Project description:Identification of murine targets of SNO-CoA-mediated S-nitrosylation following treatment of mouse kidney lysate with SNO-CoA in combination with recombinant WT or K127A mutant SNO-CoA reductase.
Project description:Human prion diseases are fatal neurodegenerative disorders characterized by neuronal damage in brain. Protein S-nitrosylation, the covalent adduction of a NO to cysteine, plays a role in human brain biology, and brain dysfunction is a prominent feature of pPrion disease, yet the direct brain targets of S-nitrosylation are largely unknown. We described the first proteomic analysis of global S-nitrosylation in brain tissues of sporadic Creutzfeldt–Jakob disease (sCJD), fatal familial insomnia (FFI) and genetic CJD with a substitution of valine for glycine at codon 114 of the prion protein gene (G114V gCJD) accompanying with normal control with isobaric tags for relative and absolute quantitation (iTRAQ) combined with a nano-HPLC/Q Exactive Mass spectrometry platform. In parallel, we used several approaches to provide quality control for the experimentally defined S-nitrosylated proteins. Total 1509 S-nitrosylated proteins (SNO-proteins) were identified, and cerebellum tissues appeared to contain more commonly differentially expressed SNO-proteins (DESPs) than cortex of sCJD, FFI and gCJD. Three selected SNO-proteins were verified by Western blots, consistent with proteomics assays. Gene ontology analysis showed that more up-regulated DESPs were involved in metabolism, cell cytoskeleton/structure and immune system both in cortex and cerebellum, while more down-regulated ones in both regions were involved in cell cytoskeleton/structure, cell-cell communication and miscelaneous function protein. Pathway analysis suggested that systemic lupus erythematosus, pathogenic Escherichia coli infection, extracellular matrix-receptor interaction were the most commonly affected pathways, which were identified from at least two different diseases. Using STRING database, the network of immune system and cell cytoskeleton and structure were commonly identified in the context of the up-regulated and down-regulated DESPs, respectively, both in cortex and cerebellum. Our study thus have implications for understanding the molecular mechanisms of human prion diseases related to abnormal protein S-nitrosylation and pave the way for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.
Project description:It is clearly established that the maternal diet during pregnancy can induce physiological and metabolic adaptations in the developing fetus which determine its susceptibility later in life to develop diabetes, obesity... The molecular and genomic mechanisms underlying the programming of the metabolic syndrome remain largely unknown but may involve resetting of epigenetic marks and fetal gene expression. We analyzed the profile of the liver methylome in 21-day-old rats born to mothers fed with a standard diet or a diet lacking methyl donor nutrients (Vitamin B12 and folates) during gestation and lactation. Modifications of DNA methylation were found in the promoter regions of 1,032 genes out of 14,981 genes. Bioinformatics analysis revealed that these genes are mainly involved in glucose and lipid metabolism, nervous system, coagulation, endoplasmic reticulum (ER) stress and mitochondrial function. MDD induced modifications of methylation in rat liver were measured in 21-day-old rats born to dams fed with standard food or food deficient in methyl group donor. Six independent experiments were performed (3 controls versus 3 MDD).
Project description:The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells.
Project description:RNA Antisense Purification and Mass Spectrometry (RAP-MS) was performed to explore the potential RNA binding proteins of lncRNA KIMAT1
Project description:The "prion-like" features of Alzheimer’s disease (AD) tauopathy and its relationship with amyloid β (Aβ) has never been experimentally studied in primates phylogenetically close to humans. We injected 17 macaques in the entorhinal cortex with nanograms of tau proteopathic seeds purified from AD brains or control extracts from aged-matched healthy brains, with or without intracerebroventricular co-injections of recombinant Aβ oligomers. After neuropathological examination of the macaque’s brain, we also performed mass spectrometry-based proteomics of macaques’ entorhinal cortex and CA1 to study the spatial response (local and distant) to tau seeds injections (and its modulation by oligomeric Aβ).
Project description:In this study, we systematically evaluated reduction and alkylation of proteins using three reducing agents (DTT, TCEP, and BME) in combination with four alkylation agents (IAA, IAC, AA, and CAA). We tested these conditions using strong anion exchange (SAX) fractionated in-solution digests and in-gel digested fractions of samples enriched for cytosolic proteins of HeLa cells. We analyzed the data using Proteome Discoverer 2 in combination with the MASCOT search engine with different combinations of variable and fixed modifications as well as error tolerant and mass tolerant searches. Furthermore, we compared IAA and AA alkylated samples by dimethyl labeling and performed multi stage activation triggered by the neutral loss of alkylated methionine side chains. This led to the identification of prominent offside alkylation due to iodine containing reagents at tyrosine, serine, threonine, histidine, lysine, methionine, aspartic and glutamic acid as well as the peptide N-terminus with varying abundances based on the reduction and alkylation reagents used.
Project description:We investigated the effect of the T4 MotB protein on E. coli gene expression. E. coli BL21 (DE3) containing either pNW129 or pNW129-MotB were grown to early log phase (OD600 ~ 0.3) then induced with 0.2% arabinose for 20 minutes. T4 phage added to the culture at MOI10. Cells were then harvested at 5 min.
Project description:Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to the organism and cause irreparable damage, for example during sepsis, when a “cytokine storm” can lead to multiple organ failure and often ends in death. One of the strongest triggers of inflammatory response is bacterial lipopolysaccharide (LPS), acting mostly through Toll-like receptor 4 (TLR4). Prior or prolonged exposure to LPS, however, can induce the state of “endotoxin tolerance” where the macrophages and monocytes do not respond to new endotoxin challenge. The cellular mechanisms regulating this phenomenon remain elusive. Our comprehensive secreted protein analysis comparing LPS-tolerant and -responsive monocyte/macrophage-like cells combined with the extracellular flux analysis reveals the robust switch from the inflammatory to metabolic protein expression as well as points to the involvement of specific proteins that may regulate the change from the responsive to the tolerant state