Kinase Peptide Research Articles

Hydrogen Attachment/Abstraction Dissociation (HAD) of Gas-Phase Peptide Ions for Tandem Mass Spectrometry.

Dissociation of gas-phase peptide ions through interaction with low-energy hydrogen (H) radical (∼0.15 eV) was observed with a quadrupole ion trap mass spectrometry. The H radical generated by thermal dissociation of H2 molecules passing through a heated tungsten capillary (∼2000 °C) was injected into the ion trap containing target peptide ions. The fragmentation spectrum showed abundant c-/z- and a-/x-type ions, attributable to H attachment/abstraction to/from peptide ion. Because the low-energy neutral H radical initiated the fragmentation, the charge state of the precursor ion was maintained during the dissociation. As a result, precursor ions of any charge state, including singly charged positive and negative ions, could be analyzed for amino acid sequence. The sequence coverage exceeding 90% was obtained for both singly protonated and singly deprotonated substance P peptide. This mass spectrometry also preserved labile post-translational modification bonds. The modification sites of triply phosphorylated peptide (kinase domain of insulin receptor) were identified with the sequence coverage exceeding 80%.

Abstract P6-08-05: Protein tyrosine kinase activity and miRNA expression profiling reveals differences according to progesterone-receptor-status in HER-2 negative breast cancer

Abstract Background: Kinases are considered as promising source of biomarkers for diagnostic, prognostic and therapeutic purposes in cancer patients. We assessed tyrosine kinase activity in 39 primary breast cancer samples that were all hormone receptor positive (ER+ and/or PR+) with differential HER-2 status, using microarray technology. Methods: Pamchip® peptide microarrays were used to measure the activity of protein tyrosine kinases in 32 breast cancer samples. The breast cancer cell lines MCF-7, BT474 and ZR75-1, was studied for kinase activity, both untreated and treated with estradiol. Results: Results showed differences in phosphorylation amongst breast cancer samples. A total of 37 peptide kinases were highly phosphorylated in a group of breast cancer samples representing 33 protein tyrosine kinases involved in cancer pathways and immunological responses. In vitro studies with breast cancer cell lines exhibited the same phosphorylation profiles, but increased phosphorylation was only observed in one cell line, ZR75-1. Eliminating HER-2 positive samples, we obtained differences in phosphorylation profiles based on PR-status only. Samples lacking PR-expression exhibited higher kinase activity of downstream kinases compared to PR-positive samples. Similar results were obtained with miRNA expression profiles of 31 breast cancer samples. Five miRNAs were identified to be significantly differentially expressed (p < 0.05) between PR-negative and PR-positive samples. This effect was even stronger when eliminating HER-2 positive samples, with 13 miRNAs exhibiting significant differential expression based on PR-status. Conclusion: Although our data are based on a small dataset, the lack of PR expression seems to have a profound effect on tyrosine kinase activity and miRNA expression in HER-2 negative breast cancers without any effect on gene expression. This indicates that regulatory and functional molecules might exhibit phenotypical features of cancer that cannot be explained by gene expression alone. Citation Format: Tahiri A, Satheesh SV, de Wijn R, Lders T, Aure MR, Quigley DA, Bukholm IR, Hurtado A, Kristensen VN, Geisler J. Protein tyrosine kinase activity and miRNA expression profiling reveals differences according to progesterone-receptor-status in HER-2 negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-05.

Are There Deleterious Cardiac Effects of Acute and Chronic Endurance Exercise?

Multiple epidemiological studies document that habitual physical activity reduces the risk of atherosclerotic cardiovascular disease (ASCVD), and most demonstrate progressively lower rates of ASCVD with progressively more physical activity. Few studies have included individuals performing high-intensity, lifelong endurance exercise, however, and recent reports suggest that prodigious amounts of exercise may increase markers for, and even the incidence of, cardiovascular disease. This review examines the evidence that extremes of endurance exercise may increase cardiovascular disease risk by reviewing the causes and incidence of exercise-related cardiac events, and the acute effects of exercise on cardiovascular function, the effect of exercise on cardiac biomarkers, including "myocardial" creatine kinase, cardiac troponins, and cardiac natriuretic peptides. This review also examines the effect of exercise on coronary atherosclerosis and calcification, the frequency of atrial fibrillation in aging athletes, and the possibility that exercise may be deleterious in individuals genetically predisposed to such cardiac abnormalities as long QT syndrome, right ventricular cardiomyopathy, and hypertrophic cardiomyopathy. This review is to our knowledge unique because it addresses all known potentially adverse cardiovascular effects of endurance exercise. The best evidence remains that physical activity and exercise training benefit the population, but it is possible that prolonged exercise and exercise training can adversely affect cardiac function in some individuals. This hypothesis warrants further examination.

