Time dependent effect of cold ischemia on the phosphoproteome and protein kinase activity in fresh-frozen colorectal cancer tissue obtained from patients.

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 < 0.01 and fold change > 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 < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 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.

Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration.

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.

Increased occurrence of protein kinase CK2 in astrocytes in Alzheimer's disease pathology.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease. In addition to the occurrence of amyloid deposits and widespread tau pathology, AD is associated with a neuroinflammatory response characterized by the activation of microglia and astrocytes. Protein kinase 2 (CK2, former casein kinase II) is involved in a wide variety of cellular processes. Previous studies on CK2 in AD showed controversial results, and the involvement of CK2 in neuroinflammation in AD remains elusive. METHODS: In this study, we used immunohistochemical and immunofluorescent staining methods to investigate the localization of CK2 in the hippocampus and temporal cortex of patients with AD and non-demented controls. We compared protein levels with Western blotting analysis, and we investigated CK2 activity in human U373 astrocytoma cells and human primary adult astrocytes stimulated with IL-1ß or TNF-α. RESULTS: We report increased levels of CK2 in the hippocampus and temporal cortex of AD patients compared to non-demented controls. Immunohistochemical analysis shows CK2 immunoreactivity in astrocytes in AD and control cases. In AD, the presence of CK2 immunoreactive astrocytes is increased. CK2 immunopositive astrocytes are associated with amyloid deposits, suggesting an involvement of CK2 in the neuroinflammatory response. In U373 cells and human primary astrocytes, the selective CK2 inhibitor CX-4945 shows a dose-dependent reduction of the IL-1ß or TNF-α induced MCP-1 and IL-6 secretion. CONCLUSIONS: This data suggests that CK2 in astrocytes is involved in the neuroinflammatory response in AD. The reduction in pro-inflammatory cytokine secretion by human astrocytes using the selective CK2 inhibitor CX-4945 indicates that CK2 could be a potential target to modulate neuroinflammation in AD.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

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.

Analysis of steady-state Förster resonance energy transfer data by avoiding pitfalls: interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides.

Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2'/3'-(N-methyl-anthraniloyl)-adenosine-5'-triphosphate (MANT-ATP) and enzymes is widely used to determine affinities for ATP-protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT-ATP, MANT-ADP [2'/3'-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT-AMP [2'/3'-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT-ATP tightly with a Kd of 15 to 25nM and excluded the presence of a second binding site. The affinity for MANT-ADP is also tight with a Kd of 50 to 80nM, whereas MANT-AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT-ATP-γ-S [2'/3'-O-(N-methylanthraniloyl) adenosine-5'-(thio)- triphosphate] yielded a Kd of 30 to 50nM. The methods demonstrated here are applicable to other enzyme-fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins.

Differentiating Benign from Malignant Thyroid Tumors by Kinase Activity Profiling and Dabrafenib BRAF V600E Targeting.

Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers.

Protein kinase activity profiling of postmortem human brain tissue.

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.

Kinase activity profiling identifies putative downstream targets of cGMP/PKG signaling in inherited retinal neurodegeneration.

Inherited retinal diseases (IRDs) are a group of neurodegenerative disorders that lead to photoreceptor cell death and eventually blindness. IRDs are characterised by a high genetic heterogeneity, making it imperative to design mutation-independent therapies. Mutations in a number of IRD disease genes have been associated with a rise of cyclic 3',5'-guanosine monophosphate (cGMP) levels in photoreceptors. Accordingly, the cGMP-dependent protein kinase (PKG) has emerged as a new potential target for the mutation-independent treatment of IRDs. However, the substrates of PKG and the downstream degenerative pathways triggered by its activity have yet to be determined. Here, we performed kinome activity profiling of different murine organotypic retinal explant cultures (diseased rd1 and wild-type controls) using multiplex peptide microarrays to identify proteins whose phosphorylation was significantly altered by PKG activity. In addition, we tested the downstream effect of a known PKG inhibitor CN03 in these organotypic retina cultures. Among the PKG substrates were potassium channels belonging to the Kv1 family (KCNA3, KCNA6), cyclic AMP-responsive element-binding protein 1 (CREB1), DNA topoisomerase 2-α (TOP2A), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (F263), and the glutamate ionotropic receptor kainate 2 (GRIK2). The retinal expression of these PKG targets was further confirmed by immunofluorescence and could be assigned to various neuronal cell types, including photoreceptors, horizontal cells, and ganglion cells. Taken together, this study confirmed the key role of PKG in photoreceptor cell death and identified new downstream targets of cGMP/PKG signalling that will improve the understanding of the degenerative mechanisms underlying IRDs.

Technological advancements to study cellular signaling pathways in inherited retinal degenerative diseases.

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.

Kinomic profiling of tumour xenografts derived from patients with non-small cell lung cancer confirms their fidelity and reveals potentially actionable pathways.

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.

KRSA: An R package and R Shiny web application for an end-to-end upstream kinase analysis of kinome array data.

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.

Evaluation of the neurotoxic effects of engineered nanomaterials in C57BL/6J mice in 28-day oral exposure studies.

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.

Erratum: Krayem, M., et al. Kinome Profiling to Predict Sensitivity to MAPK Inhibition in Melanoma and to Provide New Insights into Intrinsic and Acquired Mechanism of Resistance Short Title: Sensitivity Prediction to MAPK Inhibitors in Melanoma. Cancers 2020, 12, 512.

The authors would like to make a correction to their published paper [...].

Kinome Profiling to Predict Sensitivity to MAPK Inhibition in Melanoma and to Provide New Insights into Intrinsic and Acquired Mechanism of Resistance.

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 genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis.

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.

Development of selective bisubstrate-based inhibitors against protein kinase C (PKC) isozymes by using dynamic peptide microarrays.

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.

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A.

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.

Inactive immune pathways in triple negative breast cancers that showed resistance to neoadjuvant chemotherapy as inferred from kinase activity profiles.

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.

Biochemical characterization of the major sorghum grain peroxidase.

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.

Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.

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.