs) to its liberation in the cytoplasm. Altogether, these results strongly suggest that M2-1 interacts with viral mRNA and mRNA metabolism factors from transcription to translation, and imply that M2-1 may have an additional role in the fate of viral mRNA downstream of transcription.
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http://dx.doi.org/10.1038/s41598-019-51746-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813310PMC
October 2019

Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating.

Nat Microbiol 2019 11 14;4(11):1840-1850. Epub 2019 Oct 14.

Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, CNRS, Montpellier, France.

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify β-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.
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http://dx.doi.org/10.1038/s41564-019-0575-6DOI Listing
November 2019

LGP2 binds to PACT to regulate RIG-I- and MDA5-mediated antiviral responses.

Sci Signal 2019 10 1;12(601). Epub 2019 Oct 1.

Unité de Génomique Virale et Vaccination, Institut Pasteur, CNRS UMR-3569, Paris, France.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I- and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I-dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.
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http://dx.doi.org/10.1126/scisignal.aar3993DOI Listing
October 2019

Redox modifications of cysteine-containing proteins, cell cycle arrest and translation inhibition: Involvement in vitamin C-induced breast cancer cell death.

Redox Biol 2019 09 2;26:101290. Epub 2019 Aug 2.

Institut Curie, PSL Research University, CNRS UMR 3348, Université Paris-Sud, Université Paris-Saclay, Orsay, France. Electronic address:

Vitamin C (VitC) possesses pro-oxidant properties at high pharmacologic concentrations which favor repurposing VitC as an anti-cancer therapeutic agent. However, redox-based anticancer properties of VitC are yet partially understood. We examined the difference between the reduced and oxidized forms of VitC, ascorbic acid (AA) and dehydroascorbic acid (DHA), in terms of cytotoxicity and redox mechanisms toward breast cancer cells. Our data showed that AA displayed higher cytotoxicity towards triple-negative breast cancer (TNBC) cell lines in vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-induced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (HO) accumulation in the extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glutathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation and had less cytotoxicity. A "redoxome" approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cell cycle arrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.
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http://dx.doi.org/10.1016/j.redox.2019.101290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831881PMC
September 2019

Copper and iron metabolism in Ostreococcus tauri - the role of phytotransferrin, plastocyanin and a chloroplast copper-transporting ATPase.

Metallomics 2019 10;11(10):1657-1666

Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic.

Iron and copper are essential elements for practically all living organisms. Their metabolism is frequently interconnected, and while copper is relatively abundant in the ocean, iron is often a limiting factor for the growth of many marine microorganisms. In the present study, we aimed to elucidate the metabolisms of copper and iron and the connection of both in the marine picoalga Ostreococcus tauri. We show that O. tauri adjusts its copper economy in response to copper deficiency by downregulation of the expression of plastocyanin in favor of cytochrome c oxidase without significant changes in growth and physiology. Copper deprivation leads to increased expression of copper transporting ATPase and proteins involved in tetrapyrrole synthesis, most likely to ensure higher turnover of chlorophyll and/or heme. Elucidation of the effect of copper on the incorporation of iron into O. tauri proteins led us to identify the major iron uptake mediating protein, Ot-Fea1, whose expression and binding of iron is copper dependent. Based on our investigation of the incorporation of iron into Ot-Fea1 and ferritin, we hypothesize that O. tauri possesses another Fea1-independent iron uptake system.
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http://dx.doi.org/10.1039/c9mt00078jDOI Listing
October 2019

Label-free quantitative proteomics in Candida yeast species: technical and biological replicates to assess data reproducibility.

BMC Res Notes 2019 Aug 1;12(1):470. Epub 2019 Aug 1.

Mass Spectrometry Laboratory, CNRS, Institut Jacques Monod, UMR 7592, Université de Paris, 75205, Paris, France.

Objective: Label-free quantitative proteomics has emerged as a powerful strategy to obtain high quality quantitative measures of the proteome with only a very small quantity of total protein extract. Because our research projects were requiring the application of bottom-up shotgun mass spectrometry proteomics in the pathogenic yeasts Candida glabrata and Candida albicans, we performed preliminary experiments to (i) obtain a precise list of all the proteins for which measures of abundance could be obtained and (ii) assess the reproducibility of the results arising respectively from biological and technical replicates.