Impact of Interatrial Communication on Left Ventricle Performance in Patients with Significant Post-tricuspid Shunt.

Infants with post-tricuspid valve shunts (PTS) may benefit from interatrial communication (IAC). The effect of IAC on left ventricular (LV) performance in these patients was studied. IAC was documented prospectively in 55 patients with PTS. Clinical status, echocardiographic dimensions of LV, mitral inflow Doppler, tissue Doppler velocities and time intervals were measured. Creatinine kinase (CK), CKMB, troponin-I and NT pro-brain natriuretic peptide (NT pro-BNP) were measured. Patients were divided into four groups: (A) PTS but no IAC (n = 32); (B) PTS and IAC (n = 23); (C) VSD but no IAC (n = 16); and (D) VSD and IAC (n = 19). Group A had more frequent mitral regurgitation (p = 0.041), larger mitral annulus (1.80 vs. 1.30 cm, p < 0.0001) and larger LV systolic and diastolic dimensions (2.01 vs. 1.40 and 3.28 vs. 2.35 cm, p < 0.001) than group B. The E-wave deceleration time tended to be longer in group A (121.0 vs. 106.8 ms, p = 0.06). By tissue Doppler, group A had E'- and S-waves significantly taller (15.51 vs. 13.14 and 7.69 vs. 6.72 cm, p = 0.04 and p = 0.005, respectively) than group B. Also, NT pro-BNP was significantly higher in group A (1116.15 vs. 458.73 pg/ml, p = 0.028). Group C had significant larger mitral z-score values (1.2 vs. 0.01, p < 0.001), larger LV diameter z-score (p = 0.001) and higher NT pro-BNP level (1477.37 vs. 451.66 pg/ml, p = 0.001) than group D. There was no significant difference in the clinical status between the groups. In children with PTS, the presence of IAC could be beneficial. Their echocardiographic parameters and biomarker show better systolic and diastolic LV performance.

Phosphorylation of Nucleophosmin at Threonine 234/237 is associated with HCC metastasis

Hepatocellular carcinoma (HCC) is frequently complicated by the occurrence of intrahepatic and extrahepatic metastases, leading to poor prognosis. To improve the prognosis for HCC patients, there is an urgent need to understand the molecular mechanisms of metastasis in HCC. Since protein Serine/Threonine phosphorylation emerges to be an important posttranslational modification critical in signaling process associated with cell proliferation, survival and metastasis, we employed a pair of primary tumor-derived and corresponding lung-metastatic counterparts (PLC/PRF/5-PT and PLC/PRF/5-LM) and aimed to identify these changes using CelluSpot Serine/Threonine kinase peptide array. Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr234/237 was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wild-type NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr234/237 phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway. Identification of CDK1/p-NPM/ROCK2 signaling pathway provides a novel target for molecular therapy against HCC metastasis.

Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site.

BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa) but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild-type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases.

Neurogenic stunned myocardium - do we consider this diagnosis in patients with acute central nervous system injury and acute heart failure?

Neurogenic stunned myocardium (NSM) is defined as myocardial injury and dysfunction of a sudden onset, occurring after various types of acute brain injury as a result of an imbalance in the autonomic nervous system. The typical spectrum of clinically observed abnormalities includes acute left ventricular failure, not uncommonly progressing to cardiogenic shock with hypotension that requires inotropic agents, pulmonary oedema and various arrhythmias. Commonly-seen electrocardiographic changes include: prolonged QT interval, ST segment changes, T-wave inversion, a new Q-wave or U-wave. Echocardiography shows both an impaired both systolic and diastolic function of the left ventricle. Biochemical markers of NSM comprise metabolic acidosis and increased cardiac enzymes and markers: creatine kinase (CK), and CK-MB, troponin I and B-type natriuretic peptide. The main cause of NSM is myocardial injury induced by local catecholamine release from nerve endings within the myocardium. Recently, a theory has been proposed to classify NSM as one of the stress-related cardiomyopathies, together with Takotsubo cardiomyopathy, acute left ventricular failure in the critically ill, cardiomyopathy associated with pheochromacytoma and exogenous catecholamine administration. The occurrence of NSM increases the risk of life-threatening complications, death, and worsens neurologic outcome. As far as we know, treatment should generally focus on the underlying neurologic process in order to maximize neurologic recovery. Improvement in neurologic pathology leads to rapid improvement in cardiac function and its full recovery, as NSM is a fully reversible condition if the patient survives. Awareness of the existence of NSM and a deeper knowledge of its etiopathology may reduce diagnostic errors, optimise its treatment.

