Publications by authors named "Stephane Claverol"

81 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.
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http://dx.doi.org/10.1073/pnas.2018690118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896303PMC
February 2021

Comparative Proteomics of Ostreid Herpesvirus 1 and Pacific Oyster Interactions With Two Families Exhibiting Contrasted Susceptibility to Viral Infection.

Front Immunol 2020 18;11:621994. Epub 2021 Jan 18.

SG2M-LGPMM, Laboratoire De Génétique Et Pathologie Des Mollusques Marins, Ifremer, La Tremblade, France.

Massive mortality outbreaks affecting Pacific oysters () spat/juveniles are often associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). In this work, experimental infection trials of spat with OsHV-1 were conducted using two contrasted Pacific oyster families for their susceptibility to viral infection. Live oysters were sampled at 12, 26, and 144 h post infection (hpi) to analyze host-pathogen interactions using comparative proteomics. Shotgun proteomics allowed the detection of seven viral proteins in infected oysters, some of them with potential immunomodulatoy functions. Viral proteins were mainly detected in susceptible oysters sampled at 26 hpi, which correlates with the mortality and viral load observed in this oyster family. Concerning the Pacific oyster proteome, more than 3,000 proteins were identified and contrasted proteomic responses were observed between infected A- and P-oysters, sampled at different post-injection times. Gene ontology (GO) and KEGG pathway enrichment analysis performed on significantly modulated proteins uncover the main immune processes (such as RNA interference, interferon-like pathway, antioxidant defense) which contribute to the defense and resistance of Pacific oysters to viral infection. In the more susceptible Pacific oysters, results suggest that OsHV-1 manipulate the molecular machinery of host immune response, in particular the autophagy system. This immunomodulation may lead to weakening and consecutively triggering death of Pacific oysters. The identification of several highly modulated and defense-related Pacific oyster proteins from the most resistant oysters supports the crucial role played by the innate immune system against OsHV-1 and the viral infection. Our results confirm the implication of proteins involved in an interferon-like pathway for efficient antiviral defenses and suggest that proteins involved in RNA interference process prevent viral replication in . Overall, this study shows the interest of multi-omic approaches applied on groups of animals with differing sensitivities and provides novel insight into the interaction between Pacific oyster and OsHV-1 with key proteins involved in viral infection resistance.
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http://dx.doi.org/10.3389/fimmu.2020.621994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848083PMC
January 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.
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http://dx.doi.org/10.1007/s00439-021-02255-6DOI Listing
June 2021

Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas.

J Clin Invest 2021 Jan;131(1)

CELLOMET, Bordeaux, France.

Metabolic reprogramming is a common hallmark of cancer, but a large variability in tumor bioenergetics exists between patients. Using high-resolution respirometry on fresh biopsies of human lung adenocarcinoma, we identified 2 subgroups reflected in the histologically normal, paired, cancer-adjacent tissue: high (OX+) mitochondrial respiration and low (OX-) mitochondrial respiration. The OX+ tumors poorly incorporated [18F]fluorodeoxy-glucose and showed increased expression of the mitochondrial trifunctional fatty acid oxidation enzyme (MTP; HADHA) compared with the paired adjacent tissue. Genetic inhibition of MTP altered OX+ tumor growth in vivo. Trimetazidine, an approved drug inhibitor of MTP used in cardiology, also reduced tumor growth and induced disruption of the physical interaction between the MTP and respiratory chain complex I, leading to a cellular redox and energy crisis. MTP expression in tumors was assessed using histology scoring methods and varied in negative correlation with [18F]fluorodeoxy-glucose incorporation. These findings provide proof-of-concept data for preclinical, precision, bioenergetic medicine in oxidative lung carcinomas.
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http://dx.doi.org/10.1172/JCI133081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773363PMC
January 2021

Novel self-replicating α-synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons.

Sci Adv 2020 Oct 2;6(40). Epub 2020 Oct 2.

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France.

The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ). The generation of τ fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.
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http://dx.doi.org/10.1126/sciadv.abc4364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852382PMC
October 2020

Targeting Human Lung Adenocarcinoma with a Suppressor of Mitochondrial Superoxide Production.

