Publications by authors named "Solenne Chardonnet"

22 Publications

  • Page 1 of 1

Sublethal Exposure Effects of the Neonicotinoid Clothianidin Strongly Modify the Brain Transcriptome and Proteome in the Male Moth .

Insects 2021 Feb 11;12(2). Epub 2021 Feb 11.

Département Ecologie Sensorielle, Institut d'Ecologie et des Sciences de l'Environnement de Paris (iEES-Paris), Sorbonne Université, INRAE, CNRS, IRD, UPEC, Université de Paris, 75005 Paris, France.

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids such as clothianidin. The residual accumulation of low concentrations of these insecticides can have positive effects on target pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction and olfactory synaptic transmission is cholinergic, neonicotinoid residues could indeed modify chemical communication. We recently showed that treatments with low doses of clothianidin could induce hormetic effects on behavioral and neuronal sex pheromone responses in the male moth, . In this study, we used high-throughput RNAseq and proteomic analyses from brains of males that were intoxicated with a low dose of clothianidin to investigate the molecular mechanisms leading to the observed hormetic effect. Our results showed that clothianidin induced significant changes in transcript levels and protein quantity in the brain of treated moths: 1229 genes and 49 proteins were differentially expressed upon clothianidin exposure. In particular, our analyses highlighted a regulation in numerous enzymes as a possible detoxification response to the insecticide and also numerous changes in neuronal processes, which could act as a form of acclimatization to the insecticide-contaminated environment, both leading to enhanced neuronal and behavioral responses to sex pheromone.
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http://dx.doi.org/10.3390/insects12020152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916958PMC
February 2021

In-depth proteomic analysis of Plasmodium berghei sporozoites using trapped ion mobility spectrometry with parallel accumulation-serial fragmentation.

Proteomics 2021 Mar 23;21(6):e2000305. Epub 2021 Feb 23.

Sorbonne Université, INSERM, CNRS, Centre d'Immunologie et des Maladies Infectieuses, CIMI-Paris, Paris, France.

Sporozoites of the malaria parasite Plasmodium are transmitted by mosquitoes and infect the liver for an initial and obligatory round of replication, before exponential multiplication in the blood and onset of the disease. Sporozoites and liver stages provide attractive targets for malaria vaccines and prophylactic drugs. In this context, defining the parasite proteome is important to explore the parasite biology and to identify potential targets for antimalarial strategies. Previous studies have determined the total proteome of sporozoites from the two main human malaria parasites, P. falciparum and P. vivax, as well as P. yoelii, which infects rodents. Another murine malaria parasite, P. berghei, is widely used to investigate the parasite biology. However, a deep view of the proteome of P. berghei sporozoites is still missing. To fill this gap, we took advantage of the highly sensitive timsTOF PRO mass spectrometer, combined with three alternative methods for sporozoite purification, to identify the proteome of P. berghei sporozoites using low numbers of parasites. This study provides a reference proteome for P. berghei sporozoites, identifying a core set of proteins expressed across species, and illustrates how the unprecedented sensitivity of the timsTOF PRO system enables deep proteomic analysis from limited sample amounts.
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http://dx.doi.org/10.1002/pmic.202000305DOI Listing
March 2021

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

Immunity 2020 08;53(2):429-441.e8

Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France. Electronic address:

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.
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http://dx.doi.org/10.1016/j.immuni.2020.07.021DOI Listing
August 2020

Anti-integrin α therapy improves cardiac fibrosis after myocardial infarction by blunting cardiac PW1 stromal cells.

Sci Rep 2020 07 9;10(1):11404. Epub 2020 Jul 9.

Université de Paris, PARCC, INSERM, 56 Rue Leblanc, 75015, Paris, France.

There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1 cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1 cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1 cells (93% ± 1%), predominantly as the αVβ1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFβ at the surface of cardiac PW1 cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1CD51 cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.
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http://dx.doi.org/10.1038/s41598-020-68223-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347632PMC
July 2020

Reverse Immunology Approach to Define a New HIV-gp41-Neutralizing Epitope.

J Immunol Res 2019 24;2019:9804584. Epub 2019 Mar 24.

Sorbonne Université, INSERM, CNRS, Centre d'Immunologie et des Maladies Infectieuses-Paris (CIMI-Paris), F-75013 Paris, France.

