Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes
Project description:The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.
Project description:Nutrigenomics analysis was used to investigate the molecular responses to dietary Cu deficiency independently and in combination with 30% (w/w) sucrose in a mature rat model of NAFLD. Low Cu significantly decreased hepatic and serum Cu, and induced NAFLD-like histopathology, mild steatosis, up-regulated transcripts in inflammation and hepatic stellate cell activation, and significantly increased oxidative stress. Rats fed low Cu together with 30% sucrose also developed insulin resistance, increased ATP citrate lyase and FASN expression, and greater oxidative stress. High sucrose with adequate Cu also promoted inflammation and fibrosis, but not steatosis. This study indicates that low dietary Cu and sucrose consumption are singular and synergistic dietary factors in promotion of NAFLD and NASH that act independently of obesity or severe steatosis, likely by promoting oxidative stress and activation of inflammation and fibrosis.
Project description:RawTools is a software that provides parsing and quantification of raw Thermo Orbitrap mass spectrometer data. RawTools software was used to process a subset of injections (n = 10) from a prepared HeLa digest that were analyzed on an Orbitrap Velos to get instrument performance metrics.
Project description:RawTools is a software that provides parsing and quantification of raw Thermo Orbitrap mass spectrometer data. RawTools software was used to process a set of injections (n = 140) from a prepared HeLa digest that were analyzed on an Orbitrap Velos to get summarized instrument performance metrics for quality control.
Project description:Nutrigenomics analysis was used to investigate the molecular responses to dietary Cu deficiency independently and in combination with 30% (w/w) sucrose in a mature rat model of NAFLD. Low Cu significantly decreased hepatic and serum Cu, and induced NAFLD-like histopathology, mild steatosis, up-regulated transcripts in inflammation and hepatic stellate cell activation, and significantly increased oxidative stress. Rats fed low Cu together with 30% sucrose also developed insulin resistance, increased ATP citrate lyase and FASN expression, and greater oxidative stress. High sucrose with adequate Cu also promoted inflammation and fibrosis, but not steatosis. This study indicates that low dietary Cu and sucrose consumption are singular and synergistic dietary factors in promotion of NAFLD and NASH that act independently of obesity or severe steatosis, likely by promoting oxidative stress and activation of inflammation and fibrosis. Mature (6 months old) male Wistar Rats that had been allowed ad libitum access to Mazuri rodent pellets were used in the study. Twenty-four rats were divided into four groups and fed for 12 weeks with diets based on the Purified AIN76A formulation, modified for target sucrose and Cu content (Custom Animal Diets, Bangor, NJ). Sucrose and copper content in diets were as follows: ‘A’ CuD/30%- Cu deficient (<0.3 mg Cu/kg)/30% sucrose, ‘B’ CuA/30%- Cu adequate (125 mg/kg)/30% sucrose, ‘C’ CuD/10%- <0.3 mg/kg Cu/10% sucrose, and ‘D’ CuA (125 mg/kg Cu)/10% sucrose (control). Starch and dextrin were used to equalize carbohydrates.
Project description:Topoisomerase 1 (TOP1) poisons like camptothecin (CPT), which are used as chemotherapeutic agents in cancer, elicit DNA damage in quiescient neurons. In this study, we examined the effects of CPT and actinomycin D (ActD) on neuronal cells. Motor (MNs) and cortical (CNs) neurons were more susceptible to the toxic effects of CPT and ActD than fibroblasts. MNs and CNs exhibited a delayed DNA damage response—increase in nuclear γ-H2AX foci—relative to fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts which could explain their enhanced vulnerability to CPT and ActD toxicity. Microarray analysis was performed to identify differentially regulated transcripts in MNs treated with CPT for 2 hours. Many immediate-early genes including Fos and Egr-1 were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cells types treated with CPT; Egr-1 transcript levels, however, were reduced in CPT-treated fibroblasts even though they were elevated in treated MNs and CNs. Pathway and network analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. In conclusion, MNs were more vulnerable than fibroblasts to the damaging effects of TOP1 poisons and they elicit a unique intracellular response to CPT treatment.
Project description:We used digital gene expression (NlaIII sequence tags) and RNA-Seq to compare the transcriptomes of Cu-replete vs. Cu–deficient Chlamydomonas wild-type cells to reveal dozens of mRNAs whose abundance is modified. Half of the corresponding genes are targets of CRR1, a master regulator of nutritional copper sensing, and are associated with candidate CRR1 binding sites. The targets include many plastid-localized proteins, like FDX5 encoding a ferredoxin isoform, and CGL78, encoding a protein conserved in the green lineage, indicative of modified plastid metabolism. Immunoblot analysis and proteome profiles recapitulate the transcriptome profiles. New evidence for Cu sparing is suggested by up-regulation of AOF1 encoding a copper-independent but flavin-dependent amine oxidase and down-regulation of two metal- binding proteins. Genes encoding redox proteins, many of which function in lipid metabolism, are over-represented, which is compatible with the role of Cu in biology. Lipid profiles indicate a CRR1-dependent increase in Cu-deficient cells in the proportion of unsaturated (16:2, 16:3, 16:4, 18:2) fatty acids at the expense of the more saturated (16:0, 16:1, 18:0) precursors, especially on plastid galactolipids, which validates the increased expression of acyl-ACP and plastid-localized w-6 desaturases. CRR1-independent changes in the transcriptome suggest a role for Cu in oxygen sensing in Chlamydomonas. Sampling of Chlamydomonas CC-1021 (2137) and crr1-2, crr1:CRR1 mutant cells (the mutant is knock-down for the transcription factor crr1, which plays a key role in the transcriptional response to copper levels) cultivated in TAP or minimal medium under Cu-sufficient (control) and Cu-defficient conditions. poly-A purification, NlaIII digestion/random fragmentation