Philippe Chafey

Université René Descartes - Paris 5, Lutetia Parisorum, Île-de-France, France

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Publications (37)142.34 Total impact

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    ABSTRACT: Background: α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. Methods: We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Results: Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. Conclusions: Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. General significance: Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.
    No preview · Article · Oct 2015 · Biochimica et Biophysica Acta
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    ABSTRACT: Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
    Full-text · Article · Jul 2015 · PLoS ONE
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    ABSTRACT: TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.
    No preview · Article · Jun 2015 · Innate Immunity
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    ABSTRACT: Toxicity of pesticides towards microorganisms can have a major impact on ecosystem function. Nevertheless, some microorganisms are able to respond quickly to this stress by degrading these molecules. The edaphic Bacillus megaterium strain Mes11 can degrade the herbicide mesotrione. In order to gain insight into the cellular response involved, the intracellular proteome of Mes11 exposed to mesotrione was analyzed using the two-dimensional differential in-gel electrophoresis (2D-DIGE) approach coupled with mass spectrometry. The results showed an average of 1820 protein spots being detected. The gel profile analyses revealed 32 protein spots whose abundance is modified after treatment with mesotrione. Twenty spots could be identified, leading to 17 non redundant proteins, mainly involved in stress, metabolic and storage mechanisms. These findings clarify the pathways used by B. megaterium strain Mes11 to resist and adapt to the presence of mesotrione. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Feb 2015 · Environmental Pollution

  • No preview · Article · Dec 2014
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    ABSTRACT: Unlabelled: Hepatitis E virus (HEV) causes acute enterically transmitted hepatitis. In industrialized countries, it is a zoonotic disease, with swine being the major reservoir of human HEV contamination. The occurrence and severity of the disease are variable, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In the absence of a robust cell culture system or small-animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with three different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, two-dimensional difference in gel electrophoresis, and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the three HEV strains replicate similarly in swine and that they modulate several cellular pathways, suggesting that HEV impairs several cellular processes, which can account for the various types of disease expression. Several proteins, such as heterogeneous nuclear ribonucleoprotein K, apolipoprotein E, and prohibitin, known to be involved in other viral life cycles, were upregulated in HEV-infected livers. Some differences were observed between the three strains, suggesting that HEV's genetic variability may induce variations in pathogenesis. This comparative analysis of the liver proteome modulated during infection with three different strains of HEV genotype 3 provides an important basis for further investigations on the factors involved in HEV replication and the mechanism of HEV pathogenesis. Importance: Hepatitis E virus (HEV) is responsible for acute hepatitis, with clinical symptoms ranging from asymptomatic to self-limiting acute hepatitis, chronic infection, or fulminant hepatitis. In industrialized countries, HEV is considered an emerging zoonotic disease, with swine being the principal reservoir for human contamination. The viral and cellular factors involved in the replication and/or pathogenesis of HEV are still not fully known. Here we report that several cellular pathways involved in cholesterol and lipid metabolism or cell survival were modulated during HEV infection in the swine model. Moreover, we observed a difference between the different swine strains, suggesting that HEV's genetic variability could play a role in pathogenesis. We also identified some proteins known to be involved in other viral cycles. Our study provides insight into the mechanisms modulated during HEV infection and constitutes a useful reference for future work on HEV pathogenesis and virulence.
    Full-text · Article · Oct 2014 · Journal of Virology
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    Preview · Article · Apr 2014 · Archives of Cardiovascular Diseases Supplements
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    Full-text · Dataset · Feb 2014
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    ABSTRACT: Chronic pressure overload (PO) induces pathological left ventricular hypertrophy (LVH) leading to congestive heart failure (HF). Overexpression of FKBP12.6 (FK506-binding protein [K]) in mice should prevent Ca2+-leak during diastole and may improve overall cardiac function. In order to decipher molecular mechanisms involved in thoracic aortic constriction (TAC)-induced cardiac remodeling and the influence of gender and genotype, we performed a proteomic analysis using two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and bioinformatics techniques to identify alterations in characteristic biological networks. Wild-type (W) and K mice of both genders underwent TAC. Thirty days post-TAC, the altered cardiac remodeling was accompanied with systolic and diastolic dysfunction in all experimental groups. A gender difference in inflammatory protein expression (fibrinogen, α-1-antitrypsin isoforms) and in calreticulin occurred (males > females). Detoxification enzymes and cytoskeletal proteins were noticeably increased in K mice. Both non- and congestive failing mouse heart exhibited down- and upregulation of proteins related to mitochondrial function and purine metabolism, respectively. HF was characterized by a decrease in enzymes related to iron homeostasis, and altered mitochondrial protein expression related to fatty acid metabolism, glycolysis, and redox balance. Moreover, two distinct differential protein profiles characterized TAC-induced pathological LVH and congestive HF in all TAC mice. FKBP12.6 overexpression did not influence TAC-induced deleterious effects. Huntingtin was revealed as a potential mediator for HF. A broad dysregulation of signaling proteins associated with congestive HF suggested that different sets of proteins could be selected as useful biomarkers for HF progression and might predict outcome in PO-induced pathological LVH.
    Full-text · Article · Aug 2013
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    ABSTRACT: Citrulline (Cit) actions on muscle metabolism remain unclear. Those latter were investigated using a proteomic approach on Tibialis muscles from male Sprague-Dawley rats. At 23 months of age, rats were either fed ad libitum (AL group) or subjected to dietary restriction for 12 weeks. At the end of the restriction period, one group of rats was euthanized (R group) and 2 groups were refed for one week with a standard diet supplemented with non-essential amino acids (NEAA group) or Cit- (CIT group). Results of the proteomic approach were validated using targeted western blot analysis and assessment of gene expression of the related-genes. Maximal activities of the key-enzymes involved in mitochondrial functioning were also determined. Cit supplementation results in a significant increase in the protein expression of the main myofibrillar constituants and of a few enzymes involved in glycogenolysis and glycolysis (CIT vs AL and R, p<0.05). Conversely, the expression of oxidative enzymes from Krebs cycle and mitochondrial respiratory chain was significantly decreased (CIT vs AL, p<0.05). However, maximal activities of key-enzymes of mitochondrial metabolism were not significantly affected, except for complex 1 which presented an increased activity (CIT vs AL and R, p<0.05).. In conclusion, Cit supplementation increases expression of the main myofibrillar proteins and seems to induce a switch in muscle energy metabolism, from aerobia towards anaerobia.
    Full-text · Article · Jul 2013 · Proteomics