Data Description: Three time-courses were performed in each Candida species, and an alkaline pH stress was induced for two of them. Cells were collected 10 and 60 min after stress induction and proteins were extracted. Samples were analysed two times by mass spectrometry. Our final dataset thus comprises label-free quantitative proteomics results for 24 samples (two species, three time-courses, two time points and two runs of mass spectrometry). Statistical procedures were applied to identify proteins with differential abundances between stressed and unstressed situations. Considering that C. glabrata and C. albicans are human pathogens, which face important pH fluctuations during a human host infection, this dataset has a potential value to other researchers in the field.
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http://dx.doi.org/10.1186/s13104-019-4505-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669970PMC
August 2019

T-Cell Protein Tyrosine Phosphatase Is Irreversibly Inhibited by Etoposide-Quinone, a Reactive Metabolite of the Chemotherapy Drug Etoposide.

Mol Pharmacol 2019 08 20;96(2):297-306. Epub 2019 Jun 20.

Université de Paris, BFA, UMR 8251, CNRS, Paris, France (Q.N., J.B., W.Z., L.-C.B., R.L., F.B., J.-M.D., F.R.L.); Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China (X.X.); Université de Paris, BIGR, UMRS 1134, INSERM, Paris, France (R.D.); Université de Paris, Institut de Recherche Saint-Louis, UMRS 1131, INSERM, Paris, France (S.G., C.C., F.G.); Université de Paris, IJM, UMR 7592, CNRS, Paris, France (T.L.); and Service de Biologie Cellulaire, Assistance Publique des Hôpitaux de Paris, Hôpital Saint Louis, Paris, France (C.C.)

Etoposide is a widely prescribed anticancer drug that is, however, associated with an increased risk of secondary leukemia. Although the molecular basis underlying the development of these leukemias remains poorly understood, increasing evidence implicates the interaction of etoposide metabolites [i.e., etoposide quinone (EQ)] with topoisomerase II enzymes. However, effects of etoposide quinone on other cellular targets could also be at play. We investigated whether T-cell protein tyrosine phosphatase (TCPTP), a protein tyrosine phosphatase that plays a key role in normal and malignant hematopoiesis through regulation of Janus kinase/signal transducer and activator of transcription signaling, could be a target of EQ. We report here that EQ is an irreversible inhibitor of TCPTP phosphatase (IC = ∼7 M, second-order rate inhibition constant of ∼810 M⋅min). No inhibition was observed with the parent drug. The inhibition by EQ was found to be due to the formation of a covalent adduct at the catalytic cysteine residue in the active site of TCPTP. Exposure of human hematopoietic cells (HL60 and Jurkat) to EQ led to inhibition of endogenous TCPTP and concomitant increase in STAT1 tyrosine phosphorylation. Our results suggest that in addition to alteration of topoisomerase II functions, EQ could also contribute to etoposide-dependent leukemogenesis through impairment of key hematopoietic signaling enzymes, such as TCPTP.
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http://dx.doi.org/10.1124/mol.119.116319DOI Listing
August 2019

Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities.

Molecules 2019 Jun 6;24(11). Epub 2019 Jun 6.

Université de Paris, INSERM U1149, 16 rue Henri Huchard, 75018 Paris, France.

Mammalian protein -glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (GlcMan GlcNAc) from GlcManGlcNAc-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities.
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http://dx.doi.org/10.3390/molecules24112135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600155PMC
June 2019

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

J Virol 2019 08 30;93(16). Epub 2019 Jul 30.

UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection. Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.
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http://dx.doi.org/10.1128/JVI.00336-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675888PMC
August 2019

Adsorption of Proteins on m-CPPD and Urate Crystals Inhibits Crystal-induced Cell Responses: Study on Albumin-crystal Interaction.

J Funct Biomater 2019 Apr 25;10(2). Epub 2019 Apr 25.

CIRIMAT, Université de Toulouse, CNRS, Université Toulouse 3, Toulouse INP - ENSIACET, Toulouse 31030, France.