The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.
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http://dx.doi.org/10.1155/2019/9804584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451809PMC
August 2019

The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Cell Mol Gastroenterol Hepatol 2019 11;7(1):33-53. Epub 2018 Sep 11.

Department of Surgery, Technical University of Munich, School of Medicine, Klinikum Rechts der Isar, Munich, Germany. Electronic address:

Background & Aims: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased expression with distant metastasis formation.

Methods: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of and for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set.

Results: expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased and increased expression, associated with significantly decreased overall survival. Patients with increased expression show significantly worse response to adjuvant chemotherapy.

Conclusions: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.
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http://dx.doi.org/10.1016/j.jcmgh.2018.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251370PMC
April 2019

Identification of Plasmodium falciparum nuclear proteins by mass spectrometry and proposed protein annotation.

PLoS One 2018 31;13(10):e0205596. Epub 2018 Oct 31.

Sorbonne Université, INSERM, CNRS, Centre d'immunologie et des maladies infectieuses, CIMI-Paris, Paris, France.

The nuclear proteome of Plasmodium falciparum results from the continual shuttle of proteins between the cell cytoplasm-nucleus and vice versa. Using shotgun proteomics tools, we explored the nuclear proteins of mixed populations of Plasmodium falciparum extracted from infected erythrocytes. We combined GeLC-MS/MS and 2D-LC-MS/MS with a peptide ion exclusion procedure in order to increase the detection of low abundant proteins such as those involved in gene expression. We have identified 446 nuclear proteins covering all expected nuclear protein families involved in gene regulation. All structural ribosomal (40S and 60S) proteins were identified which is consistent with the nuclear localization of ribosomal biogenesis. Proteins involved in the translation machinery were also found suggesting that translational events might occur in the nucleus in P. falciparum as previously hypothesized in eukaryotes. These data were compared to the protein list established by PlasmoDB and submitted to Plasmobase a recently reported Plasmodium annotation website to propose new functional putative annotation of several unknown proteins found in the nuclear extracts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205596PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209197PMC
April 2019

Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation.

Hum Mol Genet 2018 09;27(17):3060-3078

Sorbonne Université, UPMC Paris 06, INSERM UMRS974, Center of Research in Myology, F-75013 Paris, France.

Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.
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http://dx.doi.org/10.1093/hmg/ddy215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097156PMC
September 2018

Protein profile of mouse ovarian follicles grown in vitro.

Mol Hum Reprod 2017 12;23(12):827-841

Université Paris VI (UPMC), Paris, France.

Study Question: Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage?

Summary Answer: From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages.

What Is Known Already: The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells.

Study Design, Size, Duration: The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics.

Participants/materials, Setting, Methods: SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 μg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 μg and 170 μg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro.

Main Results And The Role Of Chance: It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development.

Large Scale Data: Data are available via ProteomeXchange with identifier PXD006227.

Limitations, Reasons For Caution: The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant.

Wider Implications Of The Findings: This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells.

Study Funding/competing Interest(s): This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report.
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http://dx.doi.org/10.1093/molehr/gax056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909860PMC
December 2017

Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice.

Environ Health Perspect 2017 09 1;125(9):097001. Epub 2017 Sep 1.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS , Neuroscience Paris Seine - Institut de Biologie Paris Seine, Paris, France.

Background: Courtship behavior plays a critical role in attracting females and reproduction success. However, the effects of exposure to a ubiquitous contaminant di(2-ethylhexyl) phthalate (DEHP) on these behaviors and, in particular, on courtship vocalizations have not been examined.

Objective: The effects of adult exposure to DEHP on courtship and mating behaviors and gonadotropic axis and neural mechanisms involved in DEHP-induced effects were analyzed in male mice.

Methods: Adult C57BL/6J males were orally exposed to DEHP (0, 0.5, 5, and 50μg/kg/d) for 4 wk. Olfactory preference, ultrasonic vocalizations (USVs), partner preference and mating, as well as locomotor activity and motor coordination, were measured. The kisspeptin system and testosterone levels were analyzed. Proteomic and molecular studies were conducted on the hypothalamic preoptic nucleus, the key region involved in sexual motivation to vocalize and mate.

