Project description:Meningococcal sepsis is an overwhelming form of the sepsis syndrome which may cause mortality within 12-24 hours in previously healthy children and adults, where the causative infectious agent is N. meningitidis, an obligate human pathogen. The genomic changes induced by N. meningitidis are modulated by the anti-inflammatory cytokine interleukin-10 (IL-10), which is present in large quantities in plasma from patients with meningococcal sepsis. This present study investigated kinase activities in human monocytes stimulated by N. meningitidis and IL-10. The first aim was to identify array peptides that could indicate which signaling pathways were activated or inhibited by the host response to the meningococci. The second aim was to detect whether IL-10 affected N. meningitidis-nduced phosphorylation of array peptides, in order to identify potential targets of the IL-10 anti-inflammatory response. We approached this using a strategy where elutriation-purified human monocytes are stimulated in vitro with N. meningitidis and IL-10, with concentrations corresponding to previously measured levels in patients with fulminant meningococcal septicemia. This work examined activation or inhibition of signaling pathways mediated by tyrosine kinases when purified human monocytes are in vitro incubated with N. meningitidis in the presence or absence of IL-10.
Project description:Extracellular vesicles (EVs) are important mechanisms used by cells to release biomolecules. A common necroptosis effector— mixed lineage kinase like (MLKL)--- was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. We performed mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Necroptosis increased the number of EVs released and altered the protein contents within the EVs. Comparing to EVs released by healthy cells, EVs released during necroptosis contained markedly higher number of unique proteins. Receptor interacting protein kinase 3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, MEFs derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs was sensitive to Ca2+ depletion or lysosomal disruption. Neither treatments affected the RIPK3-mediated MLKL phosphorylation. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.
Project description:Nitrate nutrtition was withdrawn from Arabiodpsis plants to study early responses upon nitrate depletion. Plants were grown under full nutrition (3mM nitrate) for three weeks and then transferred to nutrient solution without nitrate for 15 minutes and 3 hours.
Project description:High yield and wide adaptation of rice is of significance for more grain supply and food security. Here, we report a type II phosphatidylinositol 4-kinase, PI4Kγ7, harboring a PI3/PI4 kinase domain, which positively regulates rice yield and affords wide adaptation of rice. We show that PI4Kγ7 interacts with a CCCH-type zinc finger transcription factor, OsLIC. Further investigations discover that PI4Kγ7 modulates rice yield by facilitating the stability and cytoplasm-to-nucleus translocation of OsLIC through phosphorylation. To identify the residue(s) being phosphorylated by PI4Kγ7, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
Project description:Grain number per panicle critically determines rice yield.GNP3 encodes a MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 22 (OsMKKK22) that phosphorylates S-adenosyl-L-methionine synthetase 1 (SAMS1), triggering its degradation to suppress ethylene biosynthesis.To identify the residue(s) being phosphorylated by GNP3, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of recombinant SAMS1 incubated with GNP3.
Project description:The number of grains per panicle is one of the important factors affecting rice yield. In this study, we found that GNP2 can phosphorylate Gnp4, a factor influencing grain number per panicle in rice. Therefore, we conducted a mass spectrometry analysis of the phosphorylation sites of Gnp4.
Project description:Translation is a tightly regulated process, and the mTORC1-S6K signaling axis plays a critical role in this control. Binding of eIF4F to the cap is hindered by eIF4E binding proteins (4EBPs), which, when hypophosphorylated, sequester eIF4E and prevent its association with eIF4G. However, in response to positive stimuli such as growth factors, mitogens, and amino acids, mTORC1 phosphorylates 4EBPs and relieves this inhibition, allowing the formation of eIF4F and subsequent initiation of translation. We are interested to know whether IBTK-mediated eIF4A1 ubiquitination is regulated by mTORC1/S6K signaling. Indeed, quantitative phosphoproteomics studies revealed that several IBTK phosphorylation sites are markedly downregulated by treatment of mTOR inhibitor, Rapamycin or Torin 1. Thus, probable phosphorylation sites of IBTK were identified by MS analysis.
Project description:Proteins play essential roles in biology, yet identifying their precise sequences and modifications remains challenging. De novo peptide sequencing offers a powerful solution by directly inferring sequences from mass spectrometry data without relying on protein databases. Recent deep learning models have significantly advanced this task but remain trapped in a major dilemma: they require labeled training data to recognize post-translational modifications (PTMs), which is unavailable for most biologically relevant but rare or unknown modifications. We solve this long-standing problem by introducing RNovA, a transformer-based de novo sequencing algorithm enhanced with relative positional embeddings and a reinforcement-learning–style sequential decision framework. RNovA enables open PTM discovery in a zero-shot settingwithout retraining or a predefined list of candidate residues—while maintaining state-of-the-art performance on standard benchmarks. Demonstrating this capability, we successfully identified peptides modified by kynurenine—an uncommon and biologically relevant PTM—in clinical samples from rheumatoid arthritis patients. RNovA overcomes key limitations of existing methods and provides a foundation for exploring previously inaccessible regions of the proteome, including peptides with unexpected or unannotated modifications. This capability is widely needed in immunology, biomarker discovery, and biomedical research.
Project description:This SuperSeries is composed of the following subset Series: GSE13863: Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa (Nimblegen) GSE19889: Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa (Illumina) Refer to individual Series