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Phosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based platforms for measuring reporter peptide signal levels allow for differential phosphorylation analysis between conditions for distinct active kinases. Peptide array technologies like the PamStation12 from PamGene allow for generating high-throughput, multi-dimensional, and complex functional proteomics data. As the adoption rate of such technologies increases, there is an imperative need for software tools that streamline the process of analyzing such data. We present Kinome Random Sampling Analyzer (KRSA), an R package and R Shiny web-application for analyzing kinome array data to help users better understand the patterns of functional proteomics in complex biological systems. KRSA is an All-In-One tool that reads, formats, fits models, analyzes, and visualizes PamStation12 kinome data. While the underlying algorithm has been experimentally validated in previous publications, we demonstrate KRSA workflow on dorsolateral prefrontal cortex (DLPFC) in male (n = 3) and female (n = 3) subjects to identify differential phosphorylation signatures and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar global phosphorylation signals patterns. ispartof: PLOS ONE vol:16 issue:12 ispartof: location:United States status: published

Background Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. Methods Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. Results Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p 2. Conclusions The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.

Inherited retinal degenerative diseases (IRDs) are rare neurodegenerative disorders with mutations in hundreds of genes leading to vision loss, primarily owing to photoreceptor cell death. This genetic diversity is impeding development of effective treatment options. Gene-based therapies have resulted in the first FDA-approved drug (Luxturna) for RPE65-specific IRD. Although currently explored in clinical trials, genomic medicines are mutation-dependent, hence suitable only for patients harboring a specific mutation. Better understanding of the pathways leading to photoreceptor degeneration may help to determine common targets and develop mutation-independent therapies for larger groups of patients with IRDs. In this review, we discuss the key pathways involved in photoreceptor cell death studied by transcriptomics, proteomics, and metabolomics techniques to identify potential therapeutic targets in IRDs.

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays. Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.

MotivationPhosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based approaches for measuring multiple kinases allow for the testing of differential phosphorylation between conditions for distinct sub-kinomes. While bioinformatics tools exist for processing and analyzing such kinome array data, current open-source tools lack the automated approach of upstream kinase prediction and network modeling. The presented tool, alongside other tools and methods designed for gene expression and protein-protein interaction network analyses, help the user better understand the complex regulation of gene and protein activities that forms biological systems and cellular signaling networks.ResultsWe present the Kinome Random Sampling Analyzer (KRSA), a web-application for kinome array analysis. While the underlying algorithm has been experimentally validated in previous publications, we tested the full KRSA application on dorsolateral prefrontal cortex (DLPFC) in male (n=3) and female (n=3) subjects to identify differential phosphorylation and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar patterns to the global phosphorylation signal. Additionally, kinase hits were compared to gene expression databases forin silicovalidation at the transcript level and showed differential gene expression of kinases.Availability and implementationKRSA as a web-based application can be found athttp://bpg-n.utoledo.edu:3838/CDRL/KRSA/. The code and data are available athttps://github.com/kalganem/KRSA.Supplementary informationSupplementary data are available online.

Introduction High fidelity between non–small cell lung cancer (NSCLC) primary tumours and patient-derived tumour xenografts (PDTXs) is of paramount relevance to spur their application. Extensive proteomic and kinomic analysis of these preclinical models are missing and may inform about their functional status, in terms of phosphopeptides and hyperactive signalling pathways. Methods We investigated tumour xenografts derived from patients with NSCLC to identify hyperactive signalling pathways. Fresh tumour fragments from 81 NSCLC surgical samples were implanted in Nod/Scid/Gamma mice, and engrafted tumours were compared with primary specimens by morphology, immunohistochemistry, gene mutation analyses, and kinase activity profiling. Four different tyrosine and serine/threonine kinase inhibitors were tested against primary tumour and PDTX lysates using the PamGene peptide microarray platform. Results The engraftment rate was 33%, with successful engraftment being more associated with poor clinical outcomes. Genomic profiles led to the recognition of hotspot mutations, some of which were initially undetected in donor samples. Kinomic analyses showed that characteristics of primary tumours were retained in PDTXs, and tyrosine kinase inhibitors (TKIs) responses of individual PDTX lines were either expected, based on the genetic status, or alternatively defined suitable targets unpredictable by single-genome fingerprints. Conclusions Collectively, PDTXs mostly resembled their parental NSCLC. Combining genomic and kinomic analyses of tumour xenografts derived from patients with NSCLC, we identified patients' specific targetable pathways, confirming PDTXs as a preclinical tool for biomarker identification and therapeutic algorithm'' improvement.