The biological effects and cellular activations triggered by monosodium urate (MSU) and calcium pyrophosphate dihydrate (monoclinic: m-CPPD) crystals might be modulated by protein coating on the crystal surface. This study is aimed at: (i) Identifying proteins adsorbed on m-CPPD crystals, and the underlying mechanisms of protein adsorption, and (ii) to understand how protein coating did modulate the inflammatory properties of m-CPPD crystals. The effects of protein coating were assessed in vitro using primary macrophages and THP1 monocytes. Physico-chemical studies on the adsorption of bovine serum albumin (BSA) upon m-CPPD crystals were performed. Adsorption of serum proteins, and BSA on MSU, as well as upon m-CPPD crystals, inhibited their capacity to induce interleukin-1-β secretions, along with a decreased ATP secretion, and a disturbance of mitochondrial membrane depolarization, suggesting an alteration of NLRP3 inflammasome activation. Proteomic analysis identified numerous m-CPPD-associated proteins including hemoglobin, complement, albumin, apolipoproteins and coagulation factors. BSA adsorption on m-CPPD crystals followed a Langmuir-Freundlich isotherm, suggesting that it could modulate m-CPPD crystal-induced cell responses through crystal/cell-membrane interaction. BSA is adsorbed on m-CPPD crystals with weak interactions, confirmed by the preliminary AFM study, but strong interactions of BSA molecules with each other occurred favoring crystal agglomeration, which might contribute to a decrease in the inflammatory properties of m-CPPD crystals. These findings give new insights into the pathogenesis of crystal-related rheumatic diseases and subsequently may open the way for new therapeutic approaches.
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http://dx.doi.org/10.3390/jfb10020018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616386PMC
April 2019

High-throughput Exploration of the Network Dependent on AKT1 in Mouse Ovarian Granulosa Cells.

Mol Cell Proteomics 2019 07 16;18(7):1307-1319. Epub 2019 Apr 16.

From the ‡Institut Jacques Monod, Université Paris-Diderot, 75013 Paris, France;; §Université Paris-Diderot, 75013 Paris, France. Electronic address:

The PI3K/AKT signaling pathway is known to regulate a broad range of cellular processes, and it is often altered in several types of cancers. Recently, somatic mutations leading to a strong activation of this kinase have been reported in juvenile granulosa cell tumors. However, the molecular role of AKT1 in the supporting cell lineage of the ovary is still poorly understood. To get insights into its function in such cells, we depleted in murine primary granulosa cells and assessed the molecular consequences at both the transcript and protein levels. We were able to corroborate the involvement of AKT1 in the regulation of metabolism, apoptosis, cell cycle, or cytoskeleton dynamics in this ovarian cell type. Consistently, we showed in established granulosa cells that depletion of provoked altered directional persistent migration and increased its velocity. This study also allowed us to put forward new direct and indirect targets of the kinase. Indeed, a series of proteins involved in intracellular transport and mitochondrial physiology were significantly affected by depletion. Using analyses, we also propose a set of kinases and transcription factors that can mediate the action of AKT1 on the deregulated transcripts and proteins. Taken altogether, our results provide a resource of direct and indirect AKT1 targets in granulosa cells and may help understand its roles in this ovarian cell type.
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http://dx.doi.org/10.1074/mcp.RA119.0014613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601207PMC
July 2019

Galectin-8 Favors the Presentation of Surface-Tethered Antigens by Stabilizing the B Cell Immune Synapse.

Cell Rep 2018 12;25(11):3110-3122.e6

Departamento de Biologia Celular y Molecular, Facultad de Ciencias Biologicas, Pontificia Universidad Católica de Chile, Region Metropolitana, Santiago 8331150, Chile. Electronic address:

Complete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8-a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins-is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo.
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http://dx.doi.org/10.1016/j.celrep.2018.11.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302547PMC
December 2018

Proteomic Landscape of Cholangiocarcinomas Reveals Three Different Subgroups According to Their Localization and the Aspect of Non-Tumor Liver.

Proteomics Clin Appl 2019 01 19;13(1):e1800128. Epub 2018 Dec 19.

Paris Diderot University, Sorbonne Paris Cité, CRI, UMR 1149, Inserm, Clichy, F-92110, France.