  • No preview · Article · Jun 2013 · Transfusion Clinique et Biologique
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    ABSTRACT: In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood-brain barrier (BBB) models. Hence, the re-induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re-induction remain largely unknown. Here, we used a differential gel electrophoresis (DIGE)-based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re-induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine (ADMA) pathway in the BBB phenotype re-induction process and we discuss ADMA's potential role in regulating endothelial function (in addition to its role as a by-product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re-induced BBB functions than in cells with limited BBB functions.
    Full-text · Article · Apr 2013 · Proteomics
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    ABSTRACT: Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.
    Full-text · Article · Jan 2013 · Journal of Virology
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    ABSTRACT: Background: Platelet concentrate (PC) functionality decreases during storage. This is referred to as the storage lesion. Pathogen inactivation may accelerate or induce lesions, potentially accounting for reduced viability. Our aim was to characterize functional and biochemical properties of platelets (PLTs) from photochemically treated buffy-coat PCs (PCT-PCs) compared to those from conventional PCs. Study design and methods: Four PCT-PCs and four conventional PCs were stored for 6.5 days and PLT function and proteomic profiles were examined at various time points during storage. To evaluate their intrinsic properties, samples of stored PLTs were taken, washed, and suspended in Tyrode's buffer before testing. Results: PLT counts and morphology were conserved although a slight increase in the PLT volume was observed after PCT. Glycoprotein (GP) IIbIIIa, IaIIa, and VI expression remained stable while GPIbα declined similarly in both types of PCs. A steep decrease (50%) in GPV occurred on Day 1.5 in PCT-PCs and Day 2.5 in control PCs. For both PCT- and control PCs, P-selectin expression and activated GPIIbIIIa remained low during storage. PCT- and control PCs were fully responsive to aggregation agonists up to Day 4.5 and exhibited similar perfusion functionality. Mitochondrial membrane potential and annexin A5 binding of PCT-PCs and control PCs were comparable. Two-dimensional differential in-gel electrophoresis and mass spectrometry profiles for 1882 protein spots revealed only three proteins selectively changed in PCT-PCs compared to control-PCs. Conclusion: Washed treated and untreated PCs have similar functional, morphologic, and proteomic characteristics provided that PLTs are suspended in an appropriate medium during testing.
    No preview · Article · Oct 2012 · Transfusion
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    ABSTRACT: Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.
    Full-text · Article · Sep 2012 · PLoS ONE
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    ABSTRACT: Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity® analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition.
    No preview · Article · Aug 2012 · Proteomics
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    ABSTRACT: Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem-like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio-chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two-dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma-derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC-conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC-conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti-HDGF antibodies. In vivo, GSC-conditioned medium induced neoangiogenesis, whereas HDGF-targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high-grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties.
    Full-text · Article · May 2012 · Stem Cells
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    ABSTRACT: We have evaluated the influence of the microfluidic environment on renal cell functionality. For that purpose, we performed a time lapse transcriptomic and proteomic analysis in which we compared gene and protein expressions of Madin-Darby canine kidney cells after 24 h and 96 h of culture in both microfluidic biochips and plates. The transcriptomic and proteomic integration revealed that the ion transporters involved in calcium, phosphate, and sodium homoeostasis and several genes involved in H(+) transporters and pH regulation were up-regulated in microfluidic biochips. Concerning drug metabolism, we found Phase I (CYP P450), Phase II enzymes (GST), various multidrug resistance genes (MRP), and Phase III transporters (SLC) were also up-regulated in the biochips. Furthermore, the study shows that those inductions were correlated with the induction of the Ahr and Nrf-2 dependent pathways, which results in a global cytoprotective response induced by the microenvironment. However, there was no apoptosis situation or cell death in the biochips. Microfluidic biochips may thus provide an important insight into exploring xenobiotic injury and transport modifications in this type of bioartificial microfluidic kidney. Finally, the investigation demonstrated that combining the transcriptomic and proteomic analyses obtained from a cell "on chip" culture would provide a pertinent new tool in the mechanistic interpretation of cellular mechanisms for predicting kidney cell toxicity and renal clearance in vitro.
    No preview · Article · Mar 2012 · Biotechnology Progress
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    ABSTRACT: Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation.
    No preview · Article · Nov 2011 · Proteomics
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    Dataset: Table S3
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    ABSTRACT: Proteins differentially expressed by the biochip versus the Petri and successfully identified by MS/MS. (DOC)
    Preview · Dataset · Aug 2011

Publication Stats

640 Citations
142.34 Total Impact Points

Institutions

  • 2009-2015
    • Université René Descartes - Paris 5
      • Faculté de Médecine
      Lutetia Parisorum, Île-de-France, France
  • 2011-2013
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2012
    • University of Strasbourg
      • Faculty of Medicine
      Strasburg, Alsace, France
  • 2004-2011
    • Institut Cochin
      Lutetia Parisorum, Île-de-France, France
  • 2010
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France