39 results on '"Domitille Schvartz"'
Search Results
2. Laser microdissection, proteomics, and multiplex immunohistochemistry: a bumpy ride into the study of paraffin-embedded fetal and pediatric lung tissues
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Luis M. Cardoso dos Santos, Yannick Avila, Domitille Schvartz, Anne-Laure Rougemont, Marie-Luce Bochaton-Piallat, and Isabelle Ruchonnet-Metrailler
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human fetal lung tissue ,formalin-fixed paraffin embedded (FFPE) lung ,human lung development ,lung proteome ,RAGE expression ,SOX-9 expression ,Medicine (General) ,R5-920 - Abstract
BackgroundKnowledge about lung development or lung disease is mainly derived from data extrapolated from mouse models. This has obvious drawbacks in developmental diseases, particularly due to species differences. Our objective is to describe the development of complementary analysis methods that will allow a better understanding of the molecular mechanisms involved in the pathogenesis of rare congenital diseases.MethodsParaffin-embedded human pediatric and fetal lung samples were laser microdissected to enrich different lung regions, namely, bronchioli or alveoli. These samples were analyzed by data-independent acquisition-based quantitative proteomics, and the lung structures were subsequently compared. To confirm the proteomic data, we employed an optimized Sequential ImmunoPeroxidase Labeling and Erasing (SIMPLE) staining for specific proteins of interest.ResultsBy quantitative proteomics, we identified typical pulmonary proteins from being differentially expressed in different regions. While the receptor for advanced glycation end products (RAGE) and the surfactant protein C (SFTPC) were downregulated, tubulin beta 4B (TUBB4B) was upregulated in bronchioli, compared to alveoli. In fetal tissues, CD31 was downregulated in fetal bronchioli compared to canaliculi. Moreover, we confirmed their presence using SIMPLE staining. Some expected proteins did not show up in the proteomic data, such as SOX-9, which was only detected by means of immunohistochemistry in the SIMPLE analysis.ConclusionOur data underline the robustness and applicability of this type of experimental approach, especially for rare paraffin-embedded tissue samples. It also strengthens the importance of these methods for future studies, particularly when considering developmental lung diseases, such as congenital lung anomalies.
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- 2023
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3. Morphine-induced modulation of Nrf2-antioxidant response element signaling pathway in primary human brain microvascular endothelial cells
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Sandrine Reymond, Tatjana Vujić, Domitille Schvartz, and Jean-Charles Sanchez
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Medicine ,Science - Abstract
Abstract Morphine is one of the most potent opioid analgesic used for pain treatment. Morphine action in the central nervous system requires crossing the blood–brain barrier. Due to the controversial relationship between morphine and oxidative stress, the potential pro- or antioxidant effects of morphine in the blood–brain barrier is important to be understood, as oxidative stress could cause its disruption and predispose to neurodegenerative diseases. However, investigation is scarce in human brain endothelial cells. Therefore, the present study evaluated the impact of morphine exposure at three different concentrations (1, 10 and 100 µM) for 24 h and 48 h on primary human brain microvascular endothelial cells. A quantitative data-independent acquisition mass spectrometry strategy was used to analyze proteome modulations. Almost 3000 proteins were quantified of which 217 were reported to be significantly regulated in at least one condition versus untreated control. Pathway enrichment analysis unveiled dysregulation of the Nrf2 pathway involved in oxidative stress response. Seahorse assay underlined mitochondria dysfunctions, which were supported by significant expression modulations of relevant mitochondrial proteins. In conclusion, our study revealed the dysregulation of the Nrf2 pathway and mitochondria dysfunctions after morphine exposure, highlighting a potential redox imbalance in human brain endothelial cells.
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- 2022
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4. Impact of peptide permeation enhancer on tight junctions opening cellular mechanisms
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Joël Brunner, Domitille Schvartz, Aurélie Gouiller, Alexandre Hainard, and Gerrit Borchard
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Tight junction ,PKC zeta ,L-R5 peptide ,Occludin ,Protein interaction ,Biology (General) ,QH301-705.5 ,Biochemistry ,QD415-436 - Abstract
The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any in vitro cytotoxicity, a proteomics study revealed that L-R5 interferes with other regulatory pathways, e.g., apoptosis and immune response. We suggest that structural modification of the peptide may increase the specificity TJ protein-peptide interaction.
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- 2022
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5. Oxidative Stress and Extracellular Matrix Remodeling Are Signature Pathways of Extracellular Vesicles Released upon Morphine Exposure on Human Brain Microvascular Endothelial Cells
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Tatjana Vujić, Domitille Schvartz, Izadora Liranço Furlani, Isabel Meister, Víctor González-Ruiz, Serge Rudaz, and Jean-Charles Sanchez
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extracellular vesicles ,endothelial cells ,morphine ,CNS ,BBB ,proteomics ,Cytology ,QH573-671 - Abstract
Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dysfunctional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the morphine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothelial cells as well as on their associated EVs. Isolation of EVs was carried out using an affinity-based method. Several orthogonal techniques (NTA, western blotting and proteomics analysis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modulations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 proteins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the “cell adhesion and extracellular matrix remodeling” process and the “HIF1 pathway”, a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings highlighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient circulation using these “signature pathways”.
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- 2022
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6. Gaining Insights Into Metabolic Networks Using Chemometrics and Bioinformatics: Chronic Kidney Disease as a Clinical Model
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Julien Boccard, Domitille Schvartz, Santiago Codesido, Mohamed Hanafi, Yoric Gagnebin, Belén Ponte, Fabien Jourdan, and Serge Rudaz
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metabolomics ,chemometrics ,bioinformatics ,integrative data analysis ,chronic kidney disease ,metabolic networks ,Biology (General) ,QH301-705.5 - Abstract
Because of its ability to generate biological hypotheses, metabolomics offers an innovative and promising approach in many fields, including clinical research. However, collecting specimens in this setting can be difficult to standardize, especially when groups of patients with different degrees of disease severity are considered. In addition, despite major technological advances, it remains challenging to measure all the compounds defining the metabolic network of a biological system. In this context, the characterization of samples based on several analytical setups is now recognized as an efficient strategy to improve the coverage of metabolic complexity. For this purpose, chemometrics proposes efficient methods to reduce the dimensionality of these complex datasets spread over several matrices, allowing the integration of different sources or structures of metabolic information. Bioinformatics databases and query tools designed to describe and explore metabolic network models offer extremely useful solutions for the contextualization of potential biomarker subsets, enabling mechanistic hypotheses to be considered rather than simple associations. In this study, network principal component analysis was used to investigate samples collected from three cohorts of patients including multiple stages of chronic kidney disease. Metabolic profiles were measured using a combination of four analytical setups involving different separation modes in liquid chromatography coupled to high resolution mass spectrometry. Based on the chemometric model, specific patterns of metabolites, such as N-acetyl amino acids, could be associated with the different subgroups of patients. Further investigation of the metabolic signatures carried out using genome-scale network modeling confirmed both tryptophan metabolism and nucleotide interconversion as relevant pathways potentially associated with disease severity. Metabolic modules composed of chemically adjacent or close compounds of biological relevance were further investigated using carbon transfer reaction paths. Overall, the proposed integrative data analysis strategy allowed deeper insights into the metabolic routes associated with different groups of patients to be gained. Because of their complementary role in the knowledge discovery process, the association of chemometrics and bioinformatics in a common workflow is therefore shown as an efficient methodology to gain meaningful insights in a clinical context.
