Project description:The aim of the study was to adapt the CysPAT technique to efficiently identify and characterize endogenous S-nitrosylation in cells under physiological conditions of an experiment from a small amount of sample. Using a macrophage cell line RAW 264.7 we compared the efficacy of the method in the identification of total endogenous S-nitrosylation and the corresponding modification of the samples treated with exogenous SNO donor (GSNO) or with DTT. We identified 999 unique formerly S-nitrosylated peptides (SIA peptides) and 965 unique SNO sites from untreated RAW cells which belong to 569 endogenous nitrosylated proteins. We discovered 579 novel S-nitrosylation sites and identified 232 proteins as new S-nitrosylation targets. A total of 1450 S-nitrosylated peptides belonging to 795 proteins were identified in samples treated with GSNO (n = 3). Interestingly, we found a large overlap (>53%) of S-nitrosylated peptides in both subsets of samples, demonstrating a high sensitivity of the method to analyze endogenous S-nitrosylation. The large number of identified endogenous S-nitrosylated peptides allowed the identification of two nitrosylation consensus sites, and to highlight protein translation and redox processes as key S-nitrosylation targets in macrophages.
Project description:Cysteine nitrosylation is emerging as important player in cellular signaling and redox homeostasis. Here we applied Cys-BOOST for quantitative analysis of nitrosylated cysteine in SNAP-treated and non-treated SH-SY5Y human neuroblastoma cells.
Project description:2-day-old soybeans were treated with flooding and flooding with aluminum oxide nanoparticles of three varying sizes. Total proteins extracted from root including hypocotyl and mitochondrial proteins extracted from root tip were analyzed by nanoLC-MS/MS.
Project description:S-nitrosothiol Resin Assisted Capture(SNORAC) coupled with mass spectrometry analysis has been used to study S-nitrosylated proteins in rat cortical neurons. Proteins were captured on the resin by covalently attaching their former S-NO moiety. Then, nistrosylated proteins were eluted by on-bead digestion. Formerly S-nitrosylated peptides were also eluted from the beads in a second step, and analyzed separately for assessment of the nitrosylation site (qualitative analysis). Out of over 600 nitrosyltated proteins, 131 proteins have never been shown to be S-nitrosylated in any system and 612 are new targets of S-nitrosylation in cortical neurons. The site(s) of Snitrosylation were also identified for 59% of the targets.
Project description:Summary The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by other posttranslational modifications, such as S-nitrosylation. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach (Palmitoylation And Nitrosylation Interplay Monitoring), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteines in a mouse model of chronic stress. Our results suggest that atypical mechanism of crosstalk between the S-palmitoylation and S-nitrosylation of synaptic proteins might be one of the major events associated with chronic stress disorders.
Project description:NO performs its biological roles mainly through protein S-nitrosylation, but pathogenic roles of protein S-nitrosylation in PDAC remains largely unexplored. In this study, we identified large number of unique S-nitrosylation sites and proteins in PDAC patients and PANC-1 cells by a site-specific proteomics.
Project description:U2OS cells were stably transfected with an ecotropic receptor expression plasmid. These cells were infected with retroviruses expressing MIZ-1 or MYC and subsequently superinfected with retroviruses expressing p14ARF. Selection was carried out 48h following superinfection and selected cells were harvested within 1 - 2 passages. For each condition around 2.5x10<superscript6> cells were pooled.<br>Using the two color Quick-Amp labelling kit (Agilent, 5190-0444) 100ng of total RNA were used for cDNA synthesis, aRNA amplification and labelling according to manufacturer's instructions.<br>Transcriptional profiling was done on a whole human genome oligo microarray (Agilent, G4112F, 014850) in a 4x44k slide format.
Project description:The activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but the precise roles of lysosomes are still unclear. Here we show the vital role of the Ragulator complex, a lysosomal protein, in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophage and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed remarkable attenuation of the severity of NLRP3-associated inflammatory diseases, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with histone deacetylase 6 (HDAC6) during NLRP3 inflammasome activation, and this interaction augmented the interaction between the Ragulator complex and NLRP3. Lack of HDAC6 attenuated the interaction between Lamtor1 and NLRP3, resulting in insufficient NLRP3 inflammasome activation. Furthermore, DL-all-rac-α-tocopherol inhibited Lamtor1–HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. Our results provide insight into the role of lysosomes in providing a platform for the activation of NLRP3 inflammasomes by the Ragulator complex.
Project description:Soyfoods have been drawn the interrest in the roles that reducing risk of cardiovascular disease. Among various components, isoflavones have been come to the attention as beneficial soy ingredients. To evaluate the effectiveness of isoflavone content in dietary soybean (Glycine max) on modulating lipid metabolism, hepatic gene expressions involved in lipid metabolism were analyzed in rats. An isoflavone-rich cultivar (Yukipirika) and a conventional cultivar (Fukuyutaka) were employed. A principal component analysis (PCA) of microarray data was used to summarize characteristics of the experimental groups. As a result, the characteristics of the diets were largely explained by the first principal component (PC1). Soybean content in the diets distinctly separated in PC1. In contrast, isoflavone content had little effect on the mRNA expression. The GeneChip data was normalized and summarized by using SuperNORM data service (Skylight Biotech Inc.). Significance of expressional change among groups was tested by 2-way ANOVA on the normalized CEL data, which was deposited in a tab-separated ASCII text format. Principal components were identified on the summarized gene data. Male Splague-Dawley rats were randomly divided into 4 groups and fed an experimental diet for 21 days. The experimental diets were as follows: a diet containing 200 g/kg casein (Cas, n=7); a diet containing two proteins derived equally from casein and Fukuyutaka (conventional cultiver soybean) (F10, n=7); a diet containing two proteins derived from casein and Yukipirika (high-isoflavone soybean) in the proportion of three parts to one (Y05, n=7); a diet containing two proteins derived equally from casein and Yukipirika (Y10, n=8). In each of the groups, five rats with average food intake were used for microarray analysis.
Project description:The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were explored through a robust investigative strategy based on joint expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, associated metabolic changes with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and mitochondrial functions and results in severe ER stress. Results are discussed in the frame of the current mechanistic knowledge existing on AF.