Project description:This project characterizes the apoplast proteome of Ryegrass (Lollium perenne) under treatment with different epiphyte strains (Epichloe festucae).
Project description:Regulated intracellular proteolysis is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. Altered proteolytic substrate turnover has been associated with various glomerular diseases ranging from diabetic nephropathy to focal and segmental glomerulosclerosis. However, thus far it has not been possible to systematically identify proteolytically cleaved proteins although some of the proteases have been characterized. Here we applied TAILS to identify N-termini in rat glomeruli and their changes on PAN-induced injury.
Project description:The project aimed to characterize the function of metacaspase 3 in Arabidopsis thaliana. This data compared MC3-knockout and MC3-overexpressing mutants to Col wild type.
Project description:For chondrogenic studies of optogenetically activated TGF-β signaling, optogenetic human iPSC-derived MSCs were encapsulated in hydrogels (20 million cells/mL of 2% agarose hydrogel). Groups received either no soluble TGF-β or optogenetic stimulation, or soluble TGF-β3 alone, or optogenetic stimulation alone. After 21 days of differentiation, we performed global quantitative proteomics on samples from two independent experiments, with n=3 replicates per group.
Project description:The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium ~100 million years ago, independently from plastids in plants and algae. Despite its more recent origin, at least one third of the chromatophore proteome consists of nucleus-encoded proteins that are imported by an unknown mechanism across the chromatophore double envelope membranes. Chromatophore-targeted proteins fall into two classes. Proteins exceeding 250 amino acids carry a conserved N-terminal sequence extension, termed the ‘chromatophore transit peptide’ (crTP), that is presumably involved in guiding these proteins into the chromatophore. Short imported proteins do not carry discernable targeting signals. To explore whether the import of protein is accompanied by their N-terminal processing, here we used a mass spectrometry-based approach to determine protein N-termini in Paulinella chromatophora and identified N-termini of 208 chromatophore-localized proteins. Our study revealed extensive N-terminal modifications by acetylation and proteolytic processing in both, the nucleus and chromatophore-encoded fraction of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Our results imply that the crTP mediates trafficking through the Golgi, is bipartite and surprisingly only the N-terminal third (‘part 1’) becomes cleaved upon import, whereas the rest (‘part 2’) remains at the mature proteins. In contrast, short imported proteins remain largely unprocessed. Finally, this work sheds light on N-terminal processing of proteins encoded in an evolutionary-early-stage photosynthetic organelle and suggests host-derived post-translationally acting factors involved in dynamic regulation of the chromatophore-encoded chromatophore proteome.
Project description:Hydrogen can be an important source of energy for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface. Nevertheless, the current knowledge of microbial hydrogen utilization in acidic environments is minimal. A multi-omics analysis was applied on Acidithiobacillus ferrooxidans growing aerobically and anaerobically (with ferric iron) on hydrogen as an electron donor, and a respiratory model proposed from the results obtained. In this model, both [NiFe] hydrogenases, cytoplasmic uptake and membrane-bound respiratory, oxidize molecular hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated [FeS]-binding proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome aa3 oxidase via cytochrome bc1 complex and cytochrome c4 or the alternate directly to cytochrome bd oxidase, resulting in proton efflux together with the reduction of molecular oxygen to water. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome c (ferric iron reductase) via cytochrome bc1 complex and a cascade of electron transporters (cytochrome c4, cytochrome c552, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux together with the reduction of ferric iron to ferrous iron. The proton gradient generated by molecular hydrogen oxidation maintains the membrane potential and allows the generation of ATP via ATP synthase and NADH via NADH-ubiquinone oxidoreductase. To a lesser extent, NADH can also be generated by another bidirectional cytoplasmic hydrogenase. ATP and NADH are further utilized in the Calvin–Benson–Bassham cycle for inorganic carbon uptake and assimilation. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition and features of the deep terrestrial subsurface from the distant past to the present.
Project description:MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in C. elegans, two conserved serine/threonine kinases - Casein Kinase 1 alpha 1 (CK1A1) and Casein Kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing
Project description:HeLa cell line is frequently used in biomedical research, however little is known about N-glycan structures expressed on individual glycoproteins of this complex sample. We characterized site-specific N-glycosylation of HeLa N-glycoproteins using a complex workflow based on high and low energy tandem mass spectrometry experiments and rigorous data evaluation. The analyses revealed high amount of bovine serum contaminants compromising previous results focusing on released glycan analysis. We reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites and 178 glycopeptides following an acetone precipitation based sample enrichment step. HeLa glycoproteins were found to be highly fucosylated and in several cases localization of the fucose (core or antenna) could also be determined based on low energy tandem mass spectra. High-mannose sugars were expressed in high amounts as expected in case of a cancer cell line. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa. Furthermore, we were the first to experimentally prove the existence of 31 glycosylation sites, where previously presence of glycosylation was only predicted based on the existence of the consensus sequon.
Project description:An amino acid exchange (P209L) in the HSPB8 binding site of the human cochaperone Bcl2-associated athanogene 3 (BAG3) gives rise to severe dominant childhood cardiomyopathy. To phenocopy the disease in mouse and gain insight into its mechanisms, we have generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates containing BAG3, components of the Z-disc, and the protein quality control system in cardiomyocytes. This was accompanied by massive fibrosis resulting in a severe, early-onset restrictive cardiomyopathy with increased mortality, as observed in patients. Here we present the shotgun proteome data of mice expressing hBAG3P209L-eGFP compared to control.
Project description:Transgenic Arabidopsis seedlings RV86-5 (contains a dexamethasone-inducible ubiquitin variant that contains Arg instead of Lys at position 48; ̈Schlögelhofer et al., 2005) were surface-sterilized and sown on Uhelon 120T (Silk & Progress, Czech Republic) mesh placed on 1% (w/v) agar containing a half-strength Murashige and Skoog medium (pH 5.7), stratified at 4 °C for 3 d, and cultivated at 21 °C/19 °C day/night temperatures, with a 16 h photoperiod (90 μmol m−2 s−1 light intensity) for 14 d. On the fourteenth day (after the first 2 h of the day period), the Uhelon mesh with the seedlings was transferred onto a new solid medium supplemented with (i) 5×10−4% (v/v) DMSO (mock); (ii) 0.7 or 7.0 μM dexamethasone (DEX) and rinsed with DMSO or DEX-supplemented water for mock and DEX-treated seedlings, respectively, and incubated for 24 hours.