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- 2021
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7. Data for Tandem Mass Tag (TMT) proteomic analysis of the pancreas during the early phase of experimental pancreatitis
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Violeta García-Hernández, Carmen Sánchez-Bernal, Domitille Schvartz, José J. Calvo, Jean-Charles Sanchez, and Jesús Sánchez-Yagüe
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Computer applications to medicine. Medical informatics ,R858-859.7 ,Science (General) ,Q1-390 - Abstract
The quantitative proteomics data reported here pertain to the research article entitled “A Tandem Mass Tag (TMT) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis” (García-Hernández et al., 2018) [1].The development of acute pancreatitis (AP, an important pathological inflammatory state of the exocrine pancreas) would be based on early changes in protein expression and signaling pathways whose unmasking would be crucial for deciphering AP at the molecular level. We reported here a Tandem Mass Tag (TMT)-based proteomics analysis of rat subcellular fractions of the pancreas during the early phase of experimental AP, using a sixplex isobaric chemical labeling technique. We identified 997 unique proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively).Accordingly, using TMT proteomics and bioinformatic tools, in García-Hernández et al., 2018- [1] we were able to detect significant changes in protein expression related to many pathobiological pathways of AP as from the early phase of the disease, including some changes never described before in this disease. Proteomics data are publicly available in ProteomeXchange via PRIDE through the identifier PXD007096. Keywords: Acute pancreatitis, Cerulein, Proteomics, Tandem Mass Tags (TMT), Shotgun proteomics
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- 2018
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8. Ubiquinone Metabolism and Transcription HIF-1 Targets Pathway Are Toxicity Signature Pathways Present in Extracellular Vesicles of Paraquat-Exposed Human Brain Microvascular Endothelial Cells
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Tatjana Vujić, Domitille Schvartz, Anton Iliuk, and Jean-Charles Sanchez
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extracellular vesicles ,endothelial cells ,paraquat ,CNS ,BBB ,proteomics ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Over the last decade, the knowledge in extracellular vesicles (EVs) biogenesis and modulation has increasingly grown. As their content reflects the physiological state of their donor cells, these “intercellular messengers” progressively became a potential source of biomarker reflecting the host cell state. However, little is known about EVs released from the human brain microvascular endothelial cells (HBMECs). The current study aimed to isolate and characterize EVs from HBMECs and to analyze their EVs proteome modulation after paraquat (PQ) stimulation, a widely used herbicide known for its neurotoxic effect. Size distribution, concentration and presence of well-known EV markers were assessed. Identification and quantification of PQ-exposed EV proteins was conducted by data-independent acquisition mass spectrometry (DIA-MS). Signature pathways of PQ-treated EVs were analyzed by gene ontology terms and pathway enrichment. Results highlighted that EVs exposed to PQ have modulated pathways, namely the ubiquinone metabolism and the transcription HIF-1 targets. These pathways may be potential molecular signatures of the PQ-induced toxicity carried by EVs that are reflecting their cell of origin by transporting with them irreversible functional changes.
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- 2021
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9. Neuroinflammatory Response to TNFα and IL1β Cytokines Is Accompanied by an Increase in Glycolysis in Human Astrocytes In Vitro
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David Pamies, Chiara Sartori, Domitille Schvartz, Víctor González-Ruiz, Luc Pellerin, Carolina Nunes, Denise Tavel, Vanille Maillard, Julien Boccard, Serge Rudaz, Jean-Charles Sanchez, and Marie-Gabrielle Zurich
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astrocytes ,astrogliosis ,reactive astrocytes ,neuroinflammation ,energy metabolism ,neuroenergetic ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1β (IL1β) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.
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- 2021
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10. Intracellular and Extracellular Markers of Lethality in Osteogenesis Imperfecta: A Quantitative Proteomic Approach
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Luca Bini, Domitille Schvartz, Chiara Carnemolla, Roberta Besio, Nadia Garibaldi, Jean-Charles Sanchez, Antonella Forlino, and Laura Bianchi
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osteogenesis imperfecta ,extracellular matrix ,cytoskeleton ,cell signaling ,bioinformatics ,REVIGO ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Osteogenesis imperfecta (OI) is a heritable disorder that mainly affects the skeleton. The inheritance is mostly autosomal dominant and associated to mutations in one of the two genes, COL1A1 and COL1A2, encoding for the type I collagen α chains. According to more than 1500 described mutation sites and to outcome spanning from very mild cases to perinatal-lethality, OI is characterized by a wide genotype/phenotype heterogeneity. In order to identify common affected molecular-pathways and disease biomarkers in OI probands with different mutations and lethal or surviving phenotypes, primary fibroblasts from dominant OI patients, carrying COL1A1 or COL1A2 defects, were investigated by applying a Tandem Mass Tag labeling-Liquid Chromatography-Tandem Mass Spectrometry (TMT LC-MS/MS) proteomics approach and bioinformatic tools for comparative protein-abundance profiling. While no difference in α1 or α2 abundance was detected among lethal (type II) and not-lethal (type III) OI patients, 17 proteins, with key effects on matrix structure and organization, cell signaling, and cell and tissue development and differentiation, were significantly different between type II and type III OI patients. Among them, some non–collagenous extracellular matrix (ECM) proteins (e.g., decorin and fibrillin-1) and proteins modulating cytoskeleton (e.g., nestin and palladin) directly correlate to the severity of the disease. Their defective presence may define proband-failure in balancing aberrances related to mutant collagen.
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- 2021
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11. Proteomic Characterization of the Olfactory Molecular Imbalance in Dementia with Lewy Bodies
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Mercedes Lachén-Montes, Naroa Mendizuri, Domitille Schvartz, Joaquín Fernández-Irigoyen, Jean Charles Sánchez, and Enrique Santamaría
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olfaction ,proteomics ,olfactory bulb ,dementia ,Lewy bodies ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Olfactory dysfunction is one of the prodromal symptoms in dementia with Lewy bodies (DLB). However, the molecular pathogenesis associated with decreased smell function remains largely undeciphered. We generated quantitative proteome maps to detect molecular alterations in olfactory bulbs (OB) derived from DLB subjects compared to neurologically intact controls. A total of 3214 olfactory proteins were quantified, and 99 proteins showed significant alterations in DLB cases. Protein interaction networks disrupted in DLB indicated an imbalance in translation and the synaptic vesicle cycle. These alterations were accompanied by alterations in AKT/MAPK/SEK1/p38 MAPK signaling pathways that showed a distinct expression profile across the OB–olfactory tract (OT) axis. Taken together, our data partially reflect the missing links in the biochemical understanding of olfactory dysfunction in DLB.
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- 2020
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12. Combined lipidomic and proteomic analysis of isolated human islets exposed to palmitate reveals time-dependent changes in insulin secretion and lipid metabolism.
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Kirsten Roomp, Hjalti Kristinsson, Domitille Schvartz, Kumari Ubhayasekera, Ernest Sargsyan, Levon Manukyan, Azazul Chowdhury, Hannes Manell, Venkata Satagopam, Karlfried Groebe, Reinhard Schneider, Jonas Bergquist, Jean-Charles Sanchez, and Peter Bergsten
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Medicine ,Science - Abstract
Studies on the pathophysiology of type 2 diabetes mellitus (T2DM) have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our findings provide insight into how saturated fatty acids contribute to islet cell dysfunction by affecting the granule maturation process and confirmation in human islets of some previous findings from rodent islet and cell-line studies.
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- 2017
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13. Fractalkine (CX3CL1), a new factor protecting β-cells against TNFα
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Sabine Rutti, Caroline Arous, Domitille Schvartz, Katharina Timper, Jean-Charles Sanchez, Emmanouil Dermitzakis, Marc Y. Donath, Philippe A. Halban, and Karim Bouzakri
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Islets ,Insulin secretion ,Myokines ,Survival ,Insulin signaling pathway ,Inflammation ,Internal medicine ,RC31-1245 - Abstract
Objective: We have previously shown the existence of a muscle–pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. Methods: Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1–50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in β-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.
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- 2014
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14. An Integrative Multi-Omics Workflow to Address Multifactorial Toxicology Experiments
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Víctor González-Ruiz, Domitille Schvartz, Jenny Sandström, Julian Pezzatti, Fabienne Jeanneret, David Tonoli, Julien Boccard, Florianne Monnet-Tschudi, Jean-Charles Sanchez, and Serge Rudaz
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metabolomics ,proteomics ,pathway analysis ,multifactorial experiments ,AMOPLS ,multiplatform omics ,toxicology ,trimethyltin ,Microbiology ,QR1-502 - Abstract
Toxicology studies can take advantage of omics approaches to better understand the phenomena underlying the phenotypic alterations induced by different types of exposure to certain toxicants. Nevertheless, in order to analyse the data generated from multifactorial omics studies, dedicated data analysis tools are needed. In this work, we propose a new workflow comprising both factor deconvolution and data integration from multiple analytical platforms. As a case study, 3D neural cell cultures were exposed to trimethyltin (TMT) and the relevance of the culture maturation state, the exposure duration, as well as the TMT concentration were simultaneously studied using a metabolomic approach combining four complementary analytical techniques (reversed-phase LC and hydrophilic interaction LC, hyphenated to mass spectrometry in positive and negative ionization modes). The ANOVA multiblock OPLS (AMOPLS) method allowed us to decompose and quantify the contribution of the different experimental factors on the outcome of the TMT exposure. Results showed that the most important contribution to the overall metabolic variability came from the maturation state and treatment duration. Even though the contribution of TMT effects represented the smallest observed modulation among the three factors, it was highly statistically significant. The MetaCore™ pathway analysis tool revealed TMT-induced alterations in biosynthetic pathways and in neuronal differentiation and signaling processes, with a predominant deleterious effect on GABAergic and glutamatergic neurons. This was confirmed by combining proteomic data, increasing the confidence on the mechanistic understanding of such a toxicant exposure.
