Project description:Nitrogen is a key factor impacting plant physiological processes and protein abundance. Although many proteins were changed substantially in poplar under N deficiency, the post-translational modifications in male and female poplars are still unclear. Therefore, we selected male and female poplars and analysed the changes of protein phosphorylation in response to N-deficient conditions.
Project description:A protocol for an improved phosphopeptide identification in tryptically digested complex peptide samples is described. The common TiO2 based phoshopeptide enrichment is coupled to a simple peptide fractionation protocol with following LC-MS analysis of the obtained fractions and proteomic identification of phosphorylated peptides. The fractions are obtained by an optimized protocol for peptide fractionation using a home-made capillary column coupled to a microgradient elution using a gastight microsyringe. The protocol leads to a substantially increased number of phosphopeptides (more than twice).
Project description:Patients with polycystic kidney disease (PKD) encounter a high risk of clear cell renal cell carcinoma (ccRCC), a malignant tumor with dysregulated lipid metabolism. SET domain–containing 2 (SETD2) has been identified as an important tumor suppressor gene in ccRCC. However, the role of SETD2 in tumorigenesis during the transition from PKD to ccRCC remains largely unexplored. Herein, we performed metabolomics, lipidomics, transcriptomics and proteomics with SETD2 loss induced PKD-ccRCC transition mouse model. To characterize biological responses triggered by SETD2 deletion during PKD-ccRCC transition at the protein level, we conducted global proteomics studies.
Project description:The aim of the proteomics experiment was to determine the specificity of the generated monobodies for the target protein STAT3. This was done in an affinity-enrichment experiment using two endogenously expressed and tandem-affinity-tagged monobodies in two different cell lines (Jurkat and K562). Eluted proteins after affinity purification were resolved by SDS-PAGE, and analysed using bottom-up proteomics workflow including tryptic digestion, LC-MS/MS, database-search and spectral counting.
Project description:Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to particular physiological characteristics, no treatments against diseases caused by oomycetes are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. The present project is focused on the molecular mechanisms that underlie the compatible plant-oomycete interaction and plant disease. The laboratory developed a novel interaction system involving the model plant, Arabidopsis thaliana, and Phytophthora parasitica, a soil-borne pathogen infecting a wide host range, thus representing the majority of Phytophthora species. A characteristic feature of the compatible Arabidopsis/P. parasitica interaction is an extended biotrophic phase, before infection becomes necrotrophic. Because the initial biotrophic phase is extremely short on natural (e.g. solanaceous) hosts, the Arabidopsis system provides the opportunity to analyze, for both interaction partners, the molecular events that determine the initiation of infection and the switch to necrotrophy. The present project aims at analyzing the compatible interaction between A. thaliana roots and P. parasitica. The Affymetrix A. thaliana full genome chip will be used to characterize modulations of the transcriptome occurring over a period of 24h from the onset of plant root infection to the beginning of necrotrophy. Parallel to this study, a custom-designed P. parasitica biochip will enable analyzing of P. parasitica gene expression during the same stages. 10 samples were used in this experiment.
Project description:A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes encoding proteins important for survival. Interestingly, under many of these conditions overall protein synthesis levels are reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in yeast, translation is rapidly and reversibly repressed, yet transcription of many stress- and glucose-repressed genes is increased. Using ribosome profiling and microscopy, we found that this transcriptionally upregulated gene set consists of two classes: (1) one producing mRNAs that are preferentially translated during glucose limitation and are diffusely localized in the cytoplasm – this class includes many heat shock protein mRNAs; and (2) another producing mRNAs that are poorly translated during glucose limitation, have high rates of translation initiation, and are concentrated in foci that co-localize with P bodies and stress granules – this class is enriched for glucose metabolism mRNAs. Remarkably, the information specifying differential localization and translation of these two classes of mRNAs is encoded in the promoter sequence – promoter responsiveness to heat shock factor (Hsf1) specifies diffuse cytoplasmic localization and preferential translation upon glucose starvation, whereas different promoter elements upstream of genes encoding poorly translated glucose metabolism mRNAs direct these mRNAs to RNA granules under glucose starvation. Thus, promoter sequences and transcription factor binding can influence not only mRNA levels, but also subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to environmental conditions. Examination of mRNA translation in S. cerevisiae upon glucose starvation.
Project description:Mass spectrometry, mutagenesis and labelling with [32P] orthophosphate identified that each of the five hydroxy-amino acids in the intracellular C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to the effectiveness of interaction of the receptor with arrestin-3. Key to such interactions was Ser303. Despite there being a greater number of hydroxy-amino acids in the C-terminal tail of both mouse and rat GPR35 the serine corresponding to residue 303 in human GPR35a also played a dominant role in arrestin-3 interactions for both rodent orthologues. Fully phospho-site deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3 and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35 a substantial proportion of the expressed protein(s) was, however, immature. Phospho-site specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35 identified only the mature forms of GPR35 and provided effective biomarkers of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programmes.
Project description:Progression through meiosis in Schizosaccharomyces pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts and also requires stage-specific, targetted proteolysis of regulatory proteins. We have used SILAC labelling to examine the relative levels of proteins in S. pombe as diploid cells undergo meiosis. We found that the relative level of 880 proteins changes at least two-fold at some stage of meiosis. Most of these proteins either increase or decrease in level during the meiotic divisions, while some show transient peaks of expression. The most notable changes are in the proteostasis network, which shows a significant increase in components involved in protein turnover concomitant to a decrease in proteins involved in ribosome biogenesis. There was also an increase in ESCRT III protein levels; biological analysis reveals a role for some ESCRT III components in chromosome segregation and spore formation. Correlation with previous studies of gene expression and ribosome occupancy through meiosis reveals that changes in steady state protein levels are mainly regulated post-transcriptionally.
Project description:Although the GPR84 activators 2-HTP and 6-OAU promoted phosphorylation of human GPR84 and its interactions with arrestin-3, PSB-16671 and DL-175 did not. Replacement of all 21 serine and threonine residues within the third intracellular loop, but not the 2 serines in the C-terminal tail, eliminated incorporation of [32P] promoted by 2-HTP and greatly reduced receptor-arrestin-3 interactions. Mass spectrometry indicated that GPR84 was phosphorylated constitutively on residues Ser221 and Ser224 whilst a range of sites became phosphorylated in response to 2-HTP. An antiserum able to identify pSer221/pSer224 recognised GPR84 from cells treated both with and without 2-THP, whilst an antiserum able to identify pThr263/pThr264 recognised GPR84 only after exposure to 2-HTP. Treatment with neither PSB-16671 nor DL-175 was able to promote receptor recognition by this antiserum. 2-HTP-mediated phosphorylation of Thr263/Thr264 was prevented by two chemically distinct GPR84 antagonists but neither affected constitutive phosphorylation of Ser221/Ser224. 2-HTP-mediated phosphorylation of Thr263/Thr264 but not constitutive phosphorylation of Ser221/Ser224 was prevented by GRK2/3 inhibition. Mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 as limited in 2-HTP-induced interactions with arrestin-3 as when all 21 serine and threonine were eliminated from the third intracellular loop. By contrast this mutant was unaffected in capacity to reduce cAMP levels in response to 2-HTP. Homology modelling and mutagenesis provided molecular insight into differences in agonist function. These studies define key residues, regulated by GRK2/3, that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.