Purpose: Cholangiocarcinomas (CCs) define a heterogeneous entity based upon their anatomic localization (intra versus extrahepatic) and, for the intrahepatic CCs, the aspect of background liver (normal versus cirrhosis). The aim of the study was to characterize the molecular heterogeneity of CCs by a global proteomic approach.

Experimental Design: Thirty-three tumor samples from 17 intrahepatic CCs (iCC) (9 developed on normal (iCC ) and 8 developed in cirrhotic liver (iCC )); 5 hilar CCs (CC ); 5 pancreatic CCs (CC ) and 6 hepatocellular carcinomas (HCC), were submitted to label-free quantitative proteomic analysis. Differential proteins were analyzed by immunohistochemistry in a validation set of 30 CCs.

Results: Unsupervised analysis revealed two main clusters: cluster 1 contained most of the iCC while cluster 2 was divided in 2 subgroups, one containing most of the iCC and the other regrouping CC and CC . Compared to iCC , iCC displayed upregulation of molecules involved in cell adhesion, motility and angiogenesis. Epithelial markers associated with secretory pathway and fibroblast markers were overexpressed in CC compared to iCC CONCLUSION AND CLINICAL RELEVANCE: This study demonstrated that iCC is a specific entity, suggesting a major impact of the background liver on tumor biology, and confirmed that extrahepatic CCs define a homogeneous subgroup.
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http://dx.doi.org/10.1002/prca.201800128DOI Listing
January 2019

Proteome characterization in various biological fluids of Trypanosoma brucei gambiense-infected subjects.

J Proteomics 2019 03 7;196:150-161. Epub 2018 Nov 7.

Institute of Neuroepidemiology and Tropical Neurology, School of Medicine, CNRS FR 3503 GEIST, University of Limoges, INSERM UMR 1094 Tropical Neuroepidemiology, Limoges, France. Electronic address:

Human African trypanosomiasis (HAT) is a neglected tropical disease that is endemic in sub-Saharan Africa. Control of the disease has been recently improved by better screening and treatment strategies, and the disease is on the WHO list of possible elimination. However, some physiopathological aspects of the disease transmission and progression remain unclear. We propose a new proteomic approach to identify new targets and thus possible new biomarkers of the disease. We also focused our attention on fluids classically associated with HAT (serum and cerebrospinal fluid (CSF)) and on the more easily accessible biological fluids urine and saliva. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) established the proteomic profile of patients with early and late stage disease. The serum, CSF, urine and saliva of 3 uninfected controls, 3 early stage patients and 4 late stage patients were analyzed. Among proteins identified, in CSF, urine and saliva, respectively, 37, 8 and 24 proteins were differentially expressed and showed particular interest with regards to their function. The most promising proteins (Neogenin, Neuroserpin, secretogranin 2 in CSF; moesin in urine and intelectin 2 in saliva) were quantified by enzyme-linked immunosorbent assay in a confirmatory cohort of 14 uninfected controls, 23 patients with early stage disease and 43 patients with late stage disease. The potential of two proteins, neuroserpin and moesin, with the latter present in urine, were further characterized. Our results showed the potential of proteomic analysis to discover new biomarkers and provide the basis of the establishment of a new proteomic catalogue applied to HAT-infected subjects and controls. SIGNIFICANCE: Sleeping sickness, also called Human African Trypanosomiasis (HAT), is a parasitic infection caused by a parasitic protozoan, Trypanosoma brucei gambiense or T. b. rhodesiense which are transmitted via an infected tsetse fly: Glossina. For both, the haemolymphatic stage (or first stage) signs and symptoms are intermittent fever, lymphadenopathy, hepatosplenomegaly, headaches, pruritus, and for T. b. rhodesiense infection a chancre is often formed at the bite site. Meningoencephalitic stage (or second stage) occurs when parasites invade the CNS, it is characterised by neurological signs and symptoms such as altered gait, tremors, neuropathy, somnolence which can lead to coma and death if untreated. first stage of the disease is characterizing by fevers, headaches, itchiness, and joint pains and progressive lethargy corresponding to the second stage with confusion, poor coordination, numbness and trouble sleeping. Actually, diagnosing HAT requires specialized expertise and significant resources such as well-equipped health centers and qualified staff. Such resources are lacking in many endemic areas that are often in rural locales, so many individuals with HAT die before the diagnosis is established. In this study, we analysed by mass spectrometry the entire proteome of serum, CSF, urine and saliva samples from infected and non-infected Angolan individuals to define new biomarkers of the disease. This work of proteomics analysis is a preliminary stage to the characterization of the whole proteome, of these 4 biological fluids, of HAT patients. We have identified 69 new biomarkers. Five of them have been thoroughly investigated by ELISA quantification. Neuroserpine and Moesin are respectively promising new biomarkers in CSF and urine's patient for a better diagnosis.
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http://dx.doi.org/10.1016/j.jprot.2018.11.005DOI Listing
March 2019

