Project description:Gene transcription was compared in blood between noninfected mice and infected mice after 6h of intraperitoneal infection by Neisseria meningitidis
Project description:Gene transcription was compared in brain between noninfected mice and infected mice after 6h of intraperitoneal infection by Neisseria meningitidis
Project description:ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues, but glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is labile and remains challenging to detect using conventional mass spectrometry (MS)-based workflows. Using synthetic peptides, we show that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, and by hydrolysis via wildtype Af1521. We developed an acidic enrichment workflow incorporating an Af1521 mutant that preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis. In cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp- and >200 Cys-ADPr sites. Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, contrasting with nuclear Ser-ADPr. Quantitative profiling revealed reproducible, cell type- and treatment-specific patterns. PARP10-mediated Glu/Asp ADPr of ubiquitin indicates direct crosstalk with ubiquitin signaling pathways. Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues. Together, our work establishes a robust MS-based workflow and provides a resource of site-specific ADPr events, revealing residue-specific ADPr in innate immune signaling.
Project description:ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues, but glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is labile and remains challenging to detect using conventional mass spectrometry (MS)-based workflows. Using synthetic peptides, we show that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, and by hydrolysis via wildtype Af1521. We developed an acidic enrichment workflow incorporating an Af1521 mutant that preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis. In cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp- and >200 Cys-ADPr sites. Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, contrasting with nuclear Ser-ADPr. Quantitative profiling revealed reproducible, cell type- and treatment-specific patterns. PARP10-mediated Glu/Asp ADPr of ubiquitin indicates direct crosstalk with ubiquitin signaling pathways. Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues. Together, our work establishes a robust MS-based workflow and provides a resource of site-specific ADPr events, revealing residue-specific ADPr in innate immune signaling.
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.
Project description:We integrated metabolome and proteome profiles of the parental cell line 143B.TK- versus ρ0, including PTM analyses such as phosphorylation and ubiquitination to characterize the impact of the absence of mtDNA for the entire cell. For quantitative proteome profiling, we used a shotgun LC-MS/MS approach including the classical SILAC labeling. For comprehensive metabolome profiling, we applied a targeted LC-MS approach, based on multiple reaction monitoring (MRM).</br></br>Our study revealed that mtDNA depletion leads to a non-uniform down-regulation of the mitochondrial energy metabolism in ρ0 cells on the proteome level. Metabolites of the TCA cycle were highly dysregulated which in turn had an impact on the amino acid levels, which were up regulated. Perturbation of the mitochondrial energy metabolism could lead to an activation of the retrograde response, indicated by sets of up-regulated signaling pathways in ρ0 cells, further supported by altered phosphorylation in signaling pathways and the cytoskeleton as well as de-ubiquitination of SLC transporters.
Project description:The transcriptome of early local kidney inflammation was profiled. Rat kidney tubules were injected with uropathogenic E. coli or PBS control. After incubation time, kidney samples were processed and transcription was assayed. Three rats were used per condition.