Antioxid Redox Signal 2020 11 29;33(13):883-902. Epub 2020 Jun 29.

CELLOMET, Functional Genomics Center (CGFB), Bordeaux, France.

REDOX signaling from reactive oxygen species (ROS) generated by the mitochondria (mitochondrial reactive oxygen species [mtROS]) has been implicated in cancer growth and survival. Here, we investigated the effect of 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (AOL), a recently characterized member of the new class of mtROS suppressors (S1QELs), on human lung adenocarcinoma proteome reprogramming, bioenergetics, and growth. AOL reduced steady-state cellular ROS levels in human lung cancer cells without altering the catalytic activity of complex I. AOL treatment induced dose-dependent inhibition of lung cancer cell proliferation and triggered a reduction in tumor growth . Molecular investigations demonstrated that AOL reprogrammed the proteome of human lung cancer cells. In particular, AOL suppressed the determinants of the Warburg effect and increased the expression of the complex I subunit NDUFV1 which was also identified as AOL binding site using molecular modeling computer simulations. Comparison of the molecular changes induced by AOL and MitoTEMPO, an mtROS scavenger that is not an S1QEL, identified a core component of 217 proteins commonly altered by the two treatments, as well as drug-specific targets. This study provides proof-of-concept data on the anticancer effect of AOL on mouse orthotopic human lung tumors. A unique dataset on proteomic reprogramming by AOL and MitoTEMPO is also provided. Lastly, our study revealed the repression of NDUFV1 by S1QEL AOL. Our findings demonstrate the preclinical anticancer properties of S1QEL AOL and delineate its mode of action on REDOX and cancer signaling.
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http://dx.doi.org/10.1089/ars.2019.7892DOI Listing
November 2020

Dysregulated pathways and differentially expressed proteins associated with adverse transfusion reactions in different types of platelet components.

J Proteomics 2020 04 20;218:103717. Epub 2020 Feb 20.

French Blood Bank (EFS) Auvergne-Rhône-Alpes, Saint-Etienne, France; GIMAP-EA3064, University of Lyon, Saint-Etienne, France. Electronic address:

Platelet components (PCs) are occasionally associated with adverse transfusion reactions (ATRs). ATRs can occur regardless of the type of PC being transfused, whether it is a single-donor apheresis PC (SDA-PC) or a pooled PC (PPCs). The purpose of this study was to investigate the proteins and dysregulated pathways in both of the main types of PCs. The proteomic profiles of platelet pellets from SDA-PCs and PPCs involved in ATRs were analysed using the label-free LC-MS/MS method. Differentially expressed proteins with fold changes >|1.5| in clinical cases versus controls were characterised using bioinformatic tools (RStudio, GeneCodis3, and Ingenuity Pathways Analysis (IPA). The proteins were confirmed by western blotting. The common primary proteins found to be dysregulated in both types of PCs were the mitochondrial carnitine/acylcarnitine carrier protein (SLC25A20), multimerin-1 (MMRN1), and calumenin (CALU), which are associated with the important enrichment of platelet activation, platelet degranulation, and mitochondrial activity. Furthermore, this analysis revealed the involvement of commonly dysregulated canonical pathways, particularly mitochondrial dysfunction, platelet activation, and acute phase response. This proteomic analysis provided an interesting contribution to our understanding of the meticulous physiopathology of PCs associated with ATR. A larger investigation would assist in delineating the most relevant proteins to target within preventive transfusion safety strategies. BIOLOGICAL SIGNIFICANCE: Within platelet transfusion strategies, the two primary types of PCs predominantly processed in Europe, include (i) single donor apheresis PCs (SDA-PCs) from one donor and (ii) pooled PCs (PPCs). The current study used PCs from five buffy coats derived from five whole blood donations that were identical in ABO, RH1 and KEL1 groups. Both PC types were shown to be associated with the onset of an ATR in the transfused patient. Several common platelet proteins were found to be dysregulated in bags associated with ATR occurrences regardless of the type of PCs transfused and of their process. The dysregulated proteins included mitochondrial carnitine/acylcarnitine carrier protein (SLC25A20), which is involved in a fatty acid oxidation disorder; calumenin (CALU); and multimerin-1 (MMRN1), which is chiefly involved in platelet activation and degranulation. Dysregulated platelet protein pathways for ATRs that occurred with SDA-PCs and PPCs could support the dysregulated functions found in association with those three proteins. Those common platelet proteins may become candidates to define biomarkers associated with the onset of an ATR from PC transfusions, including monitoring during the quality steps of PC manufacturing, provided that the results are confirmed in larger cohorts. This study enriches our knowledge of platelet proteomics in PCs under pathological conditions.
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http://dx.doi.org/10.1016/j.jprot.2020.103717DOI Listing
April 2020