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- 2019
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15. Paraquat-induced cholesterol biosynthesis proteins dysregulation in human brain microvascular endothelial cells
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Tatjana, Vujić, Domitille, Schvartz, and Jean-Charles, Sanchez
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- 2021
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16. Digoxin Induces Human Astrocyte Reaction In Vitro
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David Pamies, Tatjana Vujić, Domitille Schvartz, Julien Boccard, Cendrine Repond, Carolina Nunes, Serge Rudaz, Jean-Charles Sanchez, Víctor González-Ruiz, and Marie-Gabrielle Zurich
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Central Nervous System ,Cellular and Molecular Neuroscience ,Digoxin ,Neurology ,Tumor Necrosis Factor-alpha ,Astrocytes ,Neuroscience (miscellaneous) ,Humans ,Energy Metabolism - Abstract
Astrocyte reaction is a complex cellular process involving astrocytes in response to various types of CNS injury and a marker of neurotoxicity. It has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes have been reported with age, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected. However, the relationship between energy metabolism and astrocyte reactivity in the context of neurotoxicity is not known. We hypothesized that changes in energy metabolism of astrocytes will be coupled to their activation by xenobiotics. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics, and extracellular flux analyses after 24 h of exposure of human ReN-derived astrocytes to digoxin (1–10 µM) or TNFα (30 ng/ml) used as a positive control. Strong astrocytic reaction was observed, accompanied by increased glycolysis at low concentrations of digoxin (0.1 and 0.5 µM) and after TNFα exposure, suggesting that increased glycolysis may be a common feature of reactive astrocytes, independent of the triggering molecule. In conclusion, whether astrocyte activation is triggered by cytokines or a xenobiotic, it is strongly tied to energy metabolism in human ReN-derived astrocytes. Increased glycolysis might be considered as an endpoint to detect astrocyte activation by potentially neurotoxic compounds in vitro. Finally, ReN-derived astrocytes may help to decipher mechanisms of neurotoxicity in ascertaining the ability of chemicals to directly target astrocytes.
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- 2022
17. Impact of Peptide Permeation Enhancer on Tight Junctions Opening Cellular Mechanisms
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Joël, Brunner, Domitille, Schvartz, Aurélie, Gouiller, Alexandre, Hainard, and Gerrit, Borchard
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History ,Polymers and Plastics ,Biophysics ,Business and International Management ,Biochemistry ,Industrial and Manufacturing Engineering - Abstract
The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any
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- 2022
18. Morphine-induced modulation of Nrf2-antioxidant response element signaling pathway in primary human brain microvascular endothelial cells
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Sandrine Reymond, Tatjana Vujić, Domitille Schvartz, and Jean-Charles Sanchez
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Oxidative Stress ,Multidisciplinary ,Morphine ,NF-E2-Related Factor 2 ,Humans ,Brain ,Endothelial Cells ,Antioxidant Response Elements ,Antioxidants ,Signal Transduction - Abstract
Morphine is one of the most potent opioid analgesic used for pain treatment. Morphine action in the central nervous system requires crossing the blood–brain barrier. Due to the controversial relationship between morphine and oxidative stress, the potential pro- or antioxidant effects of morphine in the blood–brain barrier is important to be understood, as oxidative stress could cause its disruption and predispose to neurodegenerative diseases. However, investigation is scarce in human brain endothelial cells. Therefore, the present study evaluated the impact of morphine exposure at three different concentrations (1, 10 and 100 µM) for 24 h and 48 h on primary human brain microvascular endothelial cells. A quantitative data-independent acquisition mass spectrometry strategy was used to analyze proteome modulations. Almost 3000 proteins were quantified of which 217 were reported to be significantly regulated in at least one condition versus untreated control. Pathway enrichment analysis unveiled dysregulation of the Nrf2 pathway involved in oxidative stress response. Seahorse assay underlined mitochondria dysfunctions, which were supported by significant expression modulations of relevant mitochondrial proteins. In conclusion, our study revealed the dysregulation of the Nrf2 pathway and mitochondria dysfunctions after morphine exposure, highlighting a potential redox imbalance in human brain endothelial cells.
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- 2021
19. Old problem, new solutions: biomarker discovery for acetaminophen liver toxicity
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Caroline Flora Samer, Domitille Schvartz, and Yvonne Gloor
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Liver toxicity ,Apoptosis ,Toxicology ,Bioinformatics ,030226 pharmacology & pharmacy ,Extracellular vesicles ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Biomarker discovery ,Acetaminophen ,Pharmacology ,business.industry ,Liver cell ,digestive, oral, and skin physiology ,General Medicine ,Analgesics, Non-Narcotic ,Omics ,Mitochondria ,MicroRNAs ,Research Design ,030220 oncology & carcinogenesis ,Toxicity ,Biomarker (medicine) ,Chemical and Drug Induced Liver Injury ,business ,Biomarkers ,medicine.drug - Abstract
Introduction: Although the hepatotoxicity of acetaminophen is a well-known problem, the search for reliable biomarker of toxicity is still a current issue as clinical tools are missing to assess patients intoxicated following chronic use, sequential ingestion, use of modified release formulations or in case of delayed arrival to hospital. The need for new specific and robust biomarkers for acetaminophen toxicity has prompted many studies exploring the use of blood levels of acetaminophen derivatives, mitochondrial damage markers, liver cell apoptosis and/or necrosis markers and circulating microRNAs. Areas covered: In this review, we present a concise overview of the most promising biomarkers currently under evaluation including descriptions of their properties with respect to exposure type, APAP specificity, and potential clinical application. In addition, we illustrate the power of new technologies for biomarker research and describe their current application to the field of acetaminophen-induced hepatotoxicity. Expert opinion: Recently the use of extracellular vesicles isolation in combination with omics techniques has opened a new perspective to the field of biomarker research. However, the potential of those new technologies for the prediction and monitoring of hepatic diseases and acetaminophen toxicity has not yet been fully taken into consideration.
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- 2019
20. Intracellular and Extracellular Markers of Lethality in Osteogenesis Imperfecta: A Quantitative Proteomic Approach
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Jean-Charles Sanchez, Chiara Carnemolla, Domitille Schvartz, Antonella Forlino, Laura Bianchi, Roberta Besio, Luca Bini, and Nadia Garibaldi
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Male ,Proteome ,Pathway analysis ,Decorin ,osteogenesis imperfecta ,Proteomics ,medicine.disease_cause ,Extracellular matrix ,lcsh:Chemistry ,Tandem Mass Spectrometry ,skin and connective tissue diseases ,lcsh:QH301-705.5 ,Spectroscopy ,Mutation ,integumentary system ,cytoskeleton ,General Medicine ,bioinformatics ,Phenotype ,Computer Science Applications ,Osteogenesis imperfecta ,Child, Preschool ,Female ,Type I collagen ,musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,extracellular matrix ,macromolecular substances ,Biology ,Article ,Collagen Type I ,Catalysis ,Inorganic Chemistry ,Extracellular ,medicine ,Humans ,cell signaling ,Physical and Theoretical Chemistry ,Molecular Biology ,Bioinformatics ,Cell signaling ,Cytoskeleton ,REVIGO ,Organic Chemistry ,Infant, Newborn ,Infant ,medicine.disease ,Molecular biology ,Collagen Type I, alpha 1 Chain ,lcsh:Biology (General) ,lcsh:QD1-999 ,Biomarkers ,Chromatography, Liquid - Abstract
Osteogenesis imperfecta (OI) is a heritable disorder that mainly affects the skeleton. The inheritance is mostly autosomal dominant and associated to mutations in one of the two genes, COL1A1 and COL1A2, encoding for the type I collagen &alpha, chains. According to more than 1500 described mutation sites and to outcome spanning from very mild cases to perinatal-lethality, OI is characterized by a wide genotype/phenotype heterogeneity. In order to identify common affected molecular-pathways and disease biomarkers in OI probands with different mutations and lethal or surviving phenotypes, primary fibroblasts from dominant OI patients, carrying COL1A1 or COL1A2 defects, were investigated by applying a Tandem Mass Tag labeling-Liquid Chromatography-Tandem Mass Spectrometry (TMT LC-MS/MS) proteomics approach and bioinformatic tools for comparative protein-abundance profiling. While no difference in &alpha, 1 or &alpha, 2 abundance was detected among lethal (type II) and not-lethal (type III) OI patients, 17 proteins, with key effects on matrix structure and organization, cell signaling, and cell and tissue development and differentiation, were significantly different between type II and type III OI patients. Among them, some non&ndash, collagenous extracellular matrix (ECM) proteins (e.g., decorin and fibrillin-1) and proteins modulating cytoskeleton (e.g., nestin and palladin) directly correlate to the severity of the disease. Their defective presence may define proband-failure in balancing aberrances related to mutant collagen.