AAV-8 and AAV-9 Vectors Cooperate with Serum Proteins Differently Than AAV-1 and AAV-6.

Mol Ther Methods Clin Dev 2018 Sep 8;10:291-302. Epub 2018 Aug 8.

Généthon, Evry, France.

Under intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders. We show that, in contrast to rAAV-1 and rAAV-6, there was a substantial similarity in protein binding patterns between mouse and human sera for these vector serotypes. To establish an role for the vector binding of these sera proteins, we chose to study platelet factor 4 (PF4), which interacts with both vectors in both mouse and human sera. Experiments using PF4-knockout mice showed that a complete lack of PF4 did not alter skeletal muscle transduction for these vectors, whereas heart transduction was moderately improved. Our results strongly support our position that the impact of serum proteins on the transduction properties of rAAV-8 and rAAV-9, already observed in mouse models, should be similar in human preclinical trials.
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http://dx.doi.org/10.1016/j.omtm.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6111067PMC
September 2018

Quantitative proteomics in Friedreich's ataxia B-lymphocytes: A valuable approach to decipher the biochemical events responsible for pathogenesis.

Biochim Biophys Acta Mol Basis Dis 2018 Apr 9;1864(4 Pt A):997-1009. Epub 2018 Jan 9.

Université Paris Diderot, Sorbonne Paris Cité, Institut Jacques Monod, CNRS UMR 7592, 75013 Paris, France; Université Paris Diderot, Sorbonne Paris Cité, UFR Sciences du Vivant, 75013 Paris, France. Electronic address:

Friedreich's ataxia (FRDA) represents the most frequent type of autosomal-recessively inherited ataxia and is caused by the deficiency of frataxin, a mitochondrial protein. It is known that frataxin-deficiency leads to alterations in cellular and mitochondrial iron metabolism and impacts in the cell physiology at several levels. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. Currently, cellular antioxidant defense is dysregulated when frataxin is deficient, which exacerbates oxidative damage in FRDA. Moreover, alterations in lipid metabolism have been observed in several models of the disease. To better understand the biochemical sequelae of frataxin reduction, global protein expression analysis was performed using quantitative proteomic experiments in Friedreich's ataxia patient-derived B-lymphocytes as compared to controls. We were able to confirm a subset of changes in these cells and importantly, we observed previously unreported signatures of protein expression. Among the novel protein signatures that we have identified, the decrease in CHCHD4 might partly explain some aspects of the molecular pathogenesis of FRDA. The identification of a core set of proteins changing in the FRDA pathogenesis is a useful tool in trying to decipher the function(s) of frataxin in order to clarify the mitochondrial metabolic disease process.
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http://dx.doi.org/10.1016/j.bbadis.2018.01.010DOI Listing
April 2018

A Simple Light Isotope Metabolic Labeling (SLIM-labeling) Strategy: A Powerful Tool to Address the Dynamics of Proteome Variations .

Mol Cell Proteomics 2017 11 18;16(11):2017-2031. Epub 2017 Aug 18.