Treatment Options for Advanced Pancreatic Neuroendocrine Tumors: What is on the Horizon?

Abstract: Treatment options for patients with advanced or metastatic pancreatic tumors (pNET) have expanded greatly in recent years. These treatments include resection, ablation or embolization of liver metastases and systemic therapy with everolimus or sunitinib and cytotoxic agents. New investigational approaches include the use of inhibitors of multiple downstream effectors in the PI3K/Akt/mTOR pathway, novel antiangiogenics, somatostatin analogues, new tyrosine kinase inhibitors and peptide receptor radionuclide therapy. The treatment horizon for pNET patients may offer improved duration of tumor control and survival and more effective symptom control.

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition.

Efficacy of different doses of atorvastatin treatment on serum levels of 8-hydroxy-guanin (8-OHdG) and cardiac function in patients with ischemic cardiomyopathy.

Objective: To compare the efficacy of 40 mg and l0 mg atorvastatin on serum levels of 8-Hydroxy-Guanin (8-OHdG) and the cardiac function in patients with ischemic cardiomyopathy (ICM).Methods:One hundred twenty three hospitalized ICM patients and 120 healthy controls were included in this study. All subjects were randomly divided into two groups: 10 mg/d atorvastatin group (n=62) and 40 mg/d atorvastatin group (n=61). Serum levels of C-reactive protein (CRP), creatine kinase, glutamic-pyruvic transaminase, lipids and B-type natriuretic peptide (BNP) were tested in all subjects both at the initial phase and the terminal phase of this study. Adverse drug reaction events were recorded in this study. Echocardiographic method was applied to compare the cardiac function before and after treatment in the double blind study. Serum 8-OHdG levels were tested by enzyme-linked immunosorbent assay (ELISA) before and after treatment, and the results in atorvastatin treatment groups were compared with the healthy controls.Results:Serum 8-OHdG levels in ICM patients were significantly higher than that in normal control groups (p<0.05). There was significant difference of Serum 8-OHdG levels in 40 mg/d atorvastatin group (p<0.05), but was no significant difference in 10 mg/d atorvastatin group before and after the treatment. The 8-OHdG level in 40 mg/d atorvastatin group was significantly lower than that in 10 mg/d atorvastatin group before the treatment as well as after the treatment (p<0.05). The systolic and diastolic function improved significantly in 40 mg/d atorvastatin group before and after treatment, as well as in comparison with 10 mg/d atorvastatin group (p<0.05).Conclusion:Serum 8-OHdG possibly plays an important role in the pathogenesis of ICM. Atorvastatin is safe and effective in ICM treatment; furthermore atorvastatin which also has independent lipid lowering effect, is significantly better in the dose of 40 mg/day.

Study of the affinity between the protein kinase PKA and peptide substrates derived from kemptide using molecular dynamics simulations and MM/GBSA.

We have carried out a protocol in computational biochemistry including molecular dynamics (MD) simulations and MM/GBSA free energy calculations on the complex between the protein kinase A (PKA) and the specific peptide substrate Kemptide (LRRASLG). We made the same calculations on other PKA complexes that contain Kemptide derivatives (with mutations of the arginines, and with deletions of N and C-terminal amino acids). We predicted shifts in the free energy changes from the free PKA to PKA-substrate complex (ΔΔGE→ES) when Kemptide structure is modified (we consider that the calculated shifts correlate with the experimental shifts of the free energy changes from the free PKA to the transition states (ΔΔGE→TS) determined by the catalytic efficiency (kcat/KM) changes). Our results demonstrate that it is possible to predict the kinetic properties of protein kinases using simple computational biochemistry methods. As an additional benefit, these methods give detailed molecular information that permit the analysis of the atomic forces that contribute to the affinity between protein kinases and their substrates.