Results: DEHP at 50μg/kg/d reduced the emission of USVs, whereas lower doses changed the ratio of syllable categories. This was associated with diminished sexual interest of female partners toward males exposed to 5 or 50μg/kg/d and increased latency to mate, despite normal olfactory preference. The kisspeptin system and circulating testosterone levels were unaffected. In DEHP-exposed males, proteomic analysis of the preoptic nucleus identified differentially expressed proteins connected to the androgen receptor (AR). Indeed, exposure to 5 or 50μg/kg/d of DEHP induced selective AR downregulation in this nucleus and upstream chemosensory regions. The involvement of AR changes in the observed alterations was further supported by the reduced emission of courtship vocalizations in males with disrupted neural AR expression.

Conclusions: These data demonstrate the critical role of neural AR in courtship vocalizations and raises the possibility that the vulnerability of this signaling pathway to exposure to endocrine disrupters may be detrimental for courtship communication and mating in several species. https://doi.org/10.1289/EHP1443.
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http://dx.doi.org/10.1289/EHP1443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915199PMC
September 2017

Native metabotropic glutamate receptor 4 depresses synaptic transmission through an unusual Gα transduction pathway.

Neuropharmacology 2017 Jul 26;121:247-260. Epub 2017 Apr 26.

Equipe Pharmacologie et Biochimie de la Synapse, NeuroPSI - UMR 9197 « Univ Paris-sud - CNRS », Université Paris-Sud, F-91405 Orsay, France. Electronic address:

In cerebellar cortex, mGlu receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu receptors control synaptic transmission through an atypical activation of Gα proteins. First, the Gα subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu receptors and Gα subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) - Molecular Layer Interneuron (MLI) synapses involves the Gα-PLC signaling pathway. Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu receptors act by coupling to the Gα protein and PLC effector system to reduce glutamate synaptic transmission.
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http://dx.doi.org/10.1016/j.neuropharm.2017.04.036DOI Listing
July 2017

Dp71Δ78-79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1.

Proteomics 2016 05 13;16(9):1331-40. Epub 2016 Apr 13.

Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F., México.

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ78-79 dystrophin mutant is stably expressed in PC12-C11 cells. To investigate the effect of Dp71Δ78-79 overexpression on the protein profile of PC12-C11 cells, we compared the expression profiles of undifferentiated and NGF-differentiated PC12-C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ78-79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12-C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ78-79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.
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http://dx.doi.org/10.1002/pmic.201500211DOI Listing
May 2016

SASH1, a new potential link between smoking and atherosclerosis.

Atherosclerosis 2015 Oct 14;242(2):571-9. Epub 2015 Aug 14.

Sorbonne Universités, UPMC, UMR_S 1166-ICAN, Genomics and Pathophysiology of Cardiovascular Diseases, Institute of Cardiometabolism and Nutrition, ICAN, Pitié-Salpêtrière Hospital, F-75013, Paris, France. Electronic address:

Objective: We have previously reported that SASH1 expression is increased in circulating human monocytes from smokers and was positively correlated with the number of carotid atherosclerotic plaques. The aim of this study was to further validate the link between smoking, SASH1 and atherosclerosis within the vascular wall and to assess the impact of SASH1 expression on endothelial cell functions.

Method: Human carotids with atherosclerotic plaques were obtained from 58 patients (45 of them with known smoking status: smoker, non-smoker, ex-smokers), and were processed for gene expression analyses and immunostaining. To investigate its function, SASH1 was silenced in human aortic endothelial cells (HAECs) using two different siRNA and subcellular localization of SASH1 was determined by immunostaining and subcellular fractionation. Subsequently the transcriptomic analyses and functional experiments (wound healing, WST-1 proliferation or Matrigel assays) were performed to characterize SASH1 function.

Results: SASH1 was expressed in human vascular cells (HAECs, smooth muscle cells) and in monocytes/macrophages. Its tissue expression was significantly higher in the atherosclerotic carotids of smokers compared to non-smokers (p < 0.01). In HAECs, SASH1 was expressed mostly in the cytoplasm and SASH1 knockdown resulted in an increased cell migration, proliferation and angiogenesis. Transcriptomic and pathway analyses showed that SASH1 silencing results in a decreased CYP1A1 expression possibly through the inhibition of TP53 activity.

