Project description:Proteomics analysis of late human pre-40S particles purified using a catalytically-inactive RIO1 as bait. Analysis of RIO1(kd)-StHA pre-40S particles composition upon RPS26 depletion with siRNA.
Project description:The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression. Keywords: ordered
Project description:Combined therapy with anti-BRAF plus anti-MEK is currently used as first-line treatment of patients with metastatic melanomas harboring the somatic BRAF V600E mutation. However, the main issue with targeted therapy is the acquisition of tumor cell resistance. In 70% of resistant melanoma cells, the resistant process consists in epithelial-to-mesenchymal transition (EMT). This process called phenotype switching makes melanoma cells more invasive. Its signature is characterized by MITF low, AXL high, and actin cytoskeleton reorganization through RhoA activation. In parallel of this phenotype switching phase, the resistant cells exhibit an anarchic cell proliferation due to hyper-activation of the MAP kinase pathway. We show that a majority of human melanoma overexpress discoidin domain receptor 2 (DDR2) after treatment. The same result was found in resistant cell lines presenting phenotype switching compared to the corresponding sensitive cell lines. We demonstrate that DDR2 inhibition induces a decrease in AXL expression and reduces stress fiber formation in resistant melanoma cell lines. In this phenotype switching context, we report that DDR2 control cell and tumor proliferation through the MAP kinase pathway in resistant cells in vitro and in vivo. Therefore, inhibition of DDR2 could be a new and promising strategy for countering this resistance mechanism.
Project description:FBXL6 is E3 ubiquitin ligase that we suspected to be involved in mitochondrial functions. We have investigated how deletion of this E3s by CRSIPR/Cas in Hela cells affects the cellular proteome. We compared this proteome to cells treated with CRISPR Non-Targeting Control (control cells).
Project description:FBXL6 is a E3 ubiquitin ligase that we suspected to be involved in mitochondrial functions. We have investigated the specific interactome of this E3 by transfecting HEK cells with Flagged-FBXL6, or with b-gal (control). After flag immunoprecipiation, specific interacting proteome was identified by mass spectrometry and discriminate from control IP.
Project description:Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 as an efficient inducer of this mechanism. We characterized the different stages and the molecular regulators of entosis induced after silencing of Rnd3. We demonstrated that this process is dependent on RhoA/ROCK pathway, but independent on E-cadherin. The proteomic analysis of entotic cells allowed us to identify LAMP-1 as a protein implicated not only in the degradation final stage of entosis, but also in the full mechanism. By analyzing entosis in human hepatocellular carcinoma samples, we found a relationship between the presence of entotic cells and the metastatic potential of tumors. Altogether, these data suggest the involvement of entosis in liver tumor progression and highlight a new perspective for the entosis analysis in medicine research as a novel therapeutic target.
Project description:Background: Diclofenac is a non-steroidal anti-inflammatory drug frequently found in the aquatic environment. Previous studies have reported histological changes in the liver, kidney and gills of fish at concentrations similar to those measured in treated sewage effluents (~1 µg/L). Analyses or predictions of blood plasma levels in fish allow a direct comparison with human therapeutic plasma levels, and may therefore be used to indicate a risk for pharmacological effects in fish. To relate internal exposure to a pharmacological interaction we therefore investigated global hepatic gene expression together with bioconcentration to blood plasma and liver of rainbow trout (Oncorhynchus mykiss) exposed to waterborne diclofenac. Results: At the highest exposure (81.5µg/L) the fish plasma concentration reached ~88% of the human therapeutic levels (Cmax) after two weeks. Using an oligonucleotide microarray followed by quantitative PCR we found extensive effects on hepatic gene expression at this concentration, and some genes were regulated down to the lowest concentration tested (1.6 µg/L) corresponding to ~1.5% of the human Cmax. Functional analysis of differentially expressed genes revealed effects on biological processes such as inflammation and immune response, in agreement with the expected mode of action of diclofenac. In contrast to some previously reported results, the bioconcentration factor was found to be stable (4.02±0.75 for blood plasma and 2.54±0.36 for liver) regardless of the water concentration. Conclusions: Hepatic gene expression is affected in fish at blood levels similar to and below the human Cmax. This adds confidence to the use of fish blood plasma levels to predict risks for pharmacological responses in fish. The microarray results indicate similarities in the mode of action of diclofenac between humans and fish. The identified set of regulated genes may contain useful exposure biomarkers to be used in complex exposure scenarios. Juvenile rainbow trout was exposed to waterborne diclofenac at a series of different concentrations for 14 days. A flow-through system was used for eight aquaria (two per concentration), each containing 12 trouts. Nominal exposure concentrations: 1µg/L, 10 µg/L, 100 µg/L and control. Total RNA for biotinylated aRNA synthesis was extracted from the liver. Samples of aRNA from two fish from the same aquarium were pooled before the assay. In total, four biochips were analyzed corresponding to 32 microarrays. As we used two aquaria per concentration, these 32 microarrays allowed the analyses of four aRNA pools (eight fish) per aquarium, which is equal to eight pools per concentration of diclofenac.
Project description:Hepatocellular adenomas (HCA) are rare benign tumours of the liver which have been extensively classified at the clinico-pathological, genetic and proteomic levels. The b-HCA subtype has several types of mutations in the ß-catenin gene (CTNNB1) with very different clinical phenotypes and risks. Recently, we revealed a glutamine synthetase (GS) positive rim in b-HCA exon 3 S45 and b-HCA exon 7/8, indicating protein expression heterogeneity into b-HCA in comparison with the rest of the tumor. A difference in vascularization had been associated with this rim revealed by a diffuse CD34 labeling only at the center of the tumor and not in the rim. In this study, we sought to characterize this tumor heterogeneity in a tumor considered monoclonal. We used laser microdissection to analyze the rim (R) at the molecular level. We cut 1mm² areas of GS+/CD34- rim on serial cuts and we analyzed in parallel, the mutation status of CTNNB1 and the levels of protein expression by mass spectrometry. Comparison of the proteomic profiles of the rim versus the tumor center (T) revealed a sufficiently large protein expression differential to distinguish them by principal component analysis. Forty proteins were significantly differentially expressed between R and T, including a large majority of amino acid and lipid metabolism proteins whose deregulations were comparable to a perivenous expression profile in a normal hepatocyte context. Our results suggest that protein expression in the rim seems not to be dependent on the mutational status of CTNNB1 but rather on the influence of venous drainage. Thanks to laser microdissection coupled with proteomic analysis on fixed tissue, we were able to analyze this tumor heterogeneity and provide elements of biological interpretation.
Project description:Biopsies are underrated and underused and our goal was to demonstrate that their inherent expression proteomic pattern could give them an added value for diagnosis. As proof of concept, we used as model hepatocellular adenomas (HCA), well characterized benign liver tumors. From a collection of 260 cases, we selected 55 typical cases to build the first HCA proteomic database. Biopsies proteomic patterns allowed HCA classification, even for complex cases. In addition, these data gave access to a malignancy pattern identifying the HCA transformation. This pioneering work proposes a proteomic based machine learning tool, operational on fixed biopsies, to improve HCA diagnosis and therefore patient’s management.