Structural Basis for Phosphorylation and Lysine Acetylation Cross-talk in a Kinase Motif Associated with Myocardial Ischemia and Cardioprotection

Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.

Fungi on the skin: dermatophytes and Malassezia.

Several human skin diseases and disorders are associated with two groups of fungi, the dermatophytes and Malassezia. Although these skin-related problems are not generally life threatening, they are among the most common diseases and disorders of mankind. These fungi are phylogenetically divergent, with the dermatophytes within the Ascomycota and Malassezia within Basidiomycota. Genome analysis indicates that the adaptations to the skin environment are different in these two groups of fungi. Malassezia are dependent on host lipids and secrete lipases and phospholipases that likely release host fatty acids. The dermatophytes encode multiple enzymes with potential roles in modulating host interactions: polyketide synthases, nonribosomal peptide synthetases, LysM, proteases, kinases, and pseudokinases. These two fungal groups have maximized their interactions with the host using two very different mechanisms.

Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis, and Biological Evaluation.

Strategies to inhibit kinases by targeting the substrate binding site offer many advantages, including naturally evolved selectivity filters, but normally suffer from poor potency. In this work we propose a strategy to design and prepare covalent substrate-competitive kinase inhibitors as a method to improve potency. We have chosen AKT as the model kinase for this work. Using the AKT-GSK3β cocrystal structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.

Safety and Efficacy of Extracorporeal Shock Wave Myocardial Revascularization Therapy for Refractory Angina Pectoris

To assess the safety and efficacy of extracorporeal shockwave myocardial revascularization (ESMR) therapy in treating patients with refractory angina pectoris. A single-arm multicenter prospective trial to assess safety and efficacy of the ESMR therapy in patients with refractory angina (class III/IV angina) was performed. Screening exercise treadmill tests and pharmacological single-photon emission computed tomography (SPECT) were performed for all patients to assess exercise capacity and ischemic burden. Patients were treated with 9 sessions of ESMR to ischemic areas over 9 weeks. Efficacy end points were exercise capacity by using treadmill test as well as ischemic burden on pharmacological SPECT at 4 months after the last ESMR treatment. Safety measures included electrocardiography, echocardiography, troponin, creatine kinase, and brain natriuretic peptide testing, and pain questionnaires. Fifteen patients with medically refractory angina and no revascularization options were enrolled. There was a statistically significant mean increase of 122.3±156.9 seconds (38% increase compared with baseline; P=.01) in exercise treadmill time from baseline (319.8±157.2 seconds) to last follow-up after the ESMR treatment (422.1±183.3 seconds). There was no improvement in the summed stress perfusion scores after pharmacologically induced stress SPECT at 4 months after the last ESMR treatment in comparison to that at screening; however, SPECT summed stress score revealed that untreated areas had greater progression in ischemic burden vs treated areas (3.69±6.2 vs 0.31±4.5; P=.03). There was no significant change in the mean summed echo score from baseline to posttreatment (0.4±5.1; P=.70). The ESMR therapy was performed safely without any adverse events in electrocardiography, echocardiography, troponins, creatine kinase, or brain natriuretic peptide. Pain during the ESMR treatment was minimal (a score of 0.5±1.2 to 1.1±1.2 out of 10). In this multicenter feasibility study, ESMR seems to be a safe and efficacious treatment for patients with refractory angina pectoris. However, larger sham-controlled trials will be required to confirm these findings.

The Carboxy-terminus of BAK1 regulates kinase activity and is required for normal growth of Arabidopsis

Binding of brassinolide to the brassinosteroid-insenstive 1(BRI1) receptor kinase promotes interaction with its co-receptor, BRI1-associated receptor kinase 1 (BAK1). Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C)-terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1 in vitro. Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576–615 of BAK1) interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag) in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.

Synthesis of novel, peptidic kinase inhibitors with cytostatic/cytotoxic activity

The utility of a novel, chemoenzymatic procedure for the stereocontrolled synthesis of small peptides is presented in the preparation and structure optimisation of dipeptides with cytostatic/cytotoxic activity. The method uses Passerini multicomponent reaction for the preparation of racemic scaffold which is then enantioselectively hydrolysed by hydrolytic enzymes. Products of these transformations are further functionalised towards title compounds. Both activity and selectivity towards tumor cells is optimised. Final compound is shown to be an inhibitor of the protein kinase signaling pathway.