Conclusion: We showed that SASH1 expression is increased in atherosclerotic carotids in smokers and its silencing affects endothelial angiogenic functions; therefore we provide a potential link between smoking and atherosclerosis through SASH1 expression.
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http://dx.doi.org/10.1016/j.atherosclerosis.2015.08.013DOI Listing
October 2015

Evaluation of dermal extracellular matrix and epidermal-dermal junction modifications using matrix-assisted laser desorption/ionization mass spectrometric imaging, in vivo reflectance confocal microscopy, echography, and histology: effect of age and peptide applications.

J Cosmet Dermatol 2015 Jun 27;14(2):152-60. Epub 2015 Mar 27.

Laboratoire P3S, UPMC Sorbonne Universities, Paris, France.

This study was conducted to establish a new methodology for evaluating elements of dermal extracellular matrix (ECM), of epidermal-dermal junction (EDJ), and effects of molecules which can modulate their synthesis. This methodology is based on matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI). In vivo reflectance confocal microscopy (in vivo RCM) and echography were also used. Using immunohistochemistry methods on explants, age-related modification data were obtained for selected dermal ECM and EDJ proteins (collagen I, collagen IV, collagen VII, collagen XVII, nidogen I, decorin/decorunt) and used as reference for MALDI-MSI studies. A methodology was developed with MALDI-MSI to map epidermis and dermis proteins. Then MALDI-MSI was used to study age modifications. In vivo RCM and high-frequency ultrasounds were used to evaluate ECM and EDJ undulation modifications caused by aging. Anti-aging molecule evaluations were performed with a blend of palmitoyl oligopeptide and palmitoyl tetrapeptide-7. Immunohistochemistry studies demonstrated that the selected proteins were found to be less abundant in aged group explants vs. young group except for decorin. MALDI-MSI studies correlated the results obtained for decorin. In vivo RCM measurements indicated a decrease of EDJ undulation depth with age and ECM modifications in the upper part of dermis. Echography demonstrated that the peptide blend reduced subepidermal low-echogenic band thickness and improved its density. In vivo RCM studies indicated that the peptides improved the ECM structure vs. placebo. This preliminary MALDI-MSI study raised some technical difficulties that were overcome. Further studies will be conducted to identify more proteins and to demonstrate the interest of this method for cosmetic evaluations.
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http://dx.doi.org/10.1111/jocd.12135DOI Listing
June 2015

First proteomic study of S-glutathionylation in cyanobacteria.

J Proteome Res 2015 Jan 19;14(1):59-71. Epub 2014 Sep 19.

IBBMC, CNRS UMR 8619, Univ Paris-Sud , Orsay, France.

Glutathionylation, the reversible post-translational formation of a mixed disulfide between a cysteine residue and glutathione (GSH), is a crucial mechanism for signal transduction and regulation of protein function. Until now this reversible redox modification was studied mainly in eukaryotic cells. Here we report a large-scale proteomic analysis of glutathionylation in a photosynthetic prokaryote, the model cyanobacterium Synechocystis sp. PCC6803. Treatment of acellular extracts with N,N-biotinyl glutathione disulfide (BioGSSG) induced glutathionylation of numerous proteins, which were subsequently isolated by affinity chromatography on streptavidin columns and identified by nano LC-MS/MS analysis. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and mass spectrometry analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H2 production. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acids metabolism, was confirmed by biochemical studies on the purified recombinant proteins. These results suggest that glutathionylation constitutes a major mechanism of global regulation of the cyanobacterial metabolism under oxidative stress conditions.
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http://dx.doi.org/10.1021/pr500625aDOI Listing
January 2015

The purified mechanosensitive channel TREK-1 is directly sensitive to membrane tension.

J Biol Chem 2013 Sep 29;288(38):27307-27314. Epub 2013 Jul 29.

Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC), Unité Mixte de Recherche (UMR) 8619, CNRS, Université Paris-Sud, 91405 Orsay. Electronic address:

Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K(+) channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.
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http://dx.doi.org/10.1074/jbc.M113.478321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779726PMC
September 2013

The Synechocystis PCC6803 MerA-like enzyme operates in the reduction of both mercury and uranium under the control of the glutaredoxin 1 enzyme.

J Bacteriol 2013 Sep 12;195(18):4138-45. Epub 2013 Jul 12.

UMR8221, CEA, CNRS, Université Paris Sud, iBiTec-S, LBBC, Gif sur Yvette, France.

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 residues C31, which belongs to the redox active site (CX(2)C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes.
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http://dx.doi.org/10.1128/JB.00272-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754753PMC
September 2013

Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum.