From the ‡Mass Spectrometry Laboratory, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cité, F-75205 Paris Cedex 13, France;

Many quantitative proteomics strategies rely on metabolic incorporation of amino acids with modified stable isotope profiles into proteins. These methods give rise to multiple ions for each peptide, with possible distortion of the isotopolog distribution, making the overall analytical process complex. We validated an alternative strategy, simple light isotope metabolic labeling (SLIM-labeling), which alleviates many of these problems. SLIM-labeling is based on the reduction of the isotopic composition of proteins using metabolic precursors with a unique light isotope composition to label all amino acids. This brings a new dimension to in-depth, high resolution MS-based quantitative proteomics. Here, we describe a C-based SLIM-labeling strategy using U-[C]-glucose as the metabolic precursor of all amino acids in the pathogenic yeast Monoisotopic ion intensity increased exponentially following C enrichment, substantially improving peptide identification scores and protein sequence coverage in bottom-up analyses. Multiplexing samples of C composition varying from natural abundance (98.93%) to 100% makes it possible to address relative quantification issues, keeping all the critical information for each peptide within a single isotopolog cluster. We applied this method to measure, for the first time, protein turnover at the proteome scale in and its modulation by inhibitors of the proteasome and vacuolar protein degradation systems.
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http://dx.doi.org/10.1074/mcp.M117.066936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672006PMC
November 2017

Guanine glycation repair by DJ-1/Park7 and its bacterial homologs.

Science 2017 Jul 8;357(6347):208-211. Epub 2017 Jun 8.

Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), Université Paris Diderot, UMR 7086, 15 rue J.-A. de Baïf, 75013 Paris, France.

DNA damage induced by reactive carbonyls (mainly methylglyoxal and glyoxal), called DNA glycation, is quantitatively as important as oxidative damage. DNA glycation is associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. However, in contrast to guanine oxidation repair, how glycated DNA is repaired remains undetermined. Here, we found that the parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO, and YajL could repair methylglyoxal- and glyoxal-glycated nucleotides and nucleic acids. DJ-1-depleted cells displayed increased levels of glycated DNA, DNA strand breaks, and phosphorylated p53. Deglycase-deficient bacterial mutants displayed increased levels of glycated DNA and RNA and exhibited strong mutator phenotypes. Thus, DJ-1 and its prokaryotic homologs constitute a major nucleotide repair system that we name guanine glycation repair.
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http://dx.doi.org/10.1126/science.aag1095DOI Listing
July 2017

Metabolomic and proteomic investigations of impacts of titanium dioxide nanoparticles on Escherichia coli.

PLoS One 2017 1;12(6):e0178437. Epub 2017 Jun 1.

Université Paris Diderot, Sorbonne Paris Cité, IPGP, UMR 7154, Paris Cedex 13 France.

In a previous study, it was demonstrated that the toxic impact of titanium dioxide nanoparticles on Escherichia coli starts at 10 ppm and is closely related to the presence of little aggregates. It was also assumed that only a part of the bacterial population is able to adapt to this stress and attempts to survive. Proteomic analyses, supported by results from metabolomics, reveal that exposure of E. coli to nano-TiO2 induces two main effects on bacterial metabolism: firstly, the up-regulation of proteins and the increase of metabolites related to energy and growth metabolism; secondly, the down-regulation of other proteins resulting in an increase of metabolites, particularly amino acids. Some proteins, e.g. chaperonin 1 or isocitrate dehydrogenase, and some metabolites, e.g. phenylalanine or valine, might be used as biomarkers of nanoparticles stress. Astonishingly, the ATP content gradually rises in relation with the nano-TiO2 concentration in the medium, indicating a dramatic release of ATP by the damaged cells. These apparently contradictory results accredit the thesis of a heterogeneity of the bacterial population. This heterogeneity is also confirmed by SEM images which show that while some bacteria are fully covered by nano-TiO2, the major part of the bacterial population remains free from nanoparticles, resulting in a difference of proteome and metabolome. The use of combined-omics has allowed to better understand the heterogeneous bacterial response to nano-TiO2 stress due to heterogeneous contacts between the protagonists under environmental conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453534PMC
September 2017

Osteopontin and the dento-osseous pathobiology of X-linked hypophosphatemia.

Bone 2017 02 21;95:151-161. Epub 2016 Nov 21.