Structural insights into calmodulin/adenylyl cyclase 8 interaction

Calmodulin (CaM) is a highly conserved intracellular Ca(2+)-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca(2+)-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca(2+) sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

Serum dihydroxyacetone kinase peptide m/z 520.3 as predictor of disease severity in patients with compensated chronic hepatitis B

Background & aimDue to known limitations of liver biopsy, reliable non-invasive serum biomarkers for chronic liver diseases are needed. We performed serum peptidomics for such investigation in compensated chronic hepatitis B (CHB) patients.MethodsLiquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed peptides in sera from 40 CHB patients (20 with S0G0-S1G1 and 20 with S3G3-S4G4). Ion pair quantification from differentially expressed peptides in a validation set of sera from 86 CHB patients was done with multiple reaction monitoring (MRM).Results21 differentially represented peptide peaks were found through LC-MS/MS. Ion pairs generated from eleven of these peptides (m/z < 800) were quantified by MRM. Summed peak area ratios of 6 ion pairs from peptide m/z 520.3 (176.1, 353.7, 459.8, 503.3, 351.3, 593.1), which was identified as dihydroxyacetone kinase (DAK) fragment, decreased from mild to advanced stages of fibrosis or inflammation. Area Under Receiver Operating Characteristic Curves (AUROCs) of five ion models discriminating fibrosis degrees were 0.871 ~ 0.915 (S2-4 versus S0-1) and 0.804 ~ 0.924 (S3-4 versus S0-2). AUROCs discriminating inflammation grades were 0.840 ~ 0.902 (G2-4 versus G0-1) and 0.787 ~ 0.888 (G3-4 versus G0-2). The diagnostic power of these models provides improved sensitivity and specificity for predicting disease progression as compared to aspartate aminotransferase to platelet ratio index (APRI), FIB-4, Forn’s index and serum DAK protein.ConclusionsThe peptide fragment (m/z 520.3) of DAK is a promising biomarker to guide timing of antiviral treatment and to avoid liver biopsy in compensated CHB patients.

Kinase-sensing system to identify the oncogenic phospho-fingerprint of breast cancer.

23 Background: Kinases and their substrates harness myriad critical molecular pathways. A new highly valuable assay would be one detecting the true active state of phospho-signaling networks. To address this technological and knowledge gap, we developed a semi-high throughput sensing system that monitors the catalytic activity of kinases using peptides as phosphorylation targets/probes. We used this assay to evaluate the presence of dysfunctional kinases in biological extracts and identify oncogenic phospho-signatures prevalent in breast cancer. Methods: We developed a human kinase protein and peptide repertoire computationally curated from thousands of publications. Out of the 2,702 validated peptide targets and 6,173 kinase-substrate nodes we compiled, we chose a subset of 151 biological peptides predicted to specifically report on tyrosine / serine / threonine kinases, including EGFR, ERK, AKT, SRC, ABL. Along with 91 control peptides, we tested a total of 242 peptides in a luminescence ATP-consumption assay for their ability to report on the kinases’ activity. The assay was further validated using an isogenic culture model of basal like breast cancer (HMT-3522 S1 and T4-2), and cell lines harboring various oncogenic alterations (MDA-MB-231, MCF7, T47D, AU565, ZR75.1, SKBR3). Results: The differential phosphorylation activity of recombinant kinase enzymes was successfully detected in time course, dilution and inhibitor experiments. The increased presence of (hyper-) activated SRC, EGFR, AKT or ABL kinases observed by western blots in subsets of breast cancer cells, was selectively, repeatedly and specifically detected using the peptide-based kinase assay. Significantly altered peptide-phosphorylation patterns were successfully integrated into signatures using gene expression microarray-like analysis, revealing the breast cancer-associated map of phosphorylation circuits. Conclusions: This assay is suitable to comprehensively capture the activity of kinase signaling networks. We will now use this functional proteomic assay to identify the active, oncogenic kinome of breast cancer, with the objective to improve clinical guidance and individualize breast cancer patients’ therapy.