About 5% of Triple negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) experience progressive disease (PD). Few reports are published on TNBCs with PD during NAC, whereas TNBCs that respond to NAC have been well-studied. We investigated kinase activity profiles of TNBCs to explore the biological differences underlying the lack of response to NAC. Among 740 TNBCs, 20 non-responders were identified. Seven non-responders and 10 TNBCs that did not receive NAC (control group) were evaluated. No correlation was observed between NAC response and age, menopausal status, tumor size and axillary lymph node status. Tyrosine kinase activity profiles of TNBC primary tissues from NAC non-responders and the controls were determined with a peptide microarray system. Kinase activity measurements showed that 35 peptides had significantly (p < 0.05) lower phosphorylation in non-responders. ZAP70, LCK, SYK and JAK2 were identified as differentially active upstream kinases. Pathway analysis suggested lower activity in immune-related pathways in non-responders. The number of tumor infiltrating lymphocytes (TILs) was significantly lower (p = 0.0053) in non-responders. Kinases related to the immune system are less activated in non-responders. TILs evaluation suggested that the immune system is hardly active in non-responders and is not activated by NAC treatment.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

Chemical tools and methods that report on target engagement of endogenously expressed protein kinases by small molecules in human cells are highly desirable. Here, we describe a chemical genetics strategy that allows the study of non-receptor tyrosine kinase FES, a promising therapeutic target for cancer and immune disorders. Precise gene editing was used in combination with a rationally designed, complementary fluorescent probe to visualize endogenous FES kinase in HL-60 cells. We replaced a single oxygen atom by a sulphur in a serine residue at the DFG-1 position of the ATP-binding pocket in an endogenously expressed kinase, thereby sensitizing the engineered protein towards covalent labeling and inactivation by a fluorescent probe. The temporal control offered by this strategy allows acute inactivation of FES activity both during myeloid differentiation and in terminally differentiated neutrophils. Our results show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis by activation of SYK kinase, a central regulator of immune function in neutrophils. This strategy holds promise as a target validation method for kinases.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

Alzheimer’s disease (AD) is characterized by a long pre-clinical phase (20–30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value

A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases.

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP-binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate-based inhibitors that target the more selective peptide-binding site in addition to the ATP-binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate-based leads. Dynamic peptide microarrays were used to find peptide-binding site inhibitors. These were linked with chemoselective click chemistry to an ATP-binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate-based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCtheta amongst three isozymes.

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.

A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases.

Background: Identification of signal transduction pathways that are critically involved in Alzheimer’s disease (AD) is essential for the development of disease-specific biomarkers and drug therapy. Objective: This study is aimed at identifying protein kinases and signaling pathways that are activated in AD pathology. Methods: Microarray-based kinome profiling was employed for the detection of protein kinase activity in postmortem brain tissue derived from AD and age-matched nondemented control cases. Global serine/threonine kinase activity profiles are identified applying a peptide array system consisting of 140 peptides derived from known kinase substrate sequences covalently attached to porous chips, through which a protein solution is constantly pumped up and down. Peptide phosphorylation is determined by measuring the association of a mixture of fluorescently labeled antibodies, raised against phosphoserine- or phosphothreonine-containing peptides. Results: Protein lysates from freshly frozen postmortem brain tissue from nondemented controls and pathologically confirmed AD cases show ATP-dependent phosphorylation of peptides. In AD and control cases, peptides that are differentially phosphorylated are identified. Conclusion: Protein kinase activity profiling can be used to reveal novel kinases and new signaling pathways involved in AD pathology.