Faculty of Dentistry, McGill University, Montreal, QC, Canada; Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, QC, Canada. Electronic address:

Seven young patients with X-linked hypophosphatemia (XLH, having inactivating PHEX mutations) were discovered to accumulate osteopontin (OPN) at the sites of defective bone mineralization near osteocytes - the so-called hallmark periosteocytic (lacunar) "halos" of XLH. OPN was also localized in the pericanalicular matrix extending beyond the osteocyte lacunae, as well as in the hypomineralized matrix of tooth dentin. OPN, a potent inhibitor of mineralization normally degraded by PHEX, is a member of a family of acidic, phosphorylated, calcium-binding, extracellular matrix proteins known to regulate dental, skeletal, and pathologic mineralization. Associated with the increased amount of OPN (along with inhibitory OPN peptide fragments) in XLH bone matrix, we found an enlarged, hypomineralized, lacuno-canalicular network - a defective pattern of skeletal mineralization that decreases stiffness locally at: i) the cell-matrix interface in the pericellular environment of the mechanosensing osteocyte, and ii) the osteocyte's dendritic network of cell processes extending throughout the bone. Our findings of an excess of inhibitory OPN near osteocytes and their cell processes, and in dentin, spatially correlates with the defective mineralization observed at these sites in the skeleton and teeth of XLH patients. These changes likely contribute to the dento-osseous pathobiology of XLH, and participate in the aberrant bone adaptation and remodeling seen in XLH.
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http://dx.doi.org/10.1016/j.bone.2016.11.019DOI Listing
February 2017

Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein in Saccharomyces cerevisiae.

Nucleic Acids Res 2016 Oct 31;44(18):8826-8841. Epub 2016 Aug 31.

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Biologie computationnelle et quantitative - Institut de Biologie Paris Seine (LCQB - IBPS), 75005 Paris, France

The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.
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http://dx.doi.org/10.1093/nar/gkw732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062994PMC
October 2016

Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

J Biol Chem 2016 08 8;291(35):18072-83. Epub 2016 Jul 8.

From the Université Paris Diderot, Sorbonne Paris Cité, Unité BFA, CNRS UMR 8251, 75013 Paris, France, UFR Sciences du Vivant, Université Paris Diderot, 75013 Paris, France, and

Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.
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http://dx.doi.org/10.1074/jbc.M116.738898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000057PMC
August 2016

Ostreococcus tauri is a new model green alga for studying iron metabolism in eukaryotic phytoplankton.

BMC Genomics 2016 05 3;17:319. Epub 2016 May 3.

CNRS, Institut Jacques Monod, Université Paris Diderot-Paris 7, F-75013, Paris, France.

Background: Low iron bioavailability is a common feature of ocean surface water and therefore micro-algae developed original strategies to optimize iron uptake and metabolism. The marine picoeukaryotic green alga Ostreococcus tauri is a very good model for studying physiological and genetic aspects of the adaptation of the green algal lineage to the marine environment: it has a very compact genome, is easy to culture in laboratory conditions, and can be genetically manipulated by efficient homologous recombination. In this study, we aimed at characterizing the mechanisms of iron assimilation in O. tauri by combining genetics and physiological tools. Specifically, we wanted to identify and functionally characterize groups of genes displaying tightly orchestrated temporal expression patterns following the exposure of cells to iron deprivation and day/night cycles, and to highlight unique features of iron metabolism in O. tauri, as compared to the freshwater model alga Chalamydomonas reinhardtii.

Results: We used RNA sequencing to investigated the transcriptional responses to iron limitation in O. tauri and found that most of the genes involved in iron uptake and metabolism in O. tauri are regulated by day/night cycles, regardless of iron status. O. tauri lacks the classical components of a reductive iron uptake system, and has no obvious iron regulon. Iron uptake appears to be copper-independent, but is regulated by zinc. Conversely, iron deprivation resulted in the transcriptional activation of numerous genes encoding zinc-containing regulation factors. Iron uptake is likely mediated by a ZIP-family protein (Ot-Irt1) and by a new Fea1-related protein (Ot-Fea1) containing duplicated Fea1 domains. The adaptation of cells to iron limitation involved an iron-sparing response tightly coordinated with diurnal cycles to optimize cell functions and synchronize these functions with the day/night redistribution of iron orchestrated by ferritin, and a stress response based on the induction of thioredoxin-like proteins, of peroxiredoxin and of tesmin-like methallothionein rather than ascorbate. We briefly surveyed the metabolic remodeling resulting from iron deprivation.