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- 2021
21. Ubiquinone Metabolism and Transcription HIF-1 Targets Pathway Are Toxicity Signature Pathways Present in Extracellular Vesicles of Paraquat-Exposed Human Brain Microvascular Endothelial Cells
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Domitille Schvartz, Jean-Charles Sanchez, Anton Iliuk, and Tatjana Vujić
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Proteomics ,Proteome ,Ubiquinone ,Endothelial cells ,paraquat ,medicine.disease_cause ,Transcription (biology) ,oxidative stress ,Biology (General) ,Hypoxia ,Spectroscopy ,ddc:616 ,Chemistry ,Brain ,General Medicine ,Human brain ,Extracellular vesicles ,endothelial cells ,Computer Science Applications ,Cell biology ,medicine.anatomical_structure ,CNS ,extracellular vesicles ,Intracellular ,Paraquat ,Ubiquinone metabolism ,ubiquinone metabolism ,DIA-MS ,QH301-705.5 ,Article ,Catalysis ,Inorganic Chemistry ,proteomics ,medicine ,Humans ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,Herbicides ,hypoxia ,Organic Chemistry ,HIF-1 ,Hypoxia-Inducible Factor 1, alpha Subunit ,Biomarker ,Oxidative stress ,Endothelium, Vascular ,BBB ,Biomarkers ,Biogenesis - Abstract
Over the last decade, the knowledge in extracellular vesicles (EVs) biogenesis and modulation has increasingly grown. As their content reflects the physiological state of their donor cells, these “intercellular messengers” progressively became a potential source of biomarker reflecting the host cell state. However, little is known about EVs released from the human brain microvascular endothelial cells (HBMECs). The current study aimed to isolate and characterize EVs from HBMECs and to analyze their EVs proteome modulation after paraquat (PQ) stimulation, a widely used herbicide known for its neurotoxic effect. Size distribution, concentration and presence of well-known EV markers were assessed. Identification and quantification of PQ-exposed EV proteins was conducted by data-independent acquisition mass spectrometry (DIA-MS). Signature pathways of PQ-treated EVs were analyzed by gene ontology terms and pathway enrichment. Results highlighted that EVs exposed to PQ have modulated pathways, namely the ubiquinone metabolism and the transcription HIF-1 targets. These pathways may be potential molecular signatures of the PQ-induced toxicity carried by EVs that are reflecting their cell of origin by transporting with them irreversible functional changes.
- Published
- 2021
22. The olfactory bulb proteotype differs across frontotemporal dementia spectrum
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Jean-Charles Sanchez, Karina Ausín, Enrique Santamaría, María Victoria Zelaya, Andrea González-Morales, Joaquín Fernández-Irigoyen, Mercedes Lachén-Montes, and Domitille Schvartz
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0301 basic medicine ,Male ,Proteomics ,Proteome ,Biophysics ,Biology ,Biochemistry ,Progressive supranuclear palsy ,03 medical and health sciences ,Ca2+/calmodulin-dependent protein kinase ,mental disorders ,medicine ,Humans ,Protein kinase A ,Protein kinase C ,Aged ,ddc:616 ,Aged, 80 and over ,030102 biochemistry & molecular biology ,nutritional and metabolic diseases ,Frontotemporal lobar degeneration ,Middle Aged ,medicine.disease ,Olfactory Bulb ,Olfactory bulb ,nervous system diseases ,030104 developmental biology ,Frontotemporal Dementia ,Female ,Supranuclear Palsy, Progressive ,Signal transduction ,Neuroscience ,Frontotemporal dementia - Abstract
Mild olfactory dysfunction has been observed in frontotemporal dementias (FTD). However, the underlying molecular mechanisms associated to this deficit are poorly understood. We applied quantitative proteomics to analyze pathological effects on the olfactory bulb (OB) from progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration (FTLD-TDP43) subjects respect to elderly non-FTD group. Our data revealed: i) a mitochondrial and calcium homeostasis impairment in PSP and ii) a disruption of protein synthesis and vesicle trafficking in FTLD-TDP43. Although differential OB proteomes clearly differ between both FTD phenotypes, functional analyses pointed out an imbalance in survival signaling in both pathologies. A common alteration of olfactory mitogen-activated protein kinases (MAPKs), calcium/calmodulin dependent protein kinase II (CAMKII), and protein kinase C (PKC) signaling pathways was observed in PSP and FTLD subjects. In contrast, a specific shut off in mitogen-activated protein kinase kinase 4 (SEK1/MKK4)/stress-activated protein kinase (SAPK) axis was exclusively observed in PSP, whereas a specific phosphoinositide-dependent protein kinase 1 (PDK1) inactivation was observed in FTLD-TDP43. In summary, our data contribute to a better understanding of the molecular mechanisms that are modulated in PSP and FTLD-TDP43 at olfactory level, highlighting cross-disease similarities and differences in the regulation of survival pathways across FTD spectrum. SIGNIFICANCE: This work reflects differential olfactory molecular disarrangements in PSP and FTLD-TDP43, two clinically similar FTD disorders, but with different neuropathological signature. Besides FTDs present mild olfactory dysfunction, our data provide basic information for understanding the implication of the OB in the pathophysiology of FTDs.
- Published
- 2019
23. 3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles
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Jean-Charles Sanchez, Sara Rocha, Domitille Schvartz, Maren Voglstaetter, Andreas R. Thomsen, Carla Oliveira, Andreas Keller, Irina Nazarenko, Nadia Walter, Joana Carvalho, Patrícia Oliveira, Richa Khanduri, and Instituto de Investigação e Inovação em Saúde
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General Chemical Engineering ,General Physics and Astronomy ,Medicine (miscellaneous) ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,3D cell culture ,Downregulation and upregulation ,microwell arrays ,microRNA ,cancer ,General Materials Science ,ddc:616 ,Cellular architecture ,Full Paper ,Chemistry ,General Engineering ,RNA ,Full Papers ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Cell biology ,integrative network analysis ,Cancer cell ,Signal transduction ,0210 nano-technology ,extracellular vesicles ,Intracellular - Abstract
The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP-ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs. S.R. and J.C. contributed equally to this work as co-first authors. C.O. and I.N. contributed equally to this work as co-senior authors. The authors thank Celso Reis and Joy Burchell for having generously shared the anti-Mucin-1 antibody, Gabriela Almeida and Mafalda Santos for sharing the H&E images from MKN45 and MKN74 tumors in mice, Maria Lazaro for the technical support with imaging flow cytometry data, Tanja Gainey-Schleicher and Elena Grueso Navarro for the support with EV–cell association assay, and Deborah Lawrie-Blum for proofreading the manuscript. S.R., R.K., J.C., C.O., A.T., A.K., and I.N. were involved in the conception and design of the study; S.R., J.C., and P.O. were involved in the establishment and characterization of 3D microwell array culture of GC cells, characterization of EVs, small RNA sequencing experiments, data analysis and validation, and functional assays; D.S., M.V., N.W., and R.K. were involved in all proteomics studies including data analysis and validation, and the analysis of cell–EV association; S.R., J.C., P.O., C.O., D.S., A.K., J.-C.S., A.T., and I.N. analyzed the data; S.R., J.C., P.O., C.O., A.K., and I.N. wrote the manuscript; S.R., J.C., P.O., C.O., A.K., and I.N. critically reviewed the manuscript; and all authors gave final approval of the manuscript.