Analysis of fifty sorghum [Sorghum bicolor (L.) Moench] varieties used in Burkina Faso showed that they have different contents of phenolic compounds, peroxidase (POX), and polyphenol oxidase (PPO). Most of the varieties (82%) had a tannin content less than 0.25% (w/w). POX specific activity was higher than the monophenolase and o-diphenolase specific activities of PPO. For POX, there was a diversity of isoforms among varieties. No clear correlation could be made between the quantitative composition of the grain in phenolics, PPO, and POX, and resistance of plant to pathogens. In general, varieties good for a thick porridge preparation (“to”) had low phenolic compounds content and a medium POX activity. From the red varieties, those used for local beer (“dolo”) had a high content in phenolic compounds and PPO, and a low POX activity. The variety considered good for couscous had a low POX content. The characteristics might be useful as selection markers for breeding for specific applications. Keywords: Phe...

A method for the quantitative determination of riboflavin levels in beer was developed. The method is based on the quenching of riboflavin fluorescence, which occurs when riboflavin binds to the aporiboflavin-binding protein from egg white. The method does not require any pretreatment of the beer before analysis, other than dilution, and proved to be simple, reliable, and sensitive. The lowest concentration that could be detected was approximately 10 nM riboflavin. The possible interference of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) with the determination of the riboflavin content of beer was excluded, because beer contains only a very small amount of FAD (0.03 microM) and no FMN. The riboflavin levels of the types and brands of beer investigated were in the range of 0.5-1.0 microM. The origin of the riboflavin in beer proved to be the malt. Hop and yeast hardly contributed to the riboflavin content of beer. Besides its use in the determination of riboflavin levels, the aporiboflavin-binding protein also provides a way to remove riboflavin from beer, which reduces the light sensitivity and the related lightstruck off-flavor formation in beer.

Bread doughs supplemented with xylanase and xylanase plus peroxidase were fractionated into 4 insoluble and 3 soluble fractions. Chemical analysis and high-performance size-exclusion chromatography analysis of apparent molecular weight distribution indicated that xylanase acts on both cold-water-extractable arabinoxylans and on those that can be solubilized from cell wall fragments by hot water extraction. Peroxidase action increased the amount of insoluble small cell wall fragments, notably the amount of protein and arabinoxylan. Arabinoxylans were retained in the small cell wall fragments because cross-linking of arabinoxylans through ferulic acid residues to other arabinoxylans rendered them insoluble. Peroxidase did not affect the composition of gluten, nor was evidence obtained for peroxidase-catalyzed cross-linking of arabinoxylans to protein in the gluten and other fractions.

Introduction RAF-MAPK pathway inhibition with BRAF inhibitors or the combination of BRAF and MEK inhibitors has become the mainstay of therapy of metastatic melanoma harboring BRAF V600 mutations with a response rate as high as 50-75 %. A minority of patients present with primary resistance while all patients develop secondary resistance. We explored a reversed translational approach using a novel multiplex kinase assay to first identify individuals not responding to therapy using baseline biopsy samples and second to understand the mechanism of resistance using patient-derived cell lines. Method Pre-dose tissues from 4 non-responders and 3 responders to vemurafenib monotherapy, all harboring the V600E mutation, were profiled for Serine-Threonine Kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of dabrafenib. For molecular studies three melanoma cell lines, all with BRAF V600E mutations, were made resistant to vemurafenib. Protein Tyrosine Kinase (PTK) and STK activities in lysates of sensitive or resistant cells were measured +/- dabrafenib, trametinib or both. Lysates were blotted against a selected panel of (phospho)proteins. Results In tissue lysates, concentration-dependent ex vivo inhibition with dabrafenib was stronger in the patients that were clinical responders than in non-responders. This difference in inhibition of STK activity by dabrafenib was confirmed in the cell lines that were made resistant against vemurafenib. IC50s for the sensitive cell lines were below 0.5 µM and varied from 10-21 µM for cell lines with acquired resistance. PTK activity was increased in resistant cell lines. STK activity was lower, but large differences exist among the cell lines. Common features of resistance were increased activity of receptor tyrosine kinases, Src family kinases and AKT signaling. These results were confirmed by Western blot analysis. Dabrafenib (10 µM) caused inhibition of STK activity while trametinib (10 µM) gave some activation. Dabrafenib (0.5 µM) gave strong inhibition of PTK activity, both in tissue and cell lines. Trametinib had hardly any inhibitory effect. Interestingly, the combination of dabrafenib and trametinib had an antagonistic effect on the STK activity and a synergistic effect on PTK activity, resulting in a stronger inhibition of kinase activity. Conclusion Resistance of tumors and cell lines to vemurafenib can affect kinase activity profiles as detected pre-dose using a multiplex kinase assay. Dabrafenib inhibition of PTK and STK activity is stronger in sensitive cell lines. The overall activation of kinases in a lysate upon MEK inhibition suggests MEK as suppressor of kinase activity. Resistance to MAPK pathway inhibitors could be linked to an increase in tyrosine kinase activity and AKT pathway activity. Biomarkers are needed to identify patients in need of combined MAPK pathway inhibition. Citation Format: Riet Hilhorst, Mohammed Krayem, Adrienne van den Berg, Fabrice Journe, Liesbeth Hovestad-Bijl, Tim van den Hooven, Rik de Wijn, Philippe Aftimos, Ghanem Ghanem, Rob Ruijtenbeek. Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2017-3163

