Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin.

The manuscript version of this article, under a different title, is paper 3 of Anett Kristin Larsen's doctoral thesis which is available in Munin at http://hdl.handle.net/10037/2892Background: Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2). Results: During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2. Conclusion: These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2

Purified sardine and king crab trypsin stimulate IL-8 secretion and NF-κB activation, at least partly, via PAR2, but displays individual differences in transformation of the NF-κB-signal.

This article is part of Anett Kristin Larsen's doctoral thesis. Available at http://hdl.handle.net/10037/2892Respiratory symptoms occur in workers processing a great variety of seafood. Studies previously showed that salmon trypsin increases transcriptional activity of NF-κB and induces secretion of IL-8 from airway epithelial cells by activating PAR-2. The aim of this study was to explore if purified trypsins from king crab (Paralithodes camtschaticus) and sardine (Sardinops melanostictus) are able to induce similar effects in cell stimulation assays. The knowledge that crustaceans seem to display dissimilar irritant potency compared to fish inspired us to investigate if one could detect differences in intracellular signaling pathways coupled to IL-8 in human airway epithelial cells (A549). Both sardine and king crab trypsin induced secretion of IL-8 from human airway epithelial cells in a concentration-dependent manner and increased transcriptional activity of NF-κB. With the use of siRNA data indicate that these effects are both mediated, at least partly, through the activation of PAR-2. Additionally, the king crab and sardine trypsin display individual differences in transformation of the NF-κB signal into subsequent IL-8 secretion. The contribution of MEK/ERK, p38, and NF-κB to the secretion of IL-8 following stimulation with sardine and king crab trypsins were examined with the use of specific inhibitors. The results demonstrated that MEK/ERK and NF-κB are both required for sardine and king crab trypsin-induced secretion of IL-8 but via separate pathways. P38 was also found to contribute to the secretion of IL-8 seemingly by NF-κB-dependent processes

Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy

Early response evaluation by single cell signaling profiling in acute myeloid leukemia

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phosphoERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.Peer reviewe

Dual-specificity MAP kinase phosphatases in health and disease

Source at https://doi.org/10.1016/j.bbamcr.2018.09.002.It is well established that a family of dual-specificity MAP kinase phosphatases (MKPs) play key roles in the regulated dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues. MKPs provide a mechanism of spatiotemporal feedback control of these key signalling pathways, but can also mediate crosstalk between distinct MAP kinase cascades and facilitate interactions between MAP kinase pathways and other key signalling modules. As our knowledge of the regulation, substrate specificity and catalytic mechanisms of MKPs has matured, more recent work using genetic models has revealed key physiological functions for MKPs and also uncovered potentially important roles in regulating the pathophysiological outcome of signalling with relevance to human diseases. These include cancer, diabetes, inflammatory and neurodegenerative disorders. It is hoped that this understanding will reveal novel therapeutic targets and biomarkers for disease, thus contributing to more effective diagnosis and treatment for these debilitating and often fatal conditions

Purified sardine and king crab trypsin display individual differences in PAR-2-, NF-kB-, and IL-8 signaling

Respiratory symptoms occur in workers processing a great variety of seafood. Studies previously showed that salmon trypsin increases transcriptional activity of NF-κB and induces secretion of IL-8 from airway epithelial cells by activating PAR-2. The aim of this study was to explore if purified trypsins from king crab (Paralithodes camtschaticus) and sardine (Sardinops melanostictus) are able to induce similar effects in cell stimulation assays. The knowledge that crustaceans seem to display dissimilar irritant potency compared to fish inspired us to investigate if one could detect differences in intracellular signaling pathways coupled to IL-8 in human airway epithelial cells (A549). Both sardine and king crab trypsin induced secretion of IL-8 from human airway epithelial cells in a concentration-dependent manner and increased transcriptional activity of NF-κB. With the use of siRNA data indicate that these effects are both mediated, at least partly, through the activation of PAR-2. Additionally, the king crab and sardine trypsin display individual differences in transformation of the NF-κB signal into subsequent IL-8 secretion. The contribution of MEK/ERK, p38, and NF-κB to the secretion of IL-8 following stimulation with sardine and king crab trypsins were examined with the use of specific inhibitors. The results demonstrated that MEK/ERK and NF-κB are both required for sardine and king crab trypsin-induced secretion of IL-8 but via separate pathways. P38 was also found to contribute to the secretion of IL-8 seemingly by NF-κB-dependent processes