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

Is pallial mucus involved in Ostrea edulis defenses against the parasite Bonamia ostreae?

J Invertebr Pathol 2020 01 2;169:107259. Epub 2019 Dec 2.

Institut Français de Recherche pour ĺExploitation de la Mer (IFREMER), Laboratoire de Génétique et Pathologie (LGP), Avenue Mus de Loup, 17390 La Tremblade, France.

Bonamia ostreae is an intrahemocytic parasite that has been responsible for severe mortalities in the flat oyster Ostrea edulis since the 1970́s. The Pacific oyster Crassostrea gigas is considered to be resistant to the disease and appears to have mechanisms to avoid infection. Most studies carried out on the invertebrate immune system focus on the role of hemolymph, although mucus, which covers the body surface of molluscs, could also act as a barrier against pathogens. In this study, the in vitro effect of mucus from the oyster species Ostrea edulis and C. gigas on B. ostreae was investigated using flow cytometry. Results showed an increase in esterase activities and mortality rate of parasites exposed to mucus from both oyster species. In order to better understand the potential role of mucus in the defense of the oyster against parasites such as B. ostreae, liquid chromatography and tandem mass spectrometry were used to describe and compare mucus protein composition from both species. In all oyster species, pallial mucus contains a high level of proteins; however, O. edulis mucus produced a variety of proteins that could be involved in the immune response against the parasite, including Cu/Zn extracellular superoxide dismutase, thioxiredoxin, peroxiredon VI, heat shock protein 90 as well as several hydrolases. Conversely, a different set of antioxidant proteins, hydrolases and stress related proteins were identified in mucus from C. gigas. Our results suggest an innate immunity adaptation of oysters to develop a specific response against their respective pathogens. The mucosal protein composition also provides new insights for further investigations into the immune response in oysters.
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http://dx.doi.org/10.1016/j.jip.2019.107259DOI Listing
January 2020

Oxidation of methionine residues activates the high-threshold heat-sensitive ion channel TRPV2.

Proc Natl Acad Sci U S A 2019 11 12;116(48):24359-24365. Epub 2019 Nov 12.

Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, 30625 Hannover, Germany.

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of HO induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.
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http://dx.doi.org/10.1073/pnas.1904332116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883831PMC
November 2019

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules.

Nat Commun 2019 10 4;10(1):4521. Epub 2019 Oct 4.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, F-33076, Bordeaux, France.

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.
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http://dx.doi.org/10.1038/s41467-019-12528-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778148PMC
October 2019

Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis.

Nat Commun 2019 10 1;10(1):4462. Epub 2019 Oct 1.

University of Bordeaux, F-33000, Bordeaux, France.

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin's efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.
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http://dx.doi.org/10.1038/s41467-019-12434-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773865PMC
October 2019

Early defects in translation elongation factor 1α levels at excitatory synapses in α-synucleinopathy.

Acta Neuropathol 2019 12 26;138(6):971-986. Epub 2019 Aug 26.

Center for Neuropathology and Prion Research, Ludwig-Maximilians University, Munich, Germany.