- Published
- 2019
24. Data for Tandem Mass Tag (TMT) proteomic analysis of the pancreas during the early phase of experimental pancreatitis
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Carmen Sánchez-Bernal, Domitille Schvartz, Jesús Sánchez-Yagüe, José Julián Calvo, Jean-Charles Sanchez, and Violeta García-Hernández
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Proteomics ,0301 basic medicine ,Tandem Mass Tags (TMT) ,Quantitative proteomics ,Computational biology ,Biology ,Tandem mass tag ,lcsh:Computer applications to medicine. Medical informatics ,Cerulein ,03 medical and health sciences ,Shotgun proteomics ,medicine ,lcsh:Science (General) ,ddc:616 ,Multidisciplinary ,medicine.disease ,Acute pancreatitis ,030104 developmental biology ,medicine.anatomical_structure ,Physics and Astronomy ,Pancreatitis ,lcsh:R858-859.7 ,Signal transduction ,Pancreas ,lcsh:Q1-390 - Abstract
The quantitative proteomics data reported here pertain to the research article entitled “A Tandem Mass Tag (TMT) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis” (García-Hernández et al., 2018) [1].The development of acute pancreatitis (AP, an important pathological inflammatory state of the exocrine pancreas) would be based on early changes in protein expression and signaling pathways whose unmasking would be crucial for deciphering AP at the molecular level. We reported here a Tandem Mass Tag (TMT)-based proteomics analysis of rat subcellular fractions of the pancreas during the early phase of experimental AP, using a sixplex isobaric chemical labeling technique. We identified 997 unique proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively).Accordingly, using TMT proteomics and bioinformatic tools, in García-Hernández et al., 2018- [1] we were able to detect significant changes in protein expression related to many pathobiological pathways of AP as from the early phase of the disease, including some changes never described before in this disease. Proteomics data are publicly available in ProteomeXchange via PRIDE through the identifier PXD007096. Keywords: Acute pancreatitis, Cerulein, Proteomics, Tandem Mass Tags (TMT), Shotgun proteomics
- Published
- 2018
25. Proteomics of Diabetes, Obesity, and Related Disorders
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Loïc Dayon, David R. Goodlett, Robert Moulder, and Domitille Schvartz
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0301 basic medicine ,Proteomics ,Proteomics methods ,business.industry ,Genome, Human ,Clinical Biochemistry ,MEDLINE ,ta1182 ,medicine.disease ,Bioinformatics ,Global Health ,03 medical and health sciences ,030104 developmental biology ,Diabetes Mellitus, Type 1 ,Diabetes Mellitus, Type 2 ,Diabetes mellitus ,Global health ,Prevalence ,Medicine ,Humans ,ta318 ,Obesity ,business ,Diabetes obesity - Published
- 2018
26. Palmitate-Induced Insulin Hypersecretion and Later Secretory Decline Associated with Changes in Protein Expression Patterns in Human Pancreatic Islets
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Reinhard Schneider, Domitille Schvartz, Jean-Charles Sanchez, Ernest Sargsyan, Karlfried Groebe, Anders Alderborn, Peter Bergsten, Azazul Islam Chowdhury, Kirsten Roomp, and Jing Cen
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Male ,Proteomics ,0301 basic medicine ,insulin secretion ,type 2 diabetes mellitus ,medicine.medical_treatment ,Cell- och molekylärbiologi ,Palmitic Acid ,insulin hyposecretion ,Biochemistry ,Tissue Culture Techniques ,0302 clinical medicine ,Tandem Mass Spectrometry ,Insulin-Secreting Cells ,Insulin Secretion ,Child ,glucose-stimulated insulin secretion ,ddc:616 ,2. Zero hunger ,chemistry.chemical_classification ,geography.geographical_feature_category ,Chemistry ,in vitro ,Fasting ,Islet ,3. Good health ,human pancreatic islets ,medicine.anatomical_structure ,differential proteomic analysis ,Endokrinologi och diabetes ,childhood obesity ,Insulin processing ,medicine.medical_specialty ,Adolescent ,T2DM ,030209 endocrinology & metabolism ,Protein degradation ,Endocrinology and Diabetes ,insulin hypersecretion ,Islets of Langerhans ,03 medical and health sciences ,proteomics ,Internal medicine ,Autophagy ,medicine ,Humans ,Obesity ,geography ,Gene Expression Profiling ,Pancreatic islets ,Insulin ,Calcium-Binding Proteins ,Computational Biology ,Type 2 Diabetes Mellitus ,Fatty acid ,General Chemistry ,bioinform atic analysis ,GSIS ,In vitro ,Glucose ,030104 developmental biology ,Endocrinology ,Diabetes Mellitus, Type 2 ,Gene Expression Regulation ,Proteolysis ,palmitate ,Cell and Molecular Biology ,Chromatography, Liquid - Abstract
In obese children with high circulating concentrations of free fatty acid palmitate, we have observed that insulin levels at fasting and in response to a glucose challenge were several times higher than in obese children with low concentrations of the fatty acid as well as in lean controls. Declining and even insufficient insulin levels were observed in obese adolescents with high levels of the fatty acid. In isolated human islets exposed to palmitate we have observed insulin hypersecretion after 2 days exposure. In contrast, insulin secretion from the islets was reduced after 7 days culture in the presence of the fatty acid. This study aims at identifying islet-related biological events potentially linked with the observed insulin hypersecretion and later secretory decline in these obese children and adolescents using the islet model. We analyzed protein expression data obtained from human islets exposed to elevated palmitate levels for 2 and 7 days by an improved methodology for statistical analysis of differentially expressed proteins. Protein profiling of islet samples by liquid chromatography-tandem mass spectrometry identified 115 differentially expressed proteins (DEPs). Several DEPs including sorcin were associated with increased glucose-stimulated insulin secretion in islets after 2 days of exposure to palmitate. Similarly, several metabolic pathways including altered protein degradation, increased autophagy, altered redox condition, and hampered insulin processing were coupled to the functional impairment of islets after 7 days of culture in the presence of palmitate. Such biological events, once validated in the islets, may give rise to novel treatment strategies aiming at normalizing insulin levels in obese children with high palmitate levels, which may reduce or even prevent obesity-related type 2 diabetes mellitus.
- Published
- 2018
27. The human diabetes proteome project (HDPP): The 2014 update
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Loïc Dayon, Ali Tiss, Andreas Wiederkehr, Young Ah Goo, Salvatore Sechi, James Cantley, Pascale Gaudet, Pierre Fontana, Kathleen M. Snyder, A. Konvalinka, Patrik Bergsten, Ioannis Xenarios, Hindrik Mulder, Jean-Charles Sanchez, Martin Kussmann, Feliciano Priego-Capote, A. Barba De La Rosa, Francesco Finamore, Kwang-Hyun Baek, David R Goodlett, Robert Moulder, Domitille Schvartz, and J. D. Johnson
- Subjects
Engineering ,Knowledge management ,030303 biophysics ,Library science ,Disease ,ta3111 ,Biochemistry ,03 medical and health sciences ,Diabetes mellitus ,Health care ,medicine ,Human proteome project ,American population ,ta318 ,ddc:576 ,030304 developmental biology ,American diabetes association ,ddc:616 ,0303 health sciences ,business.industry ,ta1182 ,medicine.disease ,3. Good health ,Cell and molecular biology ,Proteome ,ddc:540 ,business - Abstract
Diabetes is an increasing worldwide problem leading to major associated health issues and increased health care costs. In 2012, 9.3% of the American population was affected by diabetes, according to the American Diabetes Association, with 1.7 million of new cases since during the year (www.diabetes.org). Proteome initiatives can provide a deeper understanding of the biology of this disease and help develop more effective treatments. The collaborative effort of the Human Diabetes Proteome Project (HDPP) brings together a wide variety of complementary resources to increase the existing knowledge about both type 1 and type 2 diabetes and their related complications. The goals are to identify proteins and protein isoforms associated with the pathology and to characterize underlying disease-related pathways and mechanisms. Moreover, a considerable effort is being made on data integration and network biology. Sharing these data with the scientific community will be an important part of the consortium. Here we report on: the content of the HDPP session held at the 12th HUPO meeting in Yokohama; recent achievements of the consortium; discussions of several HDPP workshops; as well as future HDPP directions as discussed at the 13th HUPO congress in Madrid, with a special attention given to the lists of prioritized, diabetes-related proteins and the proteomic means to study them. (Less)
- Published
- 2015
28. A tandem mass tag (TMT) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis
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Domitille Schvartz, Violeta García-Hernández, Jean-Charles Sanchez, Jesús Sánchez-Yagüe, José Julián Calvo, and Carmen Sánchez-Bernal
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0301 basic medicine ,Whole membrane ,Male ,Proteomics ,Proteome ,Biophysics ,Biology ,Tandem mass tag ,Biochemistry ,Mitochondrial Proteins ,03 medical and health sciences ,Animals ,Rats, Wistar ,Shotgun proteomics ,ddc:616 ,Proteomic Profiling ,Cell biology ,Transport protein ,Mitochondria ,Rats ,030104 developmental biology ,Membrane protein ,Pancreatitis ,Acute Disease ,Lysosomes ,Ceruletide - Abstract
Changes in the protein expression occurring within the initiation phase of acute pancreatitis (AP) might be vital in the development of this complex disease. However, the exact mechanisms involved in the onset of AP remains elusive and most of our knowledge about the pathobiology of AP comes from animal models. We performed in a rat pancreatitic model a high-throughput shotgun proteomic profiling of the soluble and whole membrane fractions from the pancreas during the early phase of cerulein (Cer)-induced AP. We identified 997 proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively). Gene Ontology and KEGG PATHWAY analyses revealed that these proteins were implicated in molecular mechanisms relevant to AP pathogenesis, including vesicle-mediated and protein transport, lysosomal and mitochondrial impairment or proteolysis. Numerous metabolic processes were downregulated apparently to reduce energy consumption, and a remarkable increase in inflammatory and stress responses was also highlighted. The proteomic data were verified by immunoblotting of 11 and 7 different soluble or membrane-associated proteins, either novel (VPS29 and MCTS1) or known factors in AP. Also, our first observation of the imbalance of some COP proteins during AP early phase deserves further characterization. Biological significance AP is one of the most important pathological inflammatory states of the exocrine pancreas but its pathophysiology remains incompletely understood, especially the early acinar events. Proteomic analysis of pancreatic subcellular fractions simplifies protein maps and helps in the identification of new protein alterations and biomarkers characterizing pancreatic tissue damage. Our shotgun approach has not been previously used to profile the early proteomic alterations of the disease, which are considered crucial for its development and for the founding of clinical procedures. Furthermore, our subcellular fractionation protocol allowed us to detect changes in membrane proteins so far overlooked in the proteomic study of AP. Accordingly, using TMT proteomics and bioinformatic tools, we were able to detect significant changes in protein expression related to many pathobiological pathways of acute pancreatitis as from the early phase of the disease. To our knowledge, some of these changes, such as the imbalance of some COP proteins, have never been described in this disease.
