Negative feedback regulation of MAPK signaling is an important driver of chronic lymphocytic leukemia progression

Despite available targeted treatments for the disease, drug-resistant chronic lymphocytic leukemia (CLL) poses a clinical challenge. The objective of this study is to examine whether the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate mitogen-activated protein kinases (MAPKs) and thus counterbalance excessive MAPK activity. We show that high expression of DUSP6 in CLL correlates with poor clinical prognosis. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small-molecule inhibitor reduces CLL cell survival in vitro and in vivo. Using global phospho-proteome approaches, we observe acute activation of MAPK signaling by DUSP1/6 inhibition. This promotes accumulation of mitochondrial reactive oxygen species and, thereby, DNA damage and apoptotic cell death in CLL cells. Finally, we observe that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising treatment concept to eliminate drug-resistant CLL cells

Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation

RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S > D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice

Identification of emulsifier potato peptides by bioinformatics: application to omega-3 delivery emulsions and release from potato industry side streams

We are grateful for the financial support from Innovation Fund Denmark (Grant nr: 7045-00021B, PROVIDE project). We also acknowledge K.M.C. amba (Brande, Denmark) and A.K.V. amba (Langholt, Denmark) for providing the potato samples used in this study.In this work, we developed a novel approach combining bioinformatics, testing of functionality and bottom-up proteomics to obtain peptide emulsifiers from potato side-streams. This is a significant advancement in the process to obtain emulsifier peptides and it is applicable to any type of protein. Our results indicated that structure at the interface is the major determining factor of the emulsifying activity of peptide emulsifiers. Fish oil-in-water emulsions with high physical stability were stabilized with peptides to be predicted to have facial amphiphilicity: (i) peptides with predominantly α-helix conformation at the interface and having 18–29 amino acids, and (ii) peptides with predominantly β-strand conformation at the interface and having 13–15 amino acids. In addition, high physically stable emulsions were obtained with peptides that were predicted to have axial hydrophobic/hydrophilic regions. Peptides containing the sequence FCLKVGV showed high in vitro antioxidant activity and led to emulsions with high oxidative stability. Peptide-level proteomics data and sequence analysis revealed the feasibility to obtain the potent emulsifier peptides found in this study (e.g. γ-1) by trypsin-based hydrolysis of different side streams in the potato industry.Innovation Fund Denmark 7045-00021

Signaling network dynamics investigated by quantitative phosphoproteomics

This thesis describes the application of proteomics technologies to get insight into several aspects of phosphorylation signaling dynamics. The core tool in all performed experiments is mass spectrometry (MS)-based phosphoproteomics. In Chapter 1, a general introduction is given into proteomics and MS-based proteomics workflows. In Chapter 2, we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the PKA consensus phosphorylation motif [R/K][R/K/X]X[pS/pT]. This targeted phosphoproteomic strategy, in combination with stable isotope dimethyl labeling, is used to profile temporal changes of potential PKA substrates in Jurkat T lymphocytes upon PGE2 stimulation, which increases intracellular cAMP, thereby activating PKA. It is shown that this approach is very specific and highly complementary to a large-scale phosphoproteomics approach, enabling to profile hundreds of putative PKA sites. In Chapter 3, the phosphopeptide enrichment robustness of Ti4+-IMAC is evaluated. First, we prove that Ti4+-IMAC enrichment allows a highly reproducible quantification of phosphorylation sites in HeLa cells. Subsequently, we apply this strategy to monitor the phosphoproteome of Jurkat T lymphocytes upon PGE2 stimulation, covering extended time series. We demonstrate that this enrichment strategy in combination with label-free quantification enables in-depth investigation of phosphorylation dynamics, highlighting differential regulation of different kinases over time. In Chapter 4, we employ a three-pronged MS-based proteomics strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in A431 epidermoid carcinoma cells, as a model system for skin-cancer. The integration of chemical proteomics and phosphoproteomics allows us to define a set of signaling nodes modulated by each individual drug that can be considered putative targets in epithelial cancers. In Chapter 5, we assess the beneficial use of complementary proteases, namely trypsin, LysC, GluC, AspN and chymotrypsin for the phosphoproteome analysis of Jurkat T cells, analyzed by using Ti4+-IMAC enrichment. The obtained results are a significant improvement upon data from a single protease digest. We demonstrate that nearly each phosphosite can be linked to a preferred protease forming detectable phosphopeptides, whereby the gain in using alternative proteases can be more than 10,000 fold for specific sites in intensity. Moreover, many of the identified sites are not yet reported in currently available and public depositories, demonstrating the complementary nature of these enzymes, through which different parts of the phosphoproteome can be uncovered