Cognitive decline and dementia in neurodegenerative diseases are associated with synapse dysfunction and loss, which may precede neuron loss by several years. While misfolded and aggregated α-synuclein is recognized in the disease progression of synucleinopathies, the nature of glutamatergic synapse dysfunction and loss remains incompletely understood. Using fluorescence-activated synaptosome sorting (FASS), we enriched excitatory glutamatergic synaptosomes from mice overexpressing human alpha-synuclein (h-αS) and wild-type littermates to unprecedented purity. Subsequent label-free proteomic quantification revealed a set of proteins differentially expressed upon human alpha-synuclein overexpression. These include overrepresented proteins involved in the synaptic vesicle cycle, ER-Golgi trafficking, metabolism and cytoskeleton. Unexpectedly, we found and validated a steep reduction of eukaryotic translation elongation factor 1 alpha (eEF1A1) levels in excitatory synapses at early stages of h-αS mouse model pathology. While eEF1A1 reduction correlated with the loss of postsynapses, its immunoreactivity was found on both sides of excitatory synapses. Moreover, we observed a reduction in eEF1A1 immunoreactivity in the cingulate gyrus neuropil of patients with Lewy body disease along with a reduction in PSD95 levels. Altogether, our results suggest a link between structural impairments underlying cognitive decline in neurodegenerative disorders and local synaptic defects. eEF1A1 may therefore represent a limiting factor to synapse maintenance.
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http://dx.doi.org/10.1007/s00401-019-02063-3DOI Listing
December 2019

Data from differentially expressed proteins in platelet components associated with adverse transfusion reactions.

Data Brief 2019 Aug 12;25:104013. Epub 2019 Jun 12.

French Blood Bank (EFS) Auvergne-Rhône-Alpes, Saint-Etienne, France.

The presented dataset was used for the study focused on the search for differentially expressed proteins in blood platelet components (PCs) associated with adverse transfusion reactions (ATRs). Pellets of ATR platelet components and their controls were subjected to high-throughput proteomics analysis using a Q Exactive high-resolution tandem mass spectrometer. The data reported here constitutes an extension of "Differential protein expression of blood platelet components associated with adverse transfusion reactions" article Aloui et al., 2018. The reported data herein have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD003510 for the pooled platelet components (PPCs) and PXD008886 for the apheresis platelet components (SDA-PCs) associated with ATRs.
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http://dx.doi.org/10.1016/j.dib.2019.104013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598860PMC
August 2019

Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

EMBO Rep 2019 08 9;20(8):e47182. Epub 2019 Jul 9.

Laboratoire de Biogenèse Membranaire, UMR5200, CNRS, Université de Bordeaux, Villenave d'Ornon, France.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.
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http://dx.doi.org/10.15252/embr.201847182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6680132PMC
August 2019

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

Anal Chem 2019 06 22;91(11):6953-6961. Epub 2019 May 22.

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram , The Hebrew University of Jerusalem , Jerusalem 91904 , Israel.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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http://dx.doi.org/10.1021/acs.analchem.9b00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625963PMC
June 2019

Proteomic pattern of breast milk discriminates obese mothers with infants of delayed weight gain from normal-weight mothers with infants of normal weight gain.

FEBS Open Bio 2019 04 15;9(4):736-742. Epub 2019 Mar 15.

INSERM U1069 Université François Rabelais Tours France.

We previously reported that exclusively breastfed infants born to mothers with pregestational obesity gain less weight during the first month after birth than those born to mothers of normal pregestational weight. This issue is potentially important since lower weight gain in breastfed infants of obese mothers might increase the risk of developing later obesity. Breast milk quality and quantity, together with breastfeeding practice, possibly influence infants' feeding behavior, appetite control, and regulation of growth later in life. The issue of whether breast milk protein patterns from obese mothers differ in composition from those of non-obese mothers remains largely unexplored. Here, we established a breast milk proteomic pattern that discriminates obese mothers and infants with delayed weight gain at 1 month after birth from normal-weight mothers with infants of the same age and with normal weight gain. Obese mothers were matched to normal-weight mothers ( = 26; body mass index 33.5 ± 3.2 21.5 ± 1.5 kg·m). The mean weight gain of infants in the obese group at 1 month after birth was 430.8 g lower than that of the infants in the control group. Analysis of the breast milk delipidized fraction by surface-enhanced laser desorption/ionization on CM10 and Q10 arrays was followed by MS-assisted purification and LC-MS/MS microsequencing of a selected biomarker. We identified 15 candidate protein biomarkers, seven of which were overexpressed in the obese group and eight in the normal-weight group. One of the most significant candidate biomarkers, overexpressed in the obese group, was identified as a fragment of the sixth extracellular domain of the polymeric immunoglobulin receptor. Further structural identification of these candidate biomarkers and their validation in clinical assays may facilitate the development of a predictive immunoassay.
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http://dx.doi.org/10.1002/2211-5463.12610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443869PMC
April 2019