Introduction: Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction in immune cells. While most analytical methods focus on the detection of these crucial enzymes at the RNA or protein level, a method was lacking to monitor multiple enzymatic activities in patient-derived materials like blood and (tumor) tissues. Such a substrate-specific assay is a new tool for biomarker discovery in immuno-oncology (I-O), because the I-O targets - checkpoint and immune receptors like PD1, CTLA4, LAG3, 4-1BB, CD40, CD20, OX40, TIGIT and GITR - are controlled by phosphatases. The aim of this study was to develop a multiplex PTP assay and investigate the phosphatase activity in metastatic renal cell carcinoma (mRCC) tissues with high and low levels of TILs (tumor-infiltrating lymphocytes). Methods: On the basis of hematoxylin and eosin staining of mRCC tissues, 9 tissues with high TILS-scores were selected, as well as 10 tissues without TILS, matched on basis of the percentage of viable tumor cells. The technology used is a second generation peptide microarray, which is fully automated with short turnaround times and a high throughput of clinical samples in a 96-well setup. Arrays comprise 46 unique nitrophosphotyrosine containing peptides derived from known phosphosites. When a clinical sample is applied to this peptide microarray, this will lead to a pattern of peptide dephosphorylations, which is monitored in real time (kinetic readout). Results: The PTP assay was first developed for PBMC profiling. 0.10 ug protein/array was used, corresponding to 10000 cells. The signals were concentration dependent and could be inhibited by PTP inhibitors. For tissue profiling 2.0 µg protein input (0.02 mm3 tissue) was used. A 2-group comparison analysis of the phosphatase activity in tissues with high scores of TILs (massive infiltration, N=9) vs. without (N=10), revealed that 29 of 46 peptide substrates showed significantly higher dephosphorylation (PTP) activities in the tissues with high levels of lymphocyte infiltrates. A PCA revealed a clear separation between the groups of high and low TILS scores. Discriminating phosphosites (p Conclusions: Proof of concept was shown in mRCC samples that high PTP activity correlates with high levels of TILS. As a TILS-score itself is already regarded to be a candidate biomarker for I-O therapy response, the observed correlation is a basis for the development of more mechanistic biomarkers predicting therapy response. This method can be a starting point for the development of an enzymatic and thus sensitive and quantitative TILS test. This study has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 259939. Citation Format: Rob Ruijtenbeek, Liesbeth Houkes-van Kerkhoff, Jeannette Oosterwijk, Liesbeth Hovestad-Bijl1, Riet Hilhorst, Alejandro H. Gomez, Juan F. Rodriguez, Jesus Garcia-Donas, Bart Kiemeney, Egbert Oosterwijk. Correlation of phosphatase activity with lymphocyte infiltrates in metastatic renal cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5608. doi:10.1158/1538-7445.AM2017-5608

