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Project description:Nitrate nutrtition was withdrawn from Arabiodpsis plants to study early responses upon nitrate depletion.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: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:Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L.), one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index, an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.
Project description:Progression through neuronal loss of substantia nigra pars compacta with Parkinson’s disease depends on various protein post-translational modifications mainly comprising phosphorylation, ubiquitination, acetylation, and methylation. Phosphorylation and ubiquitination regulate major physiological changes and cellular signaling pathways during dopaminergic neuronal death. Phosphorylation and ubiquitination dyshomeostasis of substantia nigra pars compacta tissue occurs earlier than movement symptom appearance. Although many phosphorylation and ubiquitination sites have been identified through site-specific methods, systematic quantitative proteomic analysis of pre-symptomatic Parkinson’s disease remains unexplored. Using quantitative proteomics, we have globally profiled ubiquitination and phosphorylation in substantia nigra pars compacta tissue of a Parkinson’s disease transgenic mouse model (A30P*A53T α-synuclein, hm2α-SYN-39 mouse strain) at pre-symptomatic stage; Our datasets of 5,351 phosphorylation sites in 2,136 proteins and 3,971 ubiquitination sites in 1,595 proteins provide valuable insight into pre-symptomatic Parkinson’s disease. After in-depth analysis of the relationship between phosphorylation and ubiquitination sites, we concluded that correlation of the relationship increased with decreasing distance. Subsequent bioinformatic analyses, including gene ontology annotation, domain annotation, subcellular localization, KEGG pathway annotation, functional cluster analysis, and motif analysis were performed to annotate quantifiable targets of phosphorylation and ubiquitination sites. Individual simultaneous phosphorylation and ubiquitination proteins that were differentially quantified were screened. The endocytosis pathway is likely regulated by both phosphorylation and ubiquitination at the molecular protein Epn2 (S439 and K135) in early-stage Parkinson’s disease. Therefore, this elucidation of the dysregulation of phosphorylation and ubiquitination has implications for understanding the pathophysiological mechanism of dopaminergic neuron degeneration and for developing novel therapeutics for Parkinson’s disease.
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.