Rho-of-plant activated root hair formation requires gene function.

Development 2019 03 11;146(5). Epub 2019 Mar 11.

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-901 83 Umeå, Sweden.

Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.
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http://dx.doi.org/10.1242/dev.168559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432664PMC
March 2019

Differential protein expression of blood platelet components associated with adverse transfusion reactions.

J Proteomics 2019 03 24;194:25-36. Epub 2018 Dec 24.

GIMAP-EA3064, University of Lyon, Saint-Etienne, France; French Blood Bank (EFS) Auvergne-Rhône-Alpes, Saint-Etienne, France. Electronic address:

Platelets found within platelet components (PCs) intended for transfusion release inflammatory molecules. Despite the implementation of leukoreduction, some of these PCs are occasionally associated with adverse transfusion reactions (ATRs). The aim of this study was to decipher the platelet proteome in two types of PCs, buffy-coat-derived pooled PCs (PPCs) and single-donor apheresis PCs (SDA-PCs), associated with ATRs. A label-free LC-MS/MS method was used for the proteomic analysis of washed platelet pellets from 3 PPCs and 3 SDA-PCs associated with ATRs, compared to matched controls. Bioinformatics tools allowed us to characterise the differentially expressed (DE) proteins between cases (ATR-PCs) and controls (no.ATR-PCs). From the PPCs and SDA-PCs, 473 and 146 proteins were DE, respectively. The functional interpretation of these proteins revealed enrichment in platelet activation and degranulation as the most important biological process. The most dysregulated pathways were integrin signaling for PPCs and acute phase response signaling for SDA-PCs. Interestingly, inflammatory disorders were found to be enriched in both PC types. Profound proteome changes were found in the platelets of PCs that led to clinical ATRs in patients. This study presents the first exploration of the platelet proteomic signature associated with ATRs and could provide clues to improving transfusion medicine. BIOLOGICAL SIGNIFICANCE: Adverse transfusion reactions (ATRs) can still occur after transfusion of platelet components (PC). This is the first report on the proteomic analysis of PCs associated with ATR. In this study, the contents of PC bags implicated in ATRs were examined. The aims of this study were to characterise molecules that could be central to the inflammation of ATRs and to highlight dysregulated mechanisms to explain the onset of ATRs. Two types of PCs were used: 3 PPCs (each from 5 donors) and 3 SDA-PCs (each from one donor). We have shown that the two types of PCs, from bags undergoing different processing (i.e., sampling, preparation), involve two types of dysregulated - pathophysiological mechanisms associated with the onset of ATRs. The most dysregulated signaling pathways were cytoskeleton and integrin regulation for PPCs, acute phase response signaling and remodelling of adherens junctions for SDA-PCs. Inflammation, platelet activation and degranulation processes were present in both PC types but were more important for PPCs. This proteomics analysis provides a better understanding of the pathophysiological mechanisms involved in ATRs and may lead to novel steps to ensure safe PC transfusion.
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http://dx.doi.org/10.1016/j.jprot.2018.12.019DOI Listing
March 2019

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

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Mar Biotechnol (NY) 2019 Feb 19;21(1):99-110. Epub 2018 Nov 19.

Department of Chemical Engineering and Center of Food Biotechnology and Bioseparations, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L and 66 mg L, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.
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http://dx.doi.org/10.1007/s10126-018-9863-zDOI Listing
February 2019

An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis.

