Project description:In recent years, more attention in systems biology has been given to the concept of protein constraints and the cell’s necessity to allocate its proteome between important processes. From this point of view, attempts to elucidate cellular maximum capacity of growth as a function of protein availability has been investigated. Elucidating the possibility of optimizing cell proliferation, by tailoring proteome allocation. To experimentally investigate this concept further we cultivated Saccharomyces cerevisiae in bioreactors with or without amino acid supplementation and performed proteomics to analyze global changes in proteome allocation, during anaerobic as well as aerobic growth on glucose. Analysis of proteomic data implies that proteome mass is mainly being re-allocated from amino acid biosynthetic processes into translation, in regard to absolute levels of change, accompanied by an increased growth rate during supplementation. Similar findings were obtained from both aerobic and anaerobic cultivations, and subsequently independent of the two examined metabolic states. Indicating the possibility of increasing growth rate through freeing up proteome mass and increasing proteome allocation towards translational machinery.
Project description:Here we quantitatively describe the influence of cell growth rate and amino acid metabolic context on gene expression in the eukaryal model organism Saccharomyces cerevisiae. We show that growth rate and metabolic cues regulate ~70% of the yeast transcriptome and proteome, thereby exerting gene expression control in a global manner. We find that the growth rate-dependent differential gene expression largely reflects changing availabilities of the mRNA and protein synthesis machineries, while metabolic cues influences gene expression through the availabilities of amino acids and nucleotides. Genes in central carbon metabolism, however, are regulated independently of these global physiological controls, demonstrating distinct mechanisms to control their expression levels.
Project description:Tandem mass tag (TMT)-based relative quantification was used in combination with the intensity-based absolute quantity estimation (IBAQ) to study of both yeast cells and isolated yeast mitochondria during fermentative growth, diauxic shift, respiratory growth.
Project description:Pseudomonas aeruginosa is a major opportunistic pathogen causing a wide range of infections and one of the most problematic bacteria regarding antibiotic resistance, with an increasing incidence of multidrug and extensively-drug resistant strains, including resistance to last resource antibiotics such as carbapenems. Resistances are often due to complex interplays of naturally and acquired resistance mechanisms which are enhanced by its remarkably large regulatory network. Thus, the use of non-targeted shotgun methodologies such as mass spectrometry-based proteomics is crucial to understand these interplays and to reveal possible strain and species-specific novel mechanisms of antibiotic resistance. The aim of this study was to determine the proteomic response of two carbapenem-resistant and extensively-drug-resistant P. aeruginosa strains to subminimal inhibitory concentrations (sub-MICs) of meropenem. The strains belonged to high-risk clones ST235 and ST395, one carrying a class 1 integron-encoded VIM-4 metallo-β-lactamase and one carrying no acquired antibiotic resistance genes. Each strain was cultivated with three different sub-MICs of meropenem, and a quantitative shotgun proteomic approach was applied, using tandem mass tag (TMT) isobaric labeling followed by nano-liquid chromatography tandem-mass spectrometry, to determine significantly up- or down-regulated proteins and enriched groups of proteins and pathways. Cultivation of both strains with ½ and ¼ of the MIC, resulted in hundreds of differentially regulated proteins, including several β-lactamases, transport-related proteins (including multiple porins and efflux pumps), proteins associated with peptidoglycan metabolism and cell wall organization and dozens of regulatory proteins. Remarkably, all components of the H1 type VI secretion system were up-regulated in one of the strains. Enrichment analyses revealed that multiple metabolic pathways were affected. Additionally, numerous proteins of unknown function were also differentially-regulated in each strain. In conclusion, high subminimal-inhibitory concentrations of meropenem cause massive changes in the proteomes of carbapenem-resistant P. aeruginosa strains, involving a wide range of common and strain-specific mechanisms and proteins, many still uncharacterized which might potentially play a role in the susceptibility of P. aeruginosa to meropenem.
Project description:The plasmids introduced into the E. coli cells affect the expression of chromosomal genes. Therefore we aimed at comparing the protein expression profiles of a strain not containing any plasmid with strains that do carry plasmids.