Background: Triple-negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) generally show a response to NAC, but about 5% experience progressive disease (PD). Although TNBCs that respond to NAC have been well-studied, reports about TNBCs with PD during NAC are absent. We aimed to compare kinase activity profiles of TNBCs of both groups to identify a biological predictor of NAC non-responders. Methods: Tyrosine kinase activity profiles of lysates of fresh frozen cancer tissues from NAC non-responders and responders, the latter represented by TNBCs who did not receive NAC, were determined with a peptide microarray system (Cancer Res. 2009; 69(14):5987-95). Tumor infiltrating lymphocytes (TILs) were counted in these tissues. Results: Among 740 TNBCs, 20 NAC non-responders were identified. For the 7 samples from PD confirmed patients and 10 TNBCs not receiving NAC, no correlation was observed between NAC response and age, menopausal status, tumor size or axillary lymph node status. Kinase activity measurements showed large differences in overall tyrosine kinase activity between responders and non-responders. 35 peptides had significantly (p < 0.05) higher phosphorylation in the responder group. Kinases that phosphorylate these peptides (upstream kinases) were identified from literature. Among others, the immune related kinases Zap70, Syk and JAK2 were identified as upstream kinases. Pathway analysis yielded evidence for increased activity in the responder group in immune-related pathways and in pathways involved in development and signal transduction. Responders had a significantly higher (p = 0.0053) TILs count. Conclusion: Kinases related to the immune system are less activated in NAC non-responders. The low TILs count in the non-responders confirmed that activation of the immune system in non-responders is lower than in NAC responders. These data merit a further investigation of the use of kinase activity profiling either alone or in combination with TIL scoring for the prediction of the response to NAC in TNBC with a larger patient cohort. Citation Format: Takeshi Sawada, Riet Hilhorst, Savithri Rangarajan, Masayuki Yoshida, Kenji Tamura, Rinie van Beuningen, Rob Ruijtenbeek, Hitoshi Tsuda, Fumiaki Koizumi. Immune system pathway activation for prediction of the response to neoadjuvant chemotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2017-1792

which function in innate immunity also suggests that innate immunity may have a significant role in AD. Given that gliosis occurs at least decades earlier than the first appearance of clinical symptoms in AD, it is conceivable that glial dysfunction and mis-activation of the innate immune system drives AD type neurodegeneration.However, very few studies have directly addressed how inflammation per se drives Ab or tau pathology.To characterize the role of innate immunefactors on amyloid b (Ab) plaque and tau pathology, we overexpressed several mouse cytokines, chemokine receptors and soluble innate immune receptors in Amyloid precursor protein (APP) expressingTgCRND8 mice or tau transgenic mice. Methods: Using adeno associated virus (AAV2/1) mediated gene delivery in neonatal mice brain, we effectively demonstrate that this technique allows us to achieve high levels of transgene expression while 1) bypassing the need to create bigenic mice to test hypotheses in a rapid cost-effective manner, 2) testing different mediators on a uniform genetic background and 3) modeling cell non autonomous signaling in vivo. Results: We show that in TgCRND8 mice brain, expression of 1) inflammatory cytokines reduce Ab plaques; 2) anti-inflammatory cytokines exacerbate Ab pathology; and 3) modulating the innate immune receptors using soluble Toll-like receptors (sTLR4 and sTLR5) decrease Ab pathology. On the other hand, over-expression of inflammatory cytokines increase soluble phosphorylated tau while differentially affecting insoluble tau levels in tau transgenic mice. Conclusions: Understanding how innate immune activation states regulate tau and Abpathology could reveal novel therapeutic approaches for AD, including re-purposing current forms of immune therapy already tested in the clinic. Our ongoing studies show that manipulating innate immunity can be a double edged sword, and that development of any innate immune therapy for AD will need to be finely tuned and extensively validated in order to be truly effective. We demonstrate that though engagement of the innate immune system early on during disease pathogenesis may be beneficial in restricting the development of Ab plaques, this may potentially exacerbate tau pathology and negatively affect proteostasis. Therefore, our study strongly argues for a cautious re-examination of unwarranted side-effects of immune based therapies and calls for exploring the therapeutic potential of decoy receptors in preclinical mouse models ofAD as a means of restricting AD pathology while simultaneously minimizing bystander toxicity.