J Biol Chem 2012 Jun 23;287(24):20176-86. Epub 2012 Apr 23.

Pharmacologie et Biochimie de la Synapse, CNRS UMR 8619, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Univ. Paris-Sud, 91405 Orsay Cedex, France.

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.
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http://dx.doi.org/10.1074/jbc.M112.347468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370200PMC
June 2012

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: low concentrations of HNE open protein-mediated leaks in the membrane.

Free Radic Biol Med 2011 Jan 23;50(2):323-36. Epub 2010 Nov 23.

Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.11.017DOI Listing
January 2011

Large-scale study of phosphoproteins involved in long-term potentiation in the rat dentate gyrus in vivo.

Eur J Neurosci 2008 Jun;27(11):2985-98

Laboratoire de Neurobiologie de l'Apprentissage, de la Mémoire et de la Communication, Université Paris-Sud, Orsay, France.

The mechanisms underlying the induction of synaptic plasticity and the formation of long-term memory involve activation of cell-signalling cascades and protein modifications such as phosphorylation and dephosphorylation. Based on a protein candidate strategy, studies have identified several protein kinases and their substrates, which show an altered phosphorylation state during the early phases of long-term potentiation (LTP), yet only a limited number of synaptic phosphoproteins are known to be implicated in LTP. To identify new phosphoproteins associated with LTP, we have undertaken a proteomic study of phosphoproteins at different time points following the induction of LTP in the dentate gyrus in vivo (0, 15 and 90 min). For each time point, proteins from the dentate gyrus were separated by two-dimensional gel electrophoresis and stained with Pro-Q Diamond, a fluorescent stain specific for phosphoproteins. Fourteen proteins whose phosphorylation state varied significantly following LTP were identified using matrix-assisted laser desorption ionization/time of flight mass spectrometry and electrospray ionization-Orbitrap tandem mass spectrometry (MS/MS). They are involved in various cellular functions implicated in synaptic plasticity, such as intracellular signalling, axonal growth, exocytosis, protein synthesis and metabolism. Our results highlight new proteins whose phosphorylation or dephosphorylation is associated with LTP induction or maintenance. Further studies focusing on the regulation of specific phosphorylation sites will lead to greater understanding of the individual implications of these proteins in LTP as well as of their molecular interactions.
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http://dx.doi.org/10.1111/j.1460-9568.2008.06280.xDOI Listing
June 2008

New mortalin and histidyl tRNA synthetase isoforms point out a pitfall in proteomic analysis of Egr1 genetically modified mice.

Proteomics 2007 Jan;7(2):289-98

Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS, Université Paris-Sud, Orsay Cedex, France.

Egr1 (Zif268) is an immediate early gene encoding an inducible transcription factor involved in synaptic plasticity and several forms of memory in rodents. Using 2-DE and MS, we compared proteomes of hippocampal subregions and cortex in Egr1-deficient and wild-type littermates. Two significant differences were identified: a shift in the pI of the molecular chaperone mortalin (mtHsp70/PBP74/Grp75) and the apparent disappearance of histidyl tRNA synthetase (HisRS). We found that the pI shift for mortalin in Egr1-deficient mice was caused by a difference in protein sequence: D626G. Using cDNA sequencing, we demonstrated for both mortalin and HisRS that protein differences were not due to a lack of Egr1 but to DNA polymorphism between the C57Bl/6J and 129/Sv strains used to generate the Egr1-deficient mice. Our results show that mortalin and HisRS genes, which map closely to the Egr1 locus, have conserved the 129/Sv haplotype despite numerous back-crossing of the null mice progeny with C57Bl/6J animals. This demonstrates that allelic differences between mouse strains can introduce variations in differential proteomic analyses of genetically modified organisms. Finally, we report the identification of new isoforms of HisRS and mortalin (mot-3) encoded by the 129/Sv haplotype.
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http://dx.doi.org/10.1002/pmic.200600513DOI Listing
January 2007

Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach.

Photosynth Res 2006 Sep 14;89(2-3):201-11. Epub 2006 Nov 14.

IBBMC, CNRS UMR 8619, Bat 430, Univ Paris-Sud, Orsay cedex, 91405, France.

A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 muM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.
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http://dx.doi.org/10.1007/s11120-006-9108-2DOI Listing
September 2006