Project description:Tandem mass tag (TMT)-based relative quantification was used to study yeast metabolism under the stepwise reduction the nitrogen content in the growth medium.
Project description:The spread of antibiotic resistance has developed to all known antibiotics. Extended spectrum β-lactamase-producing bacteria are particularly problematic, as they are resistant to a wide range of commonly used antibiotics. Resistance to β-lactams is known to be multifactorial, although the underlying mechanisms generally are poorly understood but critical factors for effective therapy against infections, especially for multi-resistant pathogenic bacteria. In the present study, a plasmid-based homologous recombination system was used to target and delete specific β-lactamase genes (i.e., the blaOXA-1, blaTEM-1 or the ESBL blaCTX-M15) of the clinical strain ESBL Escherichia coli CCUG 73778, generating three “knock-out” clone variants, each one lacking only one of the β-lactamases. The objective was to determine the genotypic impacts of each gene loss on the phenotypic antibiotic resistance and proteome of the bacterium. Quantitative proteomic analyses performed on the three clone variants and the original strain, using tandem mass tags (TMT) and bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Variation of the proteome in each clone variant was determined, to establish the relative importance of each resistance gene and better understand the genetic and proteomic responses and mechanisms of the resistance phenotypes. The knockout of blaCTX-M-15 was observed to have the greatest impact in protein expression, with the knockout of blaOXA-1 also effecting a marked but lower degree of changes. Proteins known to be associated with antibiotic resistance, cell membrane integrity, cellular stress, gene expression and hypothetical/unknown function proteins, among others, demonstrated distinct differences in expression levels (Fold change >1-5 or <-1.5), that may be related to aspects of compensation for the mutant resistance phenotypes. The present study provides a framework to study the impacts of targeted loss of antibiotic resistance genes in clinically relevant strains for understanding the mechanisms of phenotypic antibiotic resistance.
Project description:Aberrant activation of Anaplastic Lymphoma Kinase (ALK) drives neuroblastoma (NB). Previous work has identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK driven gene signature in NB. To further address the role of RET in NB, RET knock-out (KO) SK-N-AS cells were generated by CRISPR/Cas9 genome engineering. Gene expression analysis of RET KO NB cells identified a reprogramming of NB cells to a mesenchymal (MES) phenotype that was characterized by increased migration and upregulation of the AXL and MET RTKs as well as integrins and extracellular matrix components. Strikingly, the upregulation of AXL in the absence of RET reflects the development timeline observed in the neural crest as progenitor cells undergo differentiation during embryonic development. Together, these findings suggest that a MES phenotype is promoted in mesenchymal NB cells in the absence of RET, reflective of a less differentiated developmental status.
Project description:Maturation of insulin is crucial for insulin secretion and function. ENPL[1]1/GRP94/HSP90B1 plays an important role in this process. ASNA-1/TRC40/GET3 and ENPL-1/GRP94 are conserved insulin secretion regulators in Caenorhabditis elegans and mammals and mouse mutants display type 2 diabetes. ENPL-1 and GRP94 bind proinsulin and regulate proinsulin levels in C. elegans and cultured cells. Here we found that ASNA-1 and ENPL-1 co-operated to regulate insulin secretion in worms via a physical interaction that required pro-DAF-28/insulin but occurred independently of the insulin binding site of ENPL-1. ASNA-1 acted in neurons to promote DAF-28/insulin secretion. The interaction occurred in insulin expressing neurons and was sensitive to changes in pro-DAF-28 levels. The chaperone form of ASNA-1 is likely bound to ENPL-1. Loss of asna-1 disrupted Golgi trafficking pathways. ASNA-1 localization was affected in enpl-1 mutants and ENPL-1 overexpression partially bypassed ASNA[1]1 requirement. Taken together, we find a functional interaction between ENPL-1 and ASNA-1 which is necessary to maintain proper insulin secretion in C. elegans and provides insights about how their loss might produce diabetes in mammals.
Project description:Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor of atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigated its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduced lipid accumulation and suppressed activation of inflammatory and fibrotic pathways as well as lowered oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hindered the shift from a contractile to a synthetic phenotype. Together, we provide the first evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.