- Published
- 2017
29. Quantitative proteomics reveals the link between minichromosome maintenance complex and glucose-induced proliferation of rat pancreatic INS-1E β-cells
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Domitille Schvartz, Jean-Charles Sanchez, Yohann Couté, Département de science des protéines humaines [Genève], Université de Genève (UNIGE)-Faculté de médecine [Genève], Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université de Genève = University of Geneva (UNIGE)-Faculté de médecine [Genève], Laboratoire d'étude de la dynamique des protéomes (LEDyP), and Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
- Subjects
Proteomics ,[SDV]Life Sciences [q-bio] ,medicine.medical_treatment ,Quantitative proteomics ,Biophysics ,030209 endocrinology & metabolism ,Biology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Minichromosome maintenance ,Cell Line, Tumor ,Insulin-Secreting Cells ,Stable isotope labeling by amino acids in cell culture ,Insulin Secretion ,medicine ,Animals ,Insulin ,Glucose homeostasis ,Inducer ,ddc:576 ,Nuclear protein ,ComputingMilieux_MISCELLANEOUS ,Cell Proliferation ,030304 developmental biology ,0303 health sciences ,Minichromosome Maintenance Proteins ,Cell cycle ,Rats ,Glucose ,Sweetening Agents - Abstract
Proper functioning of pancreatic β-cells is a crucial for glucose homeostasis control, and therefore a main problem regarding type 2 diabetes onset and evolution. The ability of β-cells to proliferate upon certain stimuli, such as elevated glucose concentration, is an essential property to overpass a major problem of the pathology: the decrease of β-cell mass leading to a lack of insulin production. However, high glucose concentrations are also an inducer of β-cell dysfunction, when proliferation become unable to overcome insulin demand. The control of β-cell proliferation could represent an interesting target for the development of therapeutic molecules for type 2 diabetes treatment. To get new insights on β-cell replication, we investigated the modulation of nuclear proteins of INS-1E cells submitted to medium and high glucose concentrations for 24 h. Indeed, the nucleus should contain proteins responsible of proliferation-related events. The SILAC approach allowed us identifying 24 nuclear proteins whose expressions were modified by chronic high glucose. A wide Downstream Effects Analysis assigned the majority of the differentially expressed proteins to functions such as proliferation and cell cycle. Interestingly, our study linked for the first time the increase of expression of the 6 MCM components to glucose-induced stimulation in β-cells. Biological significance The current study represents a progress in the understanding of glucose-induced proliferation mechanisms in β-cells. We applied the SILAC strategy to INS-1E cells cultivated with medium or high glucose concentrations for 24 h, and we targeted nuclear proteins which have a central role in proliferation-related mechanisms. It allows quantifying 24 nuclear proteins, which are regulated by high glucose exposure. The vast majority of them are shown to be related to proliferation and cell cycle. We describe here for the first time than the 6 proteins of the MCM complex are involved in glucose-mediated proliferation in β-cells.
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- 2014
30. Characterization of the platelet granule proteome: Evidence of the presence of MHC1 in alpha-granules
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Domitille Schvartz, Pierre Fontana, Séverine Nolli, Jean-Charles Sanchez, Anne Zufferey, and Jean-Luc Reny
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Adult ,Blood Platelets ,Male ,Proteome ,Antigen presentation ,Biophysics ,Cytoplasmic Granules ,Proteomics ,Biochemistry ,Mass Spectrometry ,Antigen ,MHC class I ,Humans ,Platelet ,ddc:576 ,ddc:616 ,Antigen Presentation ,biology ,Histocompatibility Antigens Class I ,Granule (cell biology) ,Blood Proteins ,Transporter associated with antigen processing ,3. Good health ,Cell biology ,ddc:618.97 ,Immunology ,biology.protein ,Signal Transduction - Abstract
UNLABELLED In the present study we performed an extensive qualitative characterization of the platelet granule proteome using subcellular fractionation followed by mass spectrometry analysis and functional annotation. Eight hundred and twenty seven proteins were identified most of them being associated to granules and to the granule's secretory machinery. Functional pathway analysis revealed 30 pathways including the major histocompatibility complex class 1 (MHC I) presenting antigen pathway. This pathway was of particular interest for its potential interrelation between platelets and the immune system. Key proteins belonging to this metabolic route such as ß 2 microglobulin 26S protease regulatory subunit 10B from the proteasome and proteins 1 and 2 of the transporter associated with antigen processing were shown to co localize with von Willebrand factor in resting platelets and to be located on the plasma membrane when platelets were activated. Key proteins of the MHC1 antigen presenting pathway are located in platelet alpha granules. These results suggest a possible functional role of platelet granules in platelet related immune modulation. BIOLOGICAL SIGNIFICANCE In this study we described the largest dataset related to platelet granule proteins. We performed a functional pathway analysis that evidenced several expected granule related pathways. We also highlighted the "Antigen processing and presentation" pathway that has drawn our attention. Using immunofluorescence technique we confirmed the presence of several key proteins for antigen presentation in platelet granules. This study suggests a putative functional role of MHC1 and platelet granules in the immune modulation.
- Published
- 2014
31. The Human Diabetes Proteome Project (HDPP): From network biology to targets for therapies and prevention
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Pascale Gaudet, Natacha Turck, Francesco Finamore, Jean-Charles Sanchez, Peter Bergsten, David R. Goodlett, Pierre Fontana, Anne Zufferey, Domitille Schvartz, F. Topf, Feliciano Priego-Capote, Ioannis Xenarios, Pierre-Alain Binz, Martin Kussmann, and Andreas Wiederkehr
- Subjects
Islet ,Abnormal glucose ,Disease ,030204 cardiovascular system & hematology ,Biology ,Bioinformatics ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Diabetes mellitus ,Beta-Cell ,Human proteome project ,medicine ,Obesity ,ddc:576 ,Fatty acids ,030304 developmental biology ,ddc:616 ,0303 health sciences ,Diabetes ,Biomedical information ,Human proteome ,Systems approaches ,medicine.disease ,3. Good health ,Glucose ,Proteome ,Biological network - Abstract
Type 2 diabetes is a worldwide disease reaching epidemic dimensions. The rapid progression of the disease urgently calls for both a broader and deeper understanding of its pathophysiology. In line with this statement, the Human Diabetes Proteome Project (HDPP) was officially launched at the 11th HUPO meeting in Boston, 2012. A special session was dedicated to this new initiative, gathering experts in the main topics related to diabetes and its associated complications. Key issues were debated with a focus on how deranged circulating glucose and free fatty acids induce dysfunction. It has been decided that HDPP will therefore focus on studying the early stages of diabetes that lead to abnormal glucose and lipid levels. The initiative will initially focused on islets of Langerhans, insulin-producing cell lines, and blood human samples from diabetes-related cohorts. In subsequent stages HDPP will investigate target tissues in which glucose and lipids could promote protein dysfunctions. Omics-rooted systems approaches enhanced by bioinformatics will be deployed to unravel effects of lipids and glucose triggering diabetes initiation and progression. A first milestone has been defined for the 12th HUPO meeting in Yokohama, 2013: the 1000 diabetes-associated protein (the 1000-HDPP) database, i.e. a freely available internet resource ( www.HDPP.info ) of more than 1000 proteins with links to their corresponding proteotypic peptides, affinity reagents and protein-specific biological/biomedical information.