Superoxide anion-triggered chemiluminescence of Cypridina luciferin analogue (CLA), 2-methyl-6-phenyl-3,7-dohydroimidazo[1,2-alpha]pyrazin-3-one, is enhanced by non-ionic detergents such as Tween 20, Triton X-100 and Tween 80. At the concentration of 0.6% (v/v) the largest increase (2.7-fold) of CLA light intensity was obtained with Tween 20, followed by Tween 80 and Triton X-100. Using this detergent-amplified CLA chemiluminescence, the detection limits of xanthine and xanthine oxidase were examined at pH 7.4 and reinvestigated at pH 5.5. At pH 5.5, concentrations of xanthine and xanthine oxidase as low as 5 nmol/L and 3.85 x 10(-7) U/mL, respectively, could be accurately determined, whereas, under the experimental conditions used, at pH 7.4 the lowest concentrations of xanthine and xanthine oxidase detectable were 10 nmol/L and 3.85 x 10(-6) U/mL. The lowest detectable values of xanthine and xanthine oxidase obtained at pH 5.5 are about 400 and 10 times lower than those previously reported. The detection limit of xanthine (5 nmol/L) by this chemiluminescent-based method is about 200 and 20 times more sensitive than the determination of xanthine by enzymatic means or by HPLC with detection limits of 1 micromol/L and 0.1 micromol/L, respectively. Our data suggest that this chemiluminescent probe can detect concentrations of superoxide anion below the nanomolar range.

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of alpha-amylase, beta-amylase, exo-(1--3, 1--4)-beta-D-glucanase, and endo-(1--3)-beta-D-glucanase were detected in these leaves. The endo-(1--3)-beta-D-glucanase (EC 3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pIor = 10.3. Its optimal activity occurred at pH 4.5 and 50 degrees C. Kinetic analysis gave Vmax, kcat, and Km values of 659 U/mg, 395 s(-1), and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1--3)-beta-glucan chains in an endo fashion. This property is unusual for endo-acting (1--3)-beta-D-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for heterocross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed. Chemicals/CAS: Coumaric Acids; Cross-Linking Reagents; ferulic acid, 1135-24-6; Horseradish Peroxidase, EC 1.11.1.-; Hydrogen Peroxide, 7722-84-1; Tyrosine, 55520-40-6

A cerium(IV)-induced Cypridina luciferin analog (CLA) chemiluminescence is reported. This Ce(IV) triggered luciferin analog CL is enhanced about 5-fold by 0.5% (v/v) Tween 20. Using this enhanced luciferin analog-dependent chemiluminescence, a lower limit detection of 25 nM was determined for Ce(IV). Ce(III) could not generate this luciferin analog-dependent chemiluminescence. Oxidising agents such as potassium permanganate and potassium dichromate were also without effect. While the tested scavengers of reactive species such as mannitol, superoxide dismutase and catalase had no specific effect on the luminescence, this Ce(IV)-elicited luciferin analog chemiluminsecent reaction was inhibited when the reaction was performed under anaerobic condition, indicating that molecular oxygen participates in the reaction mechanism leading to the formation of the light-emitting product. A mechanism is proposed for this chemiluminescent reaction which is consistent with the presented data.

Curculigo pilosa is traditionally used in the manufacture of sorghum beer in West Africa. β-Amylase was purified 100-fold with 38% yield from a crude extract, giving final specific activities of 4850 U/mg and 5650 U/mg using soluble starch and p-nitrophenyl maltopentaoside, respectively, as substrates. The molecular mass of the monomeric enzyme was 64 kDa and its pI 4.2. Both activity and thermostability are higher than reported for other plant β-amylases. The catalytic efficiency was lower for amylose than for starches and amylopectin. In contrast to other plant amylases, the β-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In this respect, it resembles β-amylases from microbial origin. This property, and its high activity and stability, explain its traditional use in the manufacture of infant food and sorghum beer in Burkina Faso and could make it applicable for other biotechnological purposes.

