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:Preimplantation embryo development is a precisely regulated process organized by maternally inherited and newly synthesized proteins. Recently, some studies have reported that blastocyst-like structures, named blastoids, can be generated from mouse ESCs (embryonic stem cells) or EPSCs (extended pluripotent stem cells). In this study, to explore the dynamic expression characteristics of proteins and their PTMs in mouse EPS blastoids, we revealed the protein expression profile of EPS-blastoids and metabolite characteristics by TMT-based quantitative mass spectrometry (MS) strategy. Furthermore, the protein phosphorylation sites were identified to show the phosphoproteomic analysis in blastoids compared with mouse early embryos. Above all, our study revealed the protein expression profile of EPS blastoids compared with mouse embryos during preimplantation development and indicated that glucose metabolism is key to blastoid formation.
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:This project includes raw mass spectrometry data supporting the identification of phosphorylation sites on AEG-1 (MTDH), particularly S426 and T492, in gastric cancer cells(BGC823) under CXCL12 treatment, as well as PPP1R21-interacting proteins including AEG-1 in HEK293T cells. This result supports the main findings of the manuscript accepted in the Journal of Biological Chemistry.
Project description:γ-aminobutyric acid type A receptors (GABAARs) mediate fast synaptic inhibition. Phosphorylation governs GABAARs function and trafficking in α4, β and γ subunits. However, phosphorylation of other subunits has not been demonstrated. Here we show that the α2 subunit is phosphorylated on S359.
Project description:The rotation of the Earth results in predictable environmental changes. To address this daily rhythm, organisms from all kingdoms of life have evolved diverse timing mechanisms. The three proteins KaiA, KaiB, and KaiC constitute the central timing mechanism that drives circadian oscillations in cyanobacteria. In addition to this standard protein oscillator, Synechocystis sp. PCC 6803, one of the main model organisms for cyanobacterial research, harbors several, diverged clock homologs. The nonstandard KaiB3-KaiC3 system has been suggested to impact the metabolic switch in response to darkness. Here, we demonstrate the direct interaction of KaiC3 with Sll0485, a potential new chimeric KaiA homolog that we named KaiA3. At the N-terminus, KaiA3 is similar to the NarL-type response regulator receiver domain. However, its similarity to canonical NarL transcription factors drastically decreases in the C-terminal domain, which resembles the circadian clock protein, KaiA. In line with this, we detected the formation of a high molecular weight complex of KaiA3 together with KaiC3 and KaiB3, and a KaiA3-mediated stimulation of KaiC3 phosphorylation in vitro. Phosphorylation of KaiC3 was rhythmic over 48 h in vitro in the presence of KaiA3 and KaiB3 as well as in light-dark entrained cells released to free-running conditions. Deletion of the kaiA3 gene leads to KaiC3 dephosphorylation, and results in growth defects during mixotrophic growth and in darkness. Further, our analyses identified KaiA3 in other bacterial species, representing the first potential KaiA homolog outside the cyanobacterial phylum. In summary, we suggest that KaiA3 is a novel, nonstandard KaiA homolog, thereby extending the KaiB3-KaiC3 system in Cyanobacteria and potentially other prokaryotes. We suggest that KaiA3B3C3, together with the canonical KaiAB1C1 system, mediates the auto-/heterotrophic switch in the facultative heterotroph, Synechocystis.