- Published
- 2013
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32. Improved characterization of the insulin secretory granule proteomes
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Jean-Charles Sanchez, Yannick Brunner, Yohann Couté, Claes B. Wollheim, Michelangelo Foti, and Domitille Schvartz
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Proteomics ,Proteome ,Biophysics ,Biology ,Biochemistry ,Exocytosis ,03 medical and health sciences ,Cell Line, Tumor ,Insulin-Secreting Cells ,Stable isotope labeling by amino acids in cell culture ,Animals ,Insulin ,Glucose homeostasis ,Secretion ,ddc:576 ,ddc:612 ,030304 developmental biology ,0303 health sciences ,Secretory Vesicles ,030302 biochemistry & molecular biology ,virus diseases ,Rats ,Diabetes Mellitus, Type 2 ,Biogenesis ,Insulin processing - Abstract
Insulin secretory granules (ISGs) are pivotal organelles of pancreatic ß-cells and represent a key participant to glucose homeostasis. Indeed, insulin is packed and processed within these vesicles before its release by exocytosis. It is therefore crucial to acquire qualitative and quantitative data on the ISG proteome, in order to increase our knowledge on ISG biogenesis, maturation and exocytosis. Despites efforts made in the past years, the coverage of the ISG proteome is still incomplete and comprises many potential protein contaminants most likely coming from suboptimal sample preparations. We developed here a 3-step gradient purification procedure combined to Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to further characterize the ISG protein content. Our results allowed to build three complementary proteomes containing 1/ proteins which are enriched in mature ISGs, 2/ proteins sharing multiple localizations including ISGs, and finally 3/ proteins sorted out from immature ISGs and/or co-purifying contaminants. As a proof of concept, the ProSAAS, a neuronal protein found in ISGs was further characterized and its granular localization proved. ProSAAS might represent a novel potential target allowing to better understand the defaults in insulin processing and secretion observed during type 2 diabetes progression. This article is part of a special issue entitled: Translational Proteomics.
- Published
- 2012
33. Modulation of Neuronal Pentraxin 1 Expression in Rat Pancreatic β-Cells Submitted to Chronic Glucotoxic Stress
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Claes B. Wollheim, Yannick Brunner, Domitille Schvartz, Yohann Couté, and Jean-Charles Sanchez
- Subjects
Proteomics ,Nerve Tissue Proteins/genetics/metabolism ,Time Factors ,Proteome ,Pyridines ,medicine.medical_treatment ,Proteome/genetics/metabolism ,Gene Expression ,Type 2 diabetes ,medicine.disease_cause ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,Glycogen Synthase Kinase 3 ,Signal Transduction/drug effects ,Insulin-Secreting Cells ,Insulin Secretion ,Proto-Oncogene Proteins c-akt/metabolism ,Insulin ,Pyrimidines/pharmacology ,Pyridines/pharmacology ,Reverse Transcriptase Polymerase Chain Reaction ,Insulin-Secreting Cells/drug effects/metabolism/pathology ,Glucose/pharmacology ,Immunohistochemistry ,C-Reactive Protein ,Glycogen Synthase Kinase 3/antagonists & inhibitors/metabolism ,Proteomics/methods ,Signal Transduction ,Insulin processing ,medicine.medical_specialty ,Blotting, Western ,Nerve Tissue Proteins ,Gene Expression/drug effects ,Biology ,Islets of Langerhans ,Cell Line, Tumor ,Internal medicine ,Secretory Vesicles/drug effects/metabolism ,medicine ,Animals ,Hyperglycemia/genetics/metabolism ,Secretion ,ddc:576 ,Molecular Biology ,Protein kinase B ,Insulin/secretion ,Islets of Langerhans/drug effects/metabolism ,Gene Expression Profiling ,Secretory Vesicles ,Research ,C-Reactive Protein/genetics/metabolism ,medicine.disease ,Rats ,Glucose ,Pyrimidines ,Endocrinology ,Apoptosis ,Hyperglycemia ,Gene Expression Profiling/methods ,Proto-Oncogene Proteins c-akt ,Oxidative stress ,Homeostasis - Abstract
Insulin secretory granules are β-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic β-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in β-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain β-cell dysfunction. To identify the potential early molecular mechanisms underlying β-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma β-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in β-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new β-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific β-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and β-cell failure, observed after prolonged exposure to high glucose concentrations.
- Published
- 2012
34. Early activation of the fatty acid metabolism pathway by chronic high glucose exposure in rat insulin secretory β-cells
- Author
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Jean-Charles Sanchez, Domitille Schvartz, Yannick Brunner, Yohann Couté, Claes B. Wollheim, Frédérique Lisacek, Céline Hernandez, and Alexandre Masselot
- Subjects
Proteomics ,medicine.medical_specialty ,Time Factors ,Proteome ,medicine.medical_treatment ,Carbohydrate metabolism ,Biology ,Glucose/*metabolism ,Biochemistry ,Cell Line ,Proteome/*analysis/genetics/metabolism ,Insulin-Secreting Cells ,Internal medicine ,Stable isotope labeling by amino acids in cell culture ,Insulin Secretion ,medicine ,Insulin ,Animals ,Glucose homeostasis ,ddc:576 ,Molecular Biology ,Cell Proliferation ,Insulin-Secreting Cells/*chemistry/cytology/*metabolism/secretion ,Insulin/*secretion ,Cell growth ,Gene Expression Profiling ,Fatty Acids ,Fatty Acids/*metabolism ,Metabolism ,Rats ,Glucose ,Endocrinology ,Cell culture ,Lipogenesis - Abstract
Pancreatic beta-cells are responsible for insulin secretion that regulates blood glucose homeostasis. In the development of type II diabetes, a progressive impairment of insulin secretion by the pancreatic beta-cells occurs called beta-cell dysfunction or beta-cell failure. Chronic hyperglycemia has been shown being involved in beta-cell dysfunction, a phenomenon known as glucotoxicity. The molecular mechanisms underlying the impairment of insulin secretion by beta-cells induced by glucotoxicity are still not fully understood. In this work, quantitative proteomics was employed to identify early key players involved in beta-cell dysfunction induced by glucotoxicity. For this, the stable isotope labeling by amino acids in cell culture strategy was used on the slowly-growing rat beta-cell line INS-1E. We showed that the stable isotope labeling by amino acids in cell culture approach did not induce any detectable biological effects on these beta-cells, as measured at both the transcriptomic and proteomic levels. Proteins differentially expressed between control cells and cells submitted to chronic high glucose concentrations were identified and verified. The results obtained reinforce the link between glucotoxicity and lipogenesis and suggest that the fatty acid metabolism pathway may rapidly be stimulated in beta-cells submitted to chronic high glucose concentrations.
- Published
- 2010
35. Glucotoxicity and pancreatic proteomics
- Author
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Jean-Charles Sanchez, Feliciano Priego-Capote, Yohann Couté, Yannick Brunner, and Domitille Schvartz
- Subjects
Adipose Tissue/metabolism ,Liver/metabolism ,Chronic hyperglycaemia ,medicine.medical_specialty ,Proteome ,Insulin/metabolism ,Proteome/metabolism ,Biophysics ,Type 2 diabetes ,Proteomics ,Bioinformatics ,Biochemistry ,Islets of Langerhans ,Internal medicine ,Diabetes mellitus ,medicine ,Insulin ,Animals ,Brain/metabolism ,Humans ,Glucose homeostasis ,Secretion ,ddc:576 ,Glucagon/metabolism ,Pancreas ,Muscles/metabolism ,Pancreas/metabolism ,business.industry ,Muscles ,Pancreatic islets ,Brain ,Glucagon ,medicine.disease ,Glucose ,medicine.anatomical_structure ,Endocrinology ,Adipose Tissue ,Liver ,Diabetes Mellitus, Type 2 ,Glucose/metabolism/toxicity ,Islets of Langerhans/metabolism ,business - Abstract
Chronic hyperglycaemia is one of the main characteristics of a diabetic state. This is also the first cause of diabetic complications. However, it is now generally accepted that glucotoxicity also participates in the worsening of type 2 diabetes, by affecting the secretion of beta-cells. So far, different mechanisms have been proposed to explain the adverse effects of chronic hyperglycaemia. One of them suggests that the modulation of expression of several key proteins during a hyperglycaemia state, may explain the toxic effect of glucotoxicity. Therefore, proteomic analysis of biological samples represents an interesting method to study the effect of chronic hyperglycaemia on protein expression. The discovery of new proteins for which the expression could be modulated by chronic hyperglycaemia may probably help to better understand the mechanisms underlying glucotoxicity. In this review, we will first present an introduction of the different mechanisms known to be involved in the control of glucose homeostasis and in the development of glucotoxicity. In a second part, some proteomic data linked with the effect of glucotoxicity in pancreas, pancreatic islets and beta-cells will be presented and discussed.
