PK1 from Drosophila obscurin is an inactive pseudokinase with scaffolding properties.

Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.

High and individually variable enzymatic activity precludes accurate determination of pemetrexed, methotrexate and their polyglutamate metabolite concentrations in plasma.

Most drugs are metabolized in the human body. Therefore, it is essential for therapeutic drug monitoring studies to also take into account the concentrations of drug metabolites. One of the possible metabolic activities on drugs such as pemetrexed or methotrexate is (poly)glutamation. Here, we report on a series of experiments that we performed to investigate the stability of polyglutamate metabolites in plasma. Removal of glutamate residues from pemetrexed polyglutamate by most likely proteases in human plasma is influenced by temperature as it is observed at 25°C and even more strongly at 37°C, but not at 4°C. The observed protease activity is highly variable among patients; in approximately 15-20% of the patients tested it is not observed, whereas in other individuals the activity is so extensive that after 10min, more than 50% of spiked polyglutamated pemetrexed is degraded at room temperature (5-10% of the tested individuals). Similar observations also pertain to methotrexate polyglutamates. These observations do not extend to pemetrexed and methotrexate themselves which are unaffected by this activity. Due to the considerable and, among individuals, variable protease activities on polyglutamated drug metabolites in plasma, these metabolites are virtually impossible to quantify if no precautions are taken.

Pre-screening of crude peptides in a serological bead-based suspension array.

Most serological assays detect antibody responses in biological samples through affinity of serum antibodies for antigens provided in the assay. Certain antigens, however, may be difficult to produce and/or may contain unwanted epitopes. In these cases, a practical alternative may be the use of peptides as representatives for specific epitopes. Peptides can be obtained after purification in large quantities for a modest price, but screening of a large set of peptides during development may be relatively expensive. To cut costs of screening peptides for a new serological assay, the concept was investigated of using cheap non-purified (crude) peptides instead of purified peptides. Peptides were selected that represent three well-described linear epitopes of viral proteins: VP2 of canine parvovirus (CPV), gp41 of human immunodeficiency virus (HIV) and E2 of classical swine fever virus (CSFV). Crude and purified biotinylated peptides with either a short or long spacer between the biotin and the epitope were used to test their capability to bind antibodies in a bead-based suspension array. The results show that, in a bead-based suspension array, crude peptides can function as antigen for specific monoclonal antibodies, and that the acquired signals are less than with purified peptides. CSFV-derived crude peptides were also able to detect specific antibodies in swine serum, indicating the applicability of crude peptides for pre-screening large numbers of different peptides during the development of serological peptide-based assays.

A viral kinase mimics S6 kinase to enhance cell proliferation.

Viruses depend upon the host cell for manufacturing components of progeny virions. To mitigate the inextricable dependence on host cell protein synthesis, viruses can modulate protein synthesis through a variety of mechanisms. We report that the viral protein kinase (vPK) encoded by open reading frame 36 (ORF36) of Kaposi's sarcoma-associated herpesvirus (KSHV) enhances protein synthesis by mimicking the function of the cellular protein S6 kinase (S6KB1). Similar to S6KB1, vPK phosphorylates the ribosomal S6 protein and up-regulates global protein synthesis. vPK also augments cellular proliferation and anchorage-independent growth. Furthermore, we report that both vPK and S6KB1 phosphorylate the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) and that both kinases promote endothelial capillary tubule formation.

A new quantification method for assessing plasma concentrations of pemetrexed and its polyglutamate metabolites.