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. (C) 2014 Elsevier B.V. All rights reserved.

Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β (Aβ), which is associated with a neuroinflammatory response involving microglia and astrocytes. This neuroinflammatory response has detrimental effects on disease progression but also has a beneficial function on removal of excess Aβ. Microglia and astrocytes are involved in the clearance of Aβ from the brain, but neuroinflammation also promotes neurodegeneration. In order to identify signal transduction pathways critically involved in AD we analysed human brain tissue using protein kinase activity profiling. We identified increased activity of the Interleukin 1 Receptor Associated Kinase 4 (IRAK-4) in AD compared to control brain tissue. IRAK-4 is a component of the signal transduction pathway that functions downstream of the Toll-like receptors and the interleukin-1 receptor. Immunohistochemical analysis of human brain tissue revealed the presence of IRAK-4 in astrocytes and microglia. Quantification of IRAK-4 and the phosphorylated form of IRAK-1, a specific substrate for IRAK-4, revealed increased expression and activity of IRAK-4 in AD. Interestingly, IRAK-1/4 inhibitor I reduces the lipopolysaccharide-induced secretion of monocyte chemotactic protein-1 (MCP-1) by primary human microglia and the interleukin-1β-induced secretion of MCP-1 and interleukin 6 by primary human astrocytes. In contrast, the uptake of Aβ by astrocytes and microglia is not affected by IRAK-1/4 inhibition. Our data show that IRAK-4 protein kinase activity is increased in AD and selective inhibition of IRAK-1/4 inhibits a pro-inflammatory response without affecting the uptake of Aβ by glial cells, indicating that the IRAK signalling pathway is a potential target for modulating neuroinflammation in AD.

Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments.

Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments.

In the system composed of the cationic surfactant TOMAC (10 mM), the nonionic (co)surfactant Rewopal HV5 (2 mM), and octanol (0.1% v/v) in isooctane, reversed micelles are formed upon contact with an aqueous phase containing 50 mM ethylene diamine. alpha-Amylase can be transferred from the aqueous phase into reversed micelles in the pH range 9.5 to 10.5 and re-extracted into a second aqueous phase of different composition. The size of the reversed micelles (as reflected in the water content of the organic phase) can be varied by changes in percentage of octanol, type of counterion in the aqueous phase, or in the number of ethoxylate head groups of the nonionic surfactant. An increase in size results in transfer at lower pH values. Experiments in which the charge density in the reversed micellar interface was changed by incorporation of charged derivatives of the nonionic surfactant, without influencing the water content, revealed that an increased charge density facilitated transfer, resulting in a broader transfer profile. Replacement of TOMAC by other quaternary ammonium surfactants differing in number and length of tails revealed that, of the 14 surfactants tested, only 2 gave appreciable amounts of transfer. The amount of transfer is related to the dynamics of phase separation of the surfactants: those giving a poor phase separation inactivate the enzyme. This inactivation is caused by electrostatic interactions between the charged surfactant head groups and charged groups on the enzyme. Electrostatic interactions are the first step of transfer, and can result in either incorporation in a reversed micelle, or, if reversed micelle formation is slow, in enzyme inactivation. (c) 1995 John Wiley & Sons, Inc.

Background: Humoral immunity related to neuronand disease-specific cytoskeletal proteins in patients with Alzheimer’s disease (AD) is largely unknown. Methods: Antibodies against three targets [tau, light (NFL) and heavy (NFH) neurofilaments] were measured using ELISA in 80 paired serum and CSF samples from 25 patients with AD fulfilling NINCDSADRDA criteria, 13 patients with other dementias (OD), 25 cognitively normal elderly controls (NC), 17 patients with neuroinflammatory diseases (IC). We estimated intrathecal synthesis of all antibodies according to formula (CSF anti-neurocytoskeletal IgG /serum anti-neurocytoskeletal IgG)/ (CSF total IgG /serum total IgG).Results:TheAD patients had significantly higher intrathecal anti-tau antibodies than in the OD group (p 1⁄4 0.005) or the IC group (P 1⁄4 0.0003) and higher intrathecal anti-NFH antibodies than in the OD group (P 1⁄4 0.046), the NC group (p 1⁄4 0.04) or the IC group (P 1⁄4 0.0007), but not intrathecally synthesized anti-NFL antibodies. All three anti-neurocytoskeletal antibodies in serum or CSF did not differ among four groups with the exceptions for the IC group having them elevated in CSF. Conclusions: These new findings suggest some hints of specific alterations in humoral anti-neurocytoskeletal immunity and selectivity in AD. It may have diagnostic and immunotherapeutic implications.