- Published
- 2009
36. New molecular insights into modulation of platelet reactivity in aspirin-treated patients using a network-based approach
- Author
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Anne Zufferey, Domitille Schvartz, Mylène Docquier, Mark Ibberson, Séverine Nolli, Ioannis Xenarios, Pierre Fontana, Jean-Luc Reny, and Jean-Charles Sanchez
- Subjects
0301 basic medicine ,Blood Platelets ,Proteomics ,Candidate gene ,030204 cardiovascular system & hematology ,Biology ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,ddc:590 ,Genetics ,Humans ,Platelet ,ddc:576.5 ,ddc:610 ,RNA, Messenger ,ddc:576 ,Gene ,Genetics (clinical) ,Aspirin ,Phenotype ,Molecular medicine ,Human genetics ,3. Good health ,MicroRNAs ,030104 developmental biology ,ddc:540 - Abstract
Platelet reactivity (PR) is variable between individuals and modulates clinical outcome in cardiovascular (CV) patients treated with antiplatelet drugs. Although several data point to a genetic control of platelet reactivity, the genes contributing to the modulation of this phenotype are not clearly identified. Integration of data derived from high-throughput technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study is to identify candidate genes modulating platelet reactivity in aspirin-treated CV patients using an integrative network-based approach. Patients with extreme high (n = 6) or low PR (n = 6) were selected and data derived from quantitative proteomic of platelets and platelet sub-cellular fractions, as well as from transcriptomic analysis were integrated with a network biology approach. Two modules within the network containing 123 and 182 genes were identified. We then specifically assessed the level of miRNAs in these two groups of patients. Among the 12 miRNAs differentially expressed, 2 (miR-135a-5p and miR-204-5p) correlated with PR. The predicted targets of these miRNAs were mapped onto the network, allowing the identification of seven overlapping genes (THBS1, CDC42, CORO1C, SPTBN1, TPM3, GTPBP2, and MAPRE2), suggesting a synergistic effect of these two miRNAs on these predicted targets. Integration of several omics data sets allowed the identification of 2 candidate miRNAs and 7 candidate genes regulating platelet reactivity in aspirin-treated CV patients.
- Published
- 2015
37. Combined lipidomic and proteomic analysis of isolated human islets exposed to palmitate reveals time-dependent changes in insulin secretion and lipid metabolism
- Author
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Venkata P. Satagopam, Hjalti Kristinsson, Domitille Schvartz, Ernest Sargsyan, Peter Bergsten, Kumari Ubhayasekera, Reinhard Schneider, Levon Manukyan, Kirsten Roomp, Jean-Charles Sanchez, Hannes Manell, Azazul Islam Chowdhury, Karlfried Groebe, and Jonas Bergquist
- Subjects
Male ,Proteomics ,0301 basic medicine ,Time Factors ,endocrine system diseases ,Physiology ,medicine.medical_treatment ,Palmitates ,Gene Expression Regulation/drug effects ,lcsh:Medicine ,Biochemistry ,Lipid Metabolism/drug effects ,Islets of Langerhans/drug effects/metabolism/secretion ,Database and Informatics Methods ,Endocrinology ,0302 clinical medicine ,Insulin Secretion ,Medicine and Health Sciences ,Macromolecular Structure Analysis ,Insulin ,Glucose/metabolism ,lcsh:Science ,Proinsulin ,Lipid Analysis ,Multidisciplinary ,geography.geographical_feature_category ,Proteomic Databases ,Organic Compounds ,Fatty Acids ,Monosaccharides ,Middle Aged ,Islet ,Lipids ,Chemistry ,Cholesterol ,medicine.anatomical_structure ,Lipotoxicity ,Physical Sciences ,Endokrinologi och diabetes ,Female ,Palmitates/pharmacology ,Research Article ,medicine.medical_specialty ,Carbohydrates ,030209 endocrinology & metabolism ,Endocrinology and Diabetes ,Biology ,Research and Analysis Methods ,Islets of Langerhans ,03 medical and health sciences ,Internal medicine ,Lipid biosynthesis ,Lipidomics ,medicine ,Humans ,ddc:576 ,Molecular Biology ,Diabetic Endocrinology ,Insulin/secretion ,geography ,Endocrine Physiology ,Pancreatic islets ,Organic Chemistry ,lcsh:R ,Chemical Compounds ,Biology and Life Sciences ,Lipid metabolism ,Proinsulin/metabolism ,Lipid Metabolism ,Diabetes Mellitus, Type 2/metabolism ,Hormones ,Glucose ,Biological Databases ,030104 developmental biology ,Diabetes Mellitus, Type 2 ,Gene Expression Regulation ,lcsh:Q - Abstract
Studies on the pathophysiology of type 2 diabetes mellitus (T2DM) have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our findings provide insight into how saturated fatty acids contribute to islet cell dysfunction by affecting the granule maturation process and confirmation in human islets of some previous findings from rodent islet and cell-line studies.
- Published
- 2017
38. Fractalkine (CX3CL1), a new factor protecting β-cells against TNFα
- Author
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Philippe A. Halban, Marc Y. Donath, Caroline Arous, Emmanouil T. Dermitzakis, Katharina Timper, Domitille Schvartz, Sabine Rütti, Karim Bouzakri, and Jean-Charles Sanchez
- Subjects
Chemokine ,lcsh:Internal medicine ,endocrine system ,Survival ,Inflammation ,Myokine ,medicine ,ddc:576.5 ,ddc:576 ,CX3CL1 ,lcsh:RC31-1245 ,Molecular Biology ,geography ,geography.geographical_feature_category ,biology ,business.industry ,Insulin secretion ,Insulin signaling pathway ,Skeletal muscle ,Cell Biology ,Islet ,Cell biology ,medicine.anatomical_structure ,Apoptosis ,Immunology ,biology.protein ,Myokines ,Tumor necrosis factor alpha ,Original Article ,medicine.symptom ,Islets ,business - Abstract
© 2014 The Authors. Objective: We have previously shown the existence of a muscle pancreas intercommunication axis in which CX3CL1 (fractalkine) a CX3C chemokine produced by skeletal muscle cells could be implicated. It has recently been shown that the fractalkine system modulates murine ß cell function. However the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine induced apoptosis remains to be investigated. Methods: Gene expression was determined using RNA sequencing in human islets sorted ß and non ß cells. Glucose stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1 50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted ß cells exposed to CX3CL1 for 48 h alone or in the presence of TNFa (20 ng/ml). Rat and human ß cell apoptosis (TUNEL) and rat ß cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However CX3CL1 is more expressed in non ß cells than in ß cells while its receptor is more expressed in ß cells. CX3CL1 decreased human (but not rat) ß cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted ß cell GSIS. However CX3CL1 completely prevented the adverse effect of TNFa on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt AS160 paxillin) and expression (IRS2 ICAM 1 Sorcin PCSK1) of key proteins involved in these processes. Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover CX3CL1 decreases basal apoptosis of human ß cells. We further demonstrate that CX3CL1 protects ß cells from the adverse effects of TNFa on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.
- Published
- 2014
39. Inhibition of MAPKs and type IV phosphodiesterase during the early phase of acute pancreatitis: A shotgun proteomics approach
- Author
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José Julián Calvo, Jean-Charles Sanchez, Domitille Schvartz, Violeta García-Hernández, Jesús Sánchez-Yagüe, and Carmen Sánchez-Bernal
- Subjects
medicine.medical_specialty ,Hepatology ,Endocrinology, Diabetes and Metabolism ,Gastroenterology ,Phosphodiesterase ,Pharmacology ,Biology ,medicine.disease ,Pathogenesis ,Endocrinology ,Apoptosis ,Internal medicine ,medicine ,Acute pancreatitis ,cAMP-dependent pathway ,Shotgun proteomics ,Rolipram ,medicine.drug ,Calcium signaling - Abstract
Introduction: Early changes in protein expression and signaling mechanisms (MAPKs cascade or the cAMP pathway) in acute pancreatitis (AP) play important roles in the further development of the disease. Therefore, proteomic analyses of pancreatic subcellular fractions might be very useful for deciphering AP at the molecular level. Aims:WereportedhereaTandemMassTag(TMT)proteomicsanalysisof the soluble fraction of the pancreas during the early phase of experimental AP and under MAPKs or type IV phosphodiesterase (PDEIV) inhibition. Materials & methods: The early phase of AP was induced in rats by two subcutaneous injections of 20 mg of cerulein (Cer)/kg body weight at hourly intervals. PDEIV and the MAPKs cascade were inhibited by rolipram and SP600125 or PD98059, respectively, before the induction of AP. TMT 6 analysis weredesignedtocharacterizeeachexperimentalconditioninacross-sectional study. Changes in protein expression were validated by immunoblotting. Results: Out of the around 540 identified proteins, 298 were differentially expressed during the early phase of AP (Isobar, p < 0.05). Also, we identified 27 (SP600125), 39 (PD98059) or 38 (rolipram) proteins differentially regulated by the distinct pretreatments compared to the Cer class. These proteins are mainly involved in protein and membrane trafficking, apoptosis, mitochondrial function, calcium signaling and cytoskeleton impairment. We are currently characterizing novel candidates that might be relevant to AP pathogenesis. Conclusion: These data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP and might unmask new potential biomarkers of the disease.
- Published
- 2015
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