Currently no quantification method exists for potentially therapeutically relevant polyglutamate metabolites of the drug pemetrexed which is used for the treatment of lung carcinoma patients. We developed and tested an LC-MS/MS-based analytical assay that uses isotope-labeled internal standards to quantify pemetrexed and its (poly)glutamate metabolites in clinical human plasma samples of lung carcinoma patients. UHPLC chromatography and triple quadrupole mass spectrometry showed an LLOQ of 0.2nmol/L for pemetrexed and an LLOQ of 0.5nmol/L for the two metabolites (one glutamate and two glutamate moieties covalently bound to the pemetrexed molecule, for which no other quantification methods have previously been published). The recoveries for PMTX and its metabolites ranged between 30% and 67%. Precision and accuracy at a concentration of 20nmol/L for all four analytes was well below 15% CV. The precision (RSD) in the biological replicates of the separate days (within-run precision) as well as the reproducibility over several days (between-run precision), tested in the range of 5-250nmol/L, were all below 15%. Autosampler, benchtop and freeze-thaw cycle stability of the analytes was also demonstrated. To illustrate the new assay in a relevant biological context, concentrations of pemetrexed and the two metabolites were quantified in plasma samples of lung carcinoma patients treated with pemetrexed. The assay is straightforward, relatively easy to perform, and has potential for use in therapeutic drug monitoring in non-small cell lung carcinoma patients.

Peptide library-based evaluation of T-cell receptor breadth detects defects in global and regulatory activation in human immunologic diseases.

The ability of T-cells to respond to foreign antigens and to appropriately regulate this response is crucial for maintaining immune homeostasis. Using combinatorial peptide libraries, we functionally measured broad T-cell reactivity and observed impaired reactivity in established models of T-cell receptor repertoire restriction and in previously unrecognized disease contexts. By concurrently analyzing T-regulatory and T-effector cells, we show strong functional correlation between these subsets in healthy individuals and, strikingly, that alterations of this balance are associated with T helper type 2 (Th2)-mediated disease in a lymphopenic setting. Finally, we demonstrate that peptide-based priming of polyclonal naive cells with relatively low concentrations skews toward Th2 differentiation. These findings provide unique insight into the pathophysiology and functional consequences of abnormal T-cell repertoires and into differentiation of human naive T-cells.

Plasma hemopexin as a potential regulator of vascular responsiveness to angiotensin II.

This brief review focuses on the functional activities of plasma hemopexin recently recognized by several authors. In particular, the protease-like activity of hemopexin in vitro is linked with downregulation of the vascular angiotensin II receptor in vivo, leading to vascular expansion. Also a potential mechanism of inhibition of hemopexin activity by extracellular adenosine triphosphate is considered.

Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3-dependent invasion in resistant non-small cell lung cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and hampers anti-cancer activity. Here we show that whereas H460 non-small cell lung cancer (NSCLC) cells display canonical TRAIL-dependent apoptosis, A549 and SW1573 NSCLC cells are TRAIL resistant and display pro-tumorigenic activity, in particular invasion, following TRAIL treatment. We exploit this situation to contrast TRAIL effects on the kinome of apoptosis-sensitive cells to that of NSCLC cells in which non-canonical effects predominate, employing peptide arrays displaying 1024 different kinase pseudosubstrates more or less comprehensively covering the human kinome. We observed that failure of a therapeutic response to TRAIL coincides with the activation of a non-canonical TRAIL-induced signaling pathway involving, amongst others, Src, STAT3, FAK, ERK and Akt. The use of selective TRAIL variants against TRAIL-R1 or TRAIL-R2 subsequently showed that this non-canonical migration and invasion is mediated through TRAIL-R2. Short-hairpin-mediated silencing of RIP1 kinase prevented TRAIL-induced Src and STAT3 phosphorylation and reduced TRAIL-induced migration and invasion of A549 cells. Inhibition of Src or STAT3 by shRNA or chemical inhibitors including dasatinib and 5,15-diphenylporphyrin blocked TRAIL-induced invasion. FAK, AKT and ERK were activated in a RIP1-independent way and inhibition of AKT sensitized A549 cells to TRAIL-induced apoptosis. We thus identified RIP1-dependent and -independent non-canonical TRAIL kinase cascades in which Src and AKT are instrumental and could be exploited as co-targets in TRAIL therapy for NSCLC.

Multiple reaction monitoring assay for pre-eclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta.