Conclusions: The mechanisms of iron uptake and utilization by O. tauri differ fundamentally from those described in C. reinhardtii. We propose this species as a new model for investigation of iron metabolism in marine microalgae.
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http://dx.doi.org/10.1186/s12864-016-2666-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855317PMC
May 2016

The Metacaspase (Mca1p) Restricts O-glycosylation During Farnesol-induced Apoptosis in Candida albicans.

Mol Cell Proteomics 2016 07 28;15(7):2308-23. Epub 2016 Apr 28.

From the ‡Mass Spectrometry Laboratory, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cité, F-75205 Paris, France; §Mitochondria, Metals and Oxidative Stress Group, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cité, F-75205 Paris, France

Protein glycolysation is an essential posttranslational modification in eukaryotic cells. In pathogenic yeasts, it is involved in a large number of biological processes, such as protein folding quality control, cell viability and host/pathogen relationships. A link between protein glycosylation and apoptosis was established by the analysis of the phenotypes of oligosaccharyltransferase mutants in budding yeast. However, little is known about the contribution of glycosylation modifications to the adaptive response to apoptosis inducers. The cysteine protease metacaspase Mca1p plays a key role in the apoptotic response in Candida albicans triggered by the quorum sensing molecule farnesol. We subjected wild-type and mca1-deletion strains to farnesol stress and then studied the early phase of apoptosis release in quantitative glycoproteomics and glycomics experiments on cell-free extracts essentially devoid of cell walls. We identified and characterized 62 new glycosylated peptides with their glycan composition: 17 N-glycosylated, 45 O-glycosylated, and 81 additional sites of N-glycosylation. They were found to be involved in the control of protein folding, cell wall integrity and cell cycle regulation. We showed a general increase in the O-glycosylation of proteins in the mca1 deletion strain after farnesol challenge. We identified 44 new putative protein substrates of the metacaspase in the glycoprotein fraction enriched on concanavalin A. Most of these substrates are involved in protein folding or protein resolubilization and in mitochondrial functions. We show here that key Mca1p substrates, such as Cdc48p or Ssb1p, involved in degrading misfolded glycoproteins and in the protein quality control system, are themselves differentially glycosylated. We found putative substrates, such as Bgl2p (validated by immunoblot), Srb1p or Ugp1p, that are involved in the biogenesis of glycans. Our findings highlight a new role of the metacaspase in amplifying cell death processes by affecting several critical protein quality control systems through the alteration of the protein glycosylation machinery.Data are available via ProteomeXchange with identifier PXD003677.
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http://dx.doi.org/10.1074/mcp.M116.059378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937506PMC
July 2016

Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton.

Proc Natl Acad Sci U S A 2015 Nov 9;112(47):14652-7. Epub 2015 Nov 9.

Sorbonne Universités, University Pierre et Marie Curie, University of Paris VI, CNRS, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, F-66650, Banyuls-sur-Mer, France;

In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main iron-binding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.
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http://dx.doi.org/10.1073/pnas.1506074112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664360PMC
November 2015

Label-Free Quantitative Proteomics in Yeast.

Methods Mol Biol 2016 ;1361:289-307

Mass Spectrometry Laboratory, Institut Jacques Monod, UMR7592, CNRS-Univ Paris Diderot, Sorbonne Paris Cité, Paris, France.

Label-free bottom-up shotgun MS-based proteomics is an extremely powerful and simple tool to provide high quality quantitative analyses of the yeast proteome with only microgram amounts of total protein. Although the experimental design of this approach is rather straightforward and does not require the modification of growth conditions, proteins or peptides, several factors must be taken into account to benefit fully from the power of this method. Key factors include the choice of an appropriate method for the preparation of protein extracts, careful evaluation of the instrument design and available analytical capabilities, the choice of the quantification method (intensity-based vs. spectral count), and the proper manipulation of the selected quantification algorithm. The elaboration of this robust workflow for data acquisition, processing, and analysis provides unprecedented insight into the dynamics of the yeast proteome.
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http://dx.doi.org/10.1007/978-1-4939-3079-1_16DOI Listing
August 2016