The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV circular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure.

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Km for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Km for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches. Public Library of Science

Triple-negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined ‘omics’ approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi-dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches.

In “conventional” liquid-liquid extraction of protein solutions with a reversed micellar phase, the protein is transferred from an aqueous phase to the reversed micellar phase by forward extraction, and subsequently to a second aqueous phase during back extraction. This back transfer is a relatively slow process, owing to a large interfacial resistance to mass transfer. In this paper an alternative procedure is described for the back extraction, using the effect of temperature. By increasing the temperature of the reversed micellar phase, after it has been saturated with the aqueous phase during the forward extraction, a separate aqueous phase is formed in which most of the enzyme is concentrated. This excess aqueous phase can be separated easily from the reversed micellar phase by centrifugation. The enzyme can be recovered in this expelled phase at extremely high concentrations (up to 2000 times the initial concentration). This process has been performed continuously using two centrifugal separators, with the reversed micellar phase circulating between the two units. For α-amylase a recovery rate of 73% of the enzyme activity was achieved.

The rate of mass transfer in the liquid—liquid extraction of the enzyme α-amylase between an aqueous phase and a reversed micellar phase has been investigated. Mass transfer rate coefficients have been measured in a mixer/settler and in a stirred cell. The pH of the aqueous phase determines the distribution coefficient of the enzyme and thus the direction of transfer. Forward transfer of the enzyme from the aqueous to the reversed micellar phase (cationic surfactant) occurs at pH 10.0. The mass transfer rate of this process was found to be controlled by the diffusion of the enzyme in the aqueous phase boundary layer. Back transfer from the reversed micellar phase to the aqueous phase occurs in the pH range 4–6. In contrast to the forward transfer, this process was found to be controlled by the interfacial process of enzyme release from the reversed micelles instead of the boundary layer diffusion. The same effects have been observed for the transfer of the enzyme ribonuclease A to and from a reversed micellar phase with an anionic surfactant. A mechanism to explain the different mass transfer behaviour in forward and back transfer is suggested.

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.

Ethnobotanical and biochemical surveys revealed that some local plants from West Africa are novel sources of polysaccharide degrading enzymes such as amylases and glucanases. The study shows that these enzymes could be used for various biotechnological applications. In a crude extract ofCurculigo pilosa, ß-amylase was the main starch hydrolyzing enzyme. Contrary to other plant amylases, the ß-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In the bulbs of Gladiolus klattianus, activities of a-amylase and ß-amylase were found. Analysis of the enzyme action pattern showed that it released only maltose units from starch. Activities of aamylase, ß-amylase, exo-(1®3, 1®4)-ß-D-glucanase and endo-(1®3)-ß-D-glucanase were detected in the leaves of Boscia senegalensis. The combined action of a saccharogenic enzyme (ß-amylase) and a dextrinizing enzyme (a-amylase) in B. senegalensis was useful to decrease the viscosity of cerealporridges and to increase their reducing sugar contents. The effective technological utilization of these higher plants as sources of carbohydrate degrading enzymes is discussed.

Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three heterooligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented. Chemicals/CAS: Coumaric Acids; ferulic acid, 1135-24-6; Glycine, 56-40-6; Horseradish Peroxidase, EC 1.11.1.-; Hydrogen Peroxide, 7722-84-1; Oxygen, 7782-44-7; Peptides; Tyrosine, 55520-40-6