Sci Rep 2018 10 9;8(1):15016. Epub 2018 Oct 9.

CRMSB, UMR5536 CNRS, INSB, Bordeaux, 33076, France.

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis.
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http://dx.doi.org/10.1038/s41598-018-33382-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177473PMC
October 2018

The Integrative Conjugative Element (ICE) of : Key Elements Involved in Horizontal Dissemination and Influence of Coresident ICEs.

mBio 2018 07 3;9(4). Epub 2018 Jul 3.

IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France

The discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can -complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the -acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria. Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with mycoplasma ICEs.
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http://dx.doi.org/10.1128/mBio.00873-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030558PMC
July 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.
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http://dx.doi.org/10.1016/j.celrep.2018.05.060DOI Listing
June 2018

Role of Glycanation and Convertase Maturation of Soluble Glypican-3 in Inhibiting Proliferation of Hepatocellular Carcinoma Cells.

Biochemistry 2018 02 2;57(7):1201-1211. Epub 2018 Feb 2.

Univ. Bordeaux, CBMN, UMR 5248 , F-33615 Pessac, France.

Glypican 3 (GPC3) is a complex heparan sulfate proteoglycan associated with the outer surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. It is also N-glycosylated and processed by a furin-like convertase. GPC3 has numerous biological functions. Although GPC3 is undetectable in normal liver tissue, it is abnormally and highly overexpressed in hepatocellular carcinoma (HCC). Interestingly, proliferation of HCC cells such as HepG2 and HuH7 is inhibited when they express a soluble form of GPC3 after lentiviral transduction. To obtain more insight into the role of some of its post-translational modifications, we designed a mutant GPC3, sGPC3m, without its GPI anchor, convertase cleavage site, and glycosaminoglycan chains. The highly pure sGPC3m protein strongly inhibited HuH7 and HepG2 cell proliferation in vitro and induced a significant increase in their cell doubling time. It changed the morphology of HuH7 cells but not that of HepG2. It induced the enlargement of HuH7 cell nuclear area and the restructuration of adherent cell junctions. Unexpectedly, for both cell types, the levels of apoptosis, cell division, and β-catenin were not altered by sGPC3m, although growth inhibition was very efficient. Overall, our data show that glycanation and convertase maturation are not required for sGPC3m to inhibit HCC cell proliferation.
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http://dx.doi.org/10.1021/acs.biochem.7b01208DOI Listing
February 2018

A new reliable, transposable and cost-effective assay for absolute quantification of total plasmatic bevacizumab by LC-MS/MS in human plasma comparing two internal standard calibration approaches.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Dec 23;1070:43-53. Epub 2017 Oct 23.

Department of Pharmacy, Groupe hospitalier Sud, CHU de Bordeaux, Avenue de Magellan, 33604, Pessac, France; Univ. Bordeaux, EPST, Biology of Cardiovascular Diseases, U1034, 33604, Pessac, France. Electronic address:

The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC-MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%. This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC-MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies.
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http://dx.doi.org/10.1016/j.jchromb.2017.10.042DOI Listing
December 2017

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

Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions.

Front Plant Sci 2017 29;8:894. Epub 2017 May 29.

Laboratory of Membrane Biogenesis, Centre National de la Recherche Scientifique, UMR 5200Villenave d'Ornon, France.

Lipid droplets (LDs) are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of aging Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER) or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1); both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in aging leaves. We thus examined the function of AtSRP1 at this developmental stage and found that modulates the expression of in aging leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition compared to wild-type. We demonstrate that, although expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, knock-down mutants displayed fewer but larger LDs. Together our results reveal that the abundancy of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence that LDs in leaves share tenuous physical continuity as well as numerous contact sites with the ER membrane. Thus, our data suggest that leaf LDs are functionally distinct from seed LDs and that their biogenesis is strictly controlled by AtSRP1 at restricted sites of the ER.
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http://dx.doi.org/10.3389/fpls.2017.00894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447075PMC
May 2017