Although the cause of pre-eclampsia during pregnancy has not been elucidated yet, it is evident that placental and maternal endothelial dysfunction is involved. We previously demonstrated that in early onset pre-eclampsia placental calcyclin (S100A6) expression is significantly higher compared to controls ( De Groot , C. J. ; Clin. Proteomics 2007 , 1 , 325 ). In the current study, the results were confirmed and relatively quantified by using multiple reaction monitoring (MRM) on two peptide fragments of calcyclin. Cells were obtained from control (n = 5) and pre-eclamptic placental (n = 5) tissue collected by laser capture microdissection from formalin-fixed paraffin-embedded (FFPE) material treated with a solution to reverse formalin fixation. Two calcyclin peptides with an extra glycine inserted in the middle of the amino acid sequence were synthesized and used as an internal reference. Data presented show that MRM on laser microdissected material from FFPE tissue material is possible. The developed MRM assay to study quantitative levels of proteins in FFPE laser microdissected cells using nonisotopic-labeled chemical analogs of mass tagged internal references showed that in pre-eclamptic patients elevated levels of calcyclin is observed in placental trophoblast cells compared to normal trophoblast cells. By immunohistochemistry, we were able to confirm this observation in a qualitative manner.

Left ventricular diastolic function assessment from three-dimensional three-directional velocity-encoded MRI with retrospective valve tracking.

PURPOSE: To compare parameters describing left ventricular (LV) diastolic function obtained with three-dimensional (3D) three-directional velocity-encoded (VE) MRI with retrospective valve tracking and two-dimensional (2D) one-directional VE MRI in patients with ischemic heart failure. Second, to compare classification of LV diastolic function, and in particular for discriminating restrictive filling patterns, with both MRI techniques versus Doppler echocardiography. MATERIALS AND METHODS: The 3D and 2D VE MRI early (E) and atrial (A) peak flow rate indices, determined from transmitral waveform analyses, were compared. Also, net forward flow volume per cycle and transmitral regurgitation fraction were determined. Agreement in classifying diastolic filling patterns between 3D and 2D VE MRI versus Doppler echocardiography was evaluated using kappa statistics. RESULTS: The 3D three-directional VE MRI with retrospective valve tracking was statistically significantly different from 2D one-directional VE MRI for net forward flow volume and regurgitation fraction through the mitral valve and all parameters describing the diastolic waveform filling pattern, except for the E deceleration time and E/A filling ratio. Kappa-agreement between 3D three-directional VE MRI with retrospective valve tracking and echocardiography for classifying diastolic filling patterns was superior to 2D one-directional VE MRI and echocardiography (i.e., κ = 0.91 versus κ = 0.79, respectively). CONCLUSION: The 3D three-directional VE MRI with retrospective valve tracking better describes LV diastolic function as compared to 2D one-directional VE MRI in patients with ischemic heart failure.

SUMO assay with peptide arrays on solid support: insights into SUMO target sites.

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.

Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-beta (FSH-beta) using domain Scan and Matrix Scan technology.

This paper describes the application of two novel screening technologies, i.e. Domain Scan (24- and 30-mer peptides) and Matrix Scan (24-mer peptides) technology, in the mapping of a discontinuous epitope on FSH-beta for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan technology (12-mer peptides), showed selective binding to peptide-constructs corresponding to the beta3-loop of FSH in the Domain and/or Matrix Scan. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70, D71, and L73 form the core of the epitope. A Domain Scan performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three beta1-beta3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues 97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan of mAb 17 showed that peptides from four different regions on FSH (1st strand beta3-loop, alpha 1-loop, long alpha2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and Matrix Scan screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the beta3-loop showed measurable binding. Cyclic beta3-loop peptide mimics were found to bind significantly stronger (Kd approximately 5 microM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair beta1-peptide 1oIAIEKEECRFAI21 to the linear beta3-peptide *52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2-3 fold increase in Kd, thus conforming participation of the beta 1-loop in antibody binding for these mAb's.