Signaling network dynamics investigated by quantitative phosphoproteomics

This thesis describes the application of proteomics technologies to get insight into several aspects of phosphorylation signaling dynamics. The core tool in all performed experiments is mass spectrometry (MS)-based phosphoproteomics. In Chapter 1, a general introduction is given into proteomics and MS-based proteomics workflows. In Chapter 2, we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the PKA consensus phosphorylation motif [R/K][R/K/X]X[pS/pT]. This targeted phosphoproteomic strategy, in combination with stable isotope dimethyl labeling, is used to profile temporal changes of potential PKA substrates in Jurkat T lymphocytes upon PGE2 stimulation, which increases intracellular cAMP, thereby activating PKA. It is shown that this approach is very specific and highly complementary to a large-scale phosphoproteomics approach, enabling to profile hundreds of putative PKA sites. In Chapter 3, the phosphopeptide enrichment robustness of Ti4+-IMAC is evaluated. First, we prove that Ti4+-IMAC enrichment allows a highly reproducible quantification of phosphorylation sites in HeLa cells. Subsequently, we apply this strategy to monitor the phosphoproteome of Jurkat T lymphocytes upon PGE2 stimulation, covering extended time series. We demonstrate that this enrichment strategy in combination with label-free quantification enables in-depth investigation of phosphorylation dynamics, highlighting differential regulation of different kinases over time. In Chapter 4, we employ a three-pronged MS-based proteomics strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in A431 epidermoid carcinoma cells, as a model system for skin-cancer. The integration of chemical proteomics and phosphoproteomics allows us to define a set of signaling nodes modulated by each individual drug that can be considered putative targets in epithelial cancers. In Chapter 5, we assess the beneficial use of complementary proteases, namely trypsin, LysC, GluC, AspN and chymotrypsin for the phosphoproteome analysis of Jurkat T cells, analyzed by using Ti4+-IMAC enrichment. The obtained results are a significant improvement upon data from a single protease digest. We demonstrate that nearly each phosphosite can be linked to a preferred protease forming detectable phosphopeptides, whereby the gain in using alternative proteases can be more than 10,000 fold for specific sites in intensity. Moreover, many of the identified sites are not yet reported in currently available and public depositories, demonstrating the complementary nature of these enzymes, through which different parts of the phosphoproteome can be uncovered

Determinazione di ritardanti di fiamma bromurati contaminanti matrici acquose mediante LC/APPI-MS/MS

I ritardanti di fiamma sono composti chimici utilizzati come additivi, aggiunti durante o al termine della lavorazione di un materiale, per ritardare la fase iniziale del processo di combustione, per ridurre l’infiammabilità di materiali polimerici nel rispetto delle norme antincendio, per impedire o rallentare lo sviluppo di incendi. La tipologia dei ritardanti di fiamma copre un ampio spettro di composti che soddisfano le più svariate esigenze. Il gruppo dei ritardanti di fiamma a base di bromo comprende diversi composti organobromurati utilizzati per impedire la combustione e/o per ritardare la diffusione delle fiamme in diversi tipi di plastica, prodotti tessili ed altri materiali. Sono tre i gruppi chimici impiegati più comunemente: i difenileteri polibromurati (PBDE), l’esabromociclododecano (HBCD), usati come additivi, ed i bisfenoli bromurati (in particolare il TBBP-A) più comunemente usati come reagenti data la loro maggiore capacità di legarsi con i polimeri in cui vengono incorporati. Il problema ambientale dei ritardanti di fiamma nasce negli anni novanta, quando nei sedimenti del Mar Baltico è stato rilevato un aumento sensibile delle concentrazioni di PBB (difenilipolibromurati) e PBDE. Studi successivi condotti in Svezia, hanno dimostrato che tali composti sono persistenti e soggetti a notevole bioaccumulo nella catena alimentare. Infine, la Direttiva 2003/11/CE ha posto limitazioni alla produzione ed utilizzo di questo tipo di composti per le ragioni sinteticamente esposte, sebbene in presenza di caratteristiche di tossicità acuta non preoccupanti. I metodi analitici finora sviluppati per la determinazione di queste sostanze comprendono l’estrazione e la micro-estrazione in fase solida (SPE e SPME), l’estrazioni liquido-liquido con membrana microporosa (MMLLE) e la mirco-estrazione liquido-liquido (DLLME) utilizzate su campioni liquidi; l’estrazione Soxhlet utilizzata su campioni atmosferici di particolato. Le tecniche analitiche sin qui utilizzate sono: cromatografia liquida ad alte prestazioni accoppiata a rivelatore ultravioletto (HPLC-VWD), gascromatografia con rivelatore a cattura di elettroni (GC-ECD) e gascromatografia con ionizzazione chimica (CI) o elettronica (EI) e rivelazione mass-spettrometrica (GC-CI–MS, GC-EI–MS, GC-NCI-MS). Lo scopo di questo lavoro è la messa a punto di un nuovo metodo di conferma, rapido e robusto, basato sulla HPLC APPI*-MS/MS per la determinazione di basse concentrazioni di ritardanti di fiamma polibromurati in campioni di acqua; in tabella I sono riportati i composti investigati, caratteristici della classe

Six alternative proteases for mass spectrometry-based proteomics beyond trypsin

Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d

Flavonoids: chemical properties and analytical methodologies of identification and quantitation in foods and plants

Flavonoids have been recognised as one of the largest and most widespread groups of plant secondary metabolites, with marked antioxidant properties. The general name flavonoid refers to a class of more than 6500 molecules based upon a 15-carbon skeleton. In this paper a general overview of flavonoids, their classification, structures and analytical methods for their determination is presented

Recent developments in matrix solid-phase dispersion extraction

Matrix solid-phase dispersion is a sample preparation strategy widely applied to solid, semisolid or viscous samples, including animal tissues and foods with a high lipidic content. The process consists in blending the matrix onto a solid support, allowing the matrix cell disruption and the subsequent extraction of target analytes by means of a suitable elution solvent. First introduced in 1989, MSPD employment and developments are still growing because of the feasibility and versatility of the process, as evidenced by the several reviews that have been published since nineties. Therefore, the aim of the present review is to provide a general overview and an update of the last developments of MSPD. (C) 2010 